CN116327907A - An immunotherapy against Mycobacterium tuberculosis - Google Patents

Abstract

The invention relates to the fields of immunology and biomedicine, in particular to immunotherapy of tuberculosis. Specifically, the application of a biological molecule in killing mycobacterium tuberculosis is disclosed, wherein the biological molecule is preferably nucleic acid, protein, polypeptide or the like of STAT4 molecules. The biomolecule may also improve or enhance at least one property selected from the group consisting of: A. enhancing phagocytic capacity of dendritic cells; B. enhancing the migration capacity of dendritic cells; C. improving the HLA-DR surface marker expression of the dendritic cells; D. improving the expression of a CD83 surface marker of the dendritic cells; E. enhancing IFN-gamma secretion ability; F. enhancing the capability of macrophages to kill mycobacterium tuberculosis. The application of the novel biological molecule provides new possibility for the immunotherapy of tuberculosis.

Description

An immunotherapy against Mycobacterium tuberculosis

technical field

The invention relates to the fields of immunology and biomedicine, in particular to the immunotherapy of tuberculosis.

Background technique

Tuberculosis remains one of the major public health problems facing the world and is one of the ten leading causes of death. The 2022 global tuberculosis report released by WHO shows that about a quarter of the world's population is infected with tuberculosis. In 2021, there will be 10.6 million new tuberculosis patients worldwide, and 1.6 million deaths due to tuberculosis. In order to comprehensively address the challenge of tuberculosis, the World Health Organization formulated the "End TB Strategy" in 2015, aiming to reduce the incidence of tuberculosis by 90% and the mortality rate by 95% by 2035. To achieve this ambitious goal, it is not only necessary to rely on precise and standardized treatment of tuberculosis patients, but also to effectively block the spread of tuberculosis among people. Although the whole population is generally susceptible to Mycobacterium tuberculosis, the occurrence of tuberculosis depends on many factors, among which the immune status of the body is an important aspect.

JAK/STAT signal transduction starts from the binding of extracellular related cytokines or growth factors to their corresponding transmembrane receptors. In this way, JAKs can be close to each other in spatial position, and at the same time, the spatial structure of JAKs can be changed, and finally the kinase domain of JAKs can be transformed from an inhibitory form with no kinase activity to an activated form with kinase activity. This mode of binding brings JAKs and their receptors close in space, which in turn leads to phosphorylation of the tails of JAKs themselves and cytoplasmic receptors, generating potential sites necessary for STAT monomer docking. Although each cytokine/receptor is usually associated with a specific STAT, each cytokine/receptor is often associated with multiple STAT family members (heterogeneous signaling), resulting in homodimers, heteromeric and formation of higher order tetramers. They bind to DNA through sequence-consistent GAS (Gamma-nterferon activation site) in the nucleus, and also lead to activation, repression of gene expression, or neutral binding to protein-coding mRNAs or non-coding RNA target genes in a GAS-independent manner , as part of a multimolecular complex. Since the GAS motifs share the same affinity, multiple forms of STATs may co-bind to the same target site (i.e., overlapping specificities). The special sequence consensus motif suggests that STATs may compete against each other to bind to the same genomic region. It has been verified that STAT3 and STAT5 are in T cells, dendritic cells and tumor cell lines, and the transcription of IL-17 mediated by STAT3 is blocked by STAT5 (Yang XP, Ghoreschi K, Steward-Tharp SM, et al. Opposing regulation of the locus encoding IL-17through direct, reciprocal actions of STAT3 and STAT5[J].Nat Immunol,2022,12(3):247-254).

Although STATs are critical for cytokine specificity and function, stimulation of cytokine receptors can also, in part, affect STATs to generate partially STAT-independent signals. The ITAM axis receptor is a good example (immunoreceptor tyrosine-based activation motif, ITAM), which activates the receptor by regulating STATI signal transduction and interacting with tumor necrosis factor receptor-related factors. Play a role in ultimately regulating the STAT3 signaling pathway (Bezbradica JS, Rosenstein RK, DeMaeco RA, et al. A role for the ITAM signaling module in specifying cytokine-receptor functions[J]. Nat Immunol, 2014, 15(4): 333-342. Nagashima H, Okuyama Y, Asao A, et al. The adapter TRAF5 limits the differentiation of inflammatory CD4(+)T cells by an-tagonizing signaling via the receptor for IL6[J].Nat Immunol2014,15(5):449-456)) .

Four JAKs kinases (JAK1JAK2JAK3Tyk2) and seven STATS (STATI, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) in mammals can bind to more than 50 cytokines and growth factors. Then the question ensues, how do such a small number of JAKs kinases and STATs bind to their corresponding ligands (ie, cytokines and growth factors) under the premise of ensuring their specificity? One explanation is that cytokines with the same activation properties may play roles in different types of cells (or cells in different states), and STATs with different sensitivities are encoded by different STAT genes. However, this explanation does not elucidate why cytokines in "seemingly" similar STAT signaling cascades have different biological responses in the same cell type or state. For example, in myeloid cells, both IL-6 and IL-10 can effectively activate STAT3, IL-6 mainly plays a pro-inflammatory role, while IL-10 mainly plays an anti-inflammatory role. Differences in STAT3 signaling production are mainly in terms of duration and strength of STAT3 signaling, however this may only be part of the problem. Although each cytokine is usually only associated with a specific STAT, almost every STAT is a family member of multiple cytokines or growth factors, but the degree of action in different families is different, that is, heterogeneous signal transmission . For example, type I interferon (Interferon-I, IPN-I) is a typical STAT1 activator, but it can also activate STAT3 and STAT4. In some cases, cytokines participate in the activities of multiple STATS and have equal effects, such as IL-27 can strongly activate STATI and STAT3; while in other cases, the interaction between the two has a clear hierarchy, such as IFN-γ can activate Elicits a strong STAT1 response, whereas STAT3 elicits the opposite response.

STATs can directly bind to DNA and function as classical transcription factors (Transcription factors, TFs). Results of STAT-DNA interactions can be integrated into loss-of-function studies in transcriptomics and functional genomics, i.e. knockout mice, small interfering RNA (siRNA) to classify target genes, and These results were further applied to STAT binding occupancy and STAT-dependent transcriptional regulation. STATs are dispersed throughout the genome and can regulate the transcriptional processes of both protein-coding and non-coding genes. In Th cells, thousands of binding sites have been established for each family member, sometimes proximal to the transcription start site, but more often located distal to the transcription start site The ends are usually associated with enhancers or other cis-acting elements.

The JAK/STAT signaling pathway usually appears as a classical pathway, but it is full of complex factors, such as STATs also function as heteromers and higher-order tetramers. A key difference between IFN-I and IFN-II is that the former induces the formation of STAT1/STAT2 heterodimers (i.e., the ISGI3 complex), whereas the latter induces mainly STAT1 dimers (i.e., the GAF complex). In terms of function, it is believed that STAT5 is the key to transcriptional activity. STATI tetramers are important for IFN-I but not for IIFN-I. The expression of STAT proteins is strictly regulated, and the concentration of STAT proteins in the cytoplasm has important biological effects. For example, in NK cells, the relative levels of STAT1 and STAT4 determine the response level of INF-I.

In vitro studies have confirmed that phosphorylated serine phosphorylation is critical for gene transcription, such as STAT4 driving IFN-γ in T cells (morinobu A, Gadina M, Strober W, et al. STAT4serine phosphorylation is critical for IL-12-induced INF-gamma production but not for cellproliferation[J].Proc Natl Acad Sci USA,2002,99(19):12281-12286.) In vivo experiments show that STAT4 has an impact on immune function and hematopoietic system (Friedbichler K, Kerenyi MA, Kovacic B , etal. Stat5a serine 725and 779 phosphorylation is a prerequisite for hematopoietic transformation [J]. Blood, 2010,, 116 (9): 1548-1558)

In recent years, research has shown that the program of M. tuberculosis infection is determined by immune signaling pathways that either destroy the pathogen or cause tuberculosis. withaferinA (WA) stimulates the generation of T cell memory populations by eliciting STAT signaling, thereby reducing the rate of tuberculosis relapse due to reactivation and reinfection, confirming the immunotherapeutic potential of WA in enhancing the host immune response against Mycobacterium tuberculosis (Kumari Anjna; Pahuja Isha, WithaferinAProtectsagainstPrimary and RecurrentTuberculosisbyModulatingMycobacterium-SpecificHost ImmuneResponses, [J] Microbiology Spectrum2023.PP e0058323-e0058323).

The traditional concept holds that people who are exposed to the environment of Mycobacterium tuberculosis develop tuberculosis infection, but have no clinical symptoms, and become latent infections, and some latent infections develop into active tuberculosis patients in the later stage. An epidemiological survey of family close contacts from the United States showed that about 50% of the people in close contact with active tuberculosis patients in the family were not infected with tuberculosis. Early scholars attributed this non-infection to the response window period of immune response, Insufficient exposure intensity and problems with diagnostic criteria for latent infection. However, the latest research shows that among the close contacts who have been exposed to Mycobacterium tuberculosis for a long time and in large quantities (such as medical staff in tuberculosis hospitals, etc.), there are some people who can completely eliminate the invading Mycobacterium tuberculosis. Known as "tuberculosis resistance (resister)". The definition of tuberculosis resistance: (1) continuous high-intensity exposure to Mycobacterium tuberculosis (Mtb) environment; (2) persistent negative PPD and IGRA tests. This special group can eliminate Mycobacterium tuberculosis that repeatedly invades host cells, and does not produce adaptive immune responses through antigen presentation, that is, does not produce tuberculosis antigen-specific memory lymphocytes. The immune mechanism that produces tuberculosis resistance is unclear, but the lack of tuberculosis antigen-specific memory lymphocytes suggests that innate immunity rather than adaptive immunity plays a more important role in its generation. Epidemiological data suggest the existence of tuberculosis resistance, but the mechanism of Resister's resistance to Mycobacterium tuberculosis (Mtb) infection is still unclear at home and abroad.

The process of Mtb infecting the body is divided into three stages: initial infection, latent infection, and resurgence of the disease caused by massive proliferation again. The body's cellular immunity to Mtb must be mediated by antigen-presenting cells (APC). Dendritic cells (Dendritic cells, DCs) are the most effective antigen-presenting cells. After the body is infected with Mtb, a large number of immature DCs appear, which are cells specialized in presenting and processing antigens. After engulfing the antigen, DC gradually matures (IL-12p40 plays an important role in the maturation of DC after Mtb infection), migrates to the secondary draining lymph node, and chemokines and their receptors play an important role in the migration process, reaching the draining lymph node Afterwards, it highly expresses MHC class II and MHC class I molecules and presents antigens. Studies have found that only by migrating to the draining lymph nodes can DC induce Th1 cells to exert an immune response. Mature DC release a large amount of IL-12 to induce Th1 response, and removing DC in mice will aggravate Mtb infection. Therefore, DC cells play an important role in the process of Mtb infection. Studies have shown that the ingestion of different MTB antigens by DC cells will produce different clinical outcomes: when DC cells phagocytize Man-LAM (mannose-modified lipoarabinomannan) that ingests MTB, a large amount of anti-inflammatory factor IL-10 is produced, inhibiting The activation of Th-1 response hinders the clearance of Mycobacterium tuberculosis by macrophages and T cells, which is conducive to the progression of MTB infection to active tuberculosis; when DC cells ingest lipoproteins that phagocytose MTB, a large amount of pro-inflammatory factor IL12 is produced, Promote a strong Th1 response, secrete IFN-γ, activate macrophages and T cells, thereby promoting the clearance of Mycobacterium tuberculosis, and controlling the development of tuberculosis infection into latent or subclinical tuberculosis infection. These all suggest that DC cells play an important role in helping the host resist Mycobacterium tuberculosis.

By studying the proportion of peripheral blood immune cells, DC cell phagocytosis, antigen presentation, migration, cell Factor production ability, transcriptome analysis of DC cells, and further understanding of the anti-tuberculosis immune mechanism of tuberculosis-resistant people. It was found that after DCs of tuberculosis-resistant people (Resister) were stimulated by tuberculosis, they increased the secretion of IFN-γ by up-regulating STAT4, thereby improving Dendritic cells have stronger phagocytic ability, and the present invention is completed on this basis.

Contents of the invention

In a first aspect, the present invention provides an application of a biomolecule in killing Mycobacterium tuberculosis.

Further, the biomolecules are preferably nucleic acids, proteins and/or polypeptides of STAT4 molecules.

Further, the polypeptides also include polypeptides constructed from nucleic acid or protein compositions containing STAT4 molecular nucleic acids or STAT4 molecular protein compositions.

Further, the polypeptide includes one and/or a combination of multiple polypeptides.

Further, the one polypeptide may exist alone, as a multimer comprising multiple copies of the same peptide and/or as a heteromer of various peptides.

Further, the composition of one and/or more polypeptides is modified, recombined, chemically synthesized or biosynthesized.

Further, the biomolecule can also improve or enhance at least one performance selected from the following:

A. Enhance the phagocytosis of dendritic cells;

B. Enhance the migration ability of dendritic cells;

C. Increase the expression of dendritic cell HLA-DR surface markers;

D. Increase the expression of dendritic cell CD83 surface markers;

E. Enhance the secretion ability of IFN-γ;

F. Enhance the ability of macrophages to kill Mycobacterium tuberculosis.

In a second aspect, the present invention provides an application of a biomolecule in tuberculosis immunotherapy.

Further, the biomolecules are preferably nucleic acids, proteins and/or polypeptides of STAT4 molecules.

Further, the immunotherapy includes targeting antibodies, tuberculosis vaccines, secondary cell therapy, immune checkpoint inhibitors, cytokines, and immune adjuvants.

Further, the tuberculosis is tuberculosis infected by Mycobacterium conjunctiva.

Further, tuberculosis includes primary pulmonary tuberculosis, hematogenously disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tuberculous pleurisy, and extrapulmonary tuberculosis.

In the third aspect, the application of a biomolecule of the present invention in the preparation of a pharmaceutical preparation for treating tuberculosis.

Further, the biomolecules are preferably nucleic acids, proteins and/or polypeptides of STAT4 molecules.

Further, the pharmaceutical preparation includes a drug route selected from: oral administration, sublingual administration, gastric or intestinal administration, local administration, injection administration, intravenous injection, subcutaneous injection, intramuscular injection, transdermal administration and/or inhalation administration, etc.

Further, the pharmaceutical preparation can be used for patients infected with Mycobacterium tuberculosis, including humans and animals;

Further, the animal may be selected from bats, mustelidae, canines, cats, deer, poultry and livestock, rodents and the like.

In the fourth aspect, the present invention provides an application of a biomolecule in the preparation of an immunodiagnostic reagent for detecting the susceptibility of Mycobacterium tuberculosis in a population.

Further, the immunodiagnosis includes the diagnosis of the expression level of STAT4 molecule in the detected object.

Further, the expression level is manifested by gene mutation of STAT4 molecule without expression or significantly lower expression level of STAT4 molecule.

In the fifth aspect, the present invention provides an application of a biomolecule in constructing a cell model resistant to Mycobacterium tuberculosis.

Further, the biomolecules are preferably nucleic acids, proteins and/or polypeptides of STAT4 molecules. Expression of the biomolecule is enhanced in the cell.

Further, the cell model is preferably dendritic cells.

Further, the biomolecule can also improve or enhance at least one performance selected from the following:

A. Enhance the phagocytosis of dendritic cells;

B. Enhance the migration ability of dendritic cells;

C. Increase the expression of dendritic cell HLA-DR surface markers;

D. Increase the expression of dendritic cell CD83 surface markers;

E. Enhance the secretion ability of IFN-γ;

F. Enhance the ability of macrophages to kill Mycobacterium tuberculosis.

In a sixth aspect, the present invention provides a dendritic cell line capable of killing Mycobacterium tuberculosis.

Further, the expression level and/or phosphorylation level of STAT4 in the dendritic cells is higher than the expression level and/or phosphorylation level of STAT4 in normal dendritic cells.

Further, the dendritic cell line may have at least one of the following functions:

G. Stronger phagocytic ability.

H. The ability to migrate is stronger.

I. High expression of HLA-DR surface markers.

J. High expression of CD83 surface marker.

K. The secretion ability of IFN-γ is stronger.

L. has the ability to kill Mycobacterium tuberculosis.

Description of drawings

Figure 1 Experimental flow chart

Figure 2 Flow cytometry analysis of the distribution of PBMCs immune cell subsets in the HC group, Resister group, LTBI group and TB group, the percentage in the figure is the percentage of immune cells in the total viable PBMCs. The number of people included in HC group, Resister group, LTBI group and TB group was n=6, 5, 7 and 5, respectively. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test).

Figure 3 Identification of dendritic cells (Mo-DCs) induced by human monocytes. (A) The morphological changes of monocytes before and after induction were observed under a 400X light microscope. (B) Mo-DCs were identified by flow cytometry. (C) Expression of Mo-DCs surface markers detected by flow cytometry. (D) Comparison of the purity of dendritic cells induced by monocytes from four groups of people. The number of people included in HC group, Resister group, LTBI group and TB group was n=3, 3, 3, 3, respectively. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test).

Figure 4 Flow cytometry to detect the phagocytic ability of different populations of MO-DCs; the left panel shows representative images, and the statistical graph in the right panel indicates the proportion of DC cells that phagocytose latex beads to the total cells. The number of people included in HC group, Resister group, LTBI group and TB group was n=6, 5, 7, 5, respectively. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test).

Figure 5 uses the transwell migration assay to detect the migration ability of MO-DCs from different populations. Cells were counted after staining with 0.1% crystal violet. Scale bar = 100um. The left picture is a representative image, and the right picture is a statistical chart of the proportion of migrated cells to the total cells; the number of people included in the HC group, the Resister group, the LTBI group and the TB group was n=5, 5, 5, and 5, respectively. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test).

Figure 6 The expression levels of Mo-DCs surface markers in different populations. The number of people included in HC group, Resister group, LTBI group and TB group was n=5, 5, 5, 5, respectively. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test). MFI: mean fluorescence intensity, average fluorescence intensity.

Fig. 7 Comparison of cytokine production ability of Mo-DCs in different populations (A and B). (A) Quantitative PCR analysis of the expressions of IFN-γmRNA, TNF-αmRNA, IL1βmRNA, IL6mRNA, IL10mRNA, IL12B mRNA, IL23mRNA, and TGF-βmRNA at different time points when Mo-DCs were stimulated by tuberculosis lysates in each group, HC group, Resister group, The numbers of people included in the LTBI group and the TB group were n=6, 5, 6, and 5, respectively. (B) ELISA detection of IFN-γ in the supernatant of Mo-DCs stimulated by tuberculosis lysate at different time points in each group, the number of people included in the HC group, Resister group, LTBI group and TB group were n=5, 4, 5, respectively , 4. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test).

Figure 8 detects the survival of intracellular Mtb after Mtb-infected macrophages were co-cultured with different groups of Mo-DCs. At each designated time point, the macrophages were lysed, and several series of serially diluted lysates were prepared, then cultured on 7H10 agar plates, and bacterial CFU counts were performed. The number of people included in HC group, Resister group, LTBI group and TB group were n=4, 3, 4, 3 respectively. Data are presented as mean ± standard error. P>0.05, not significant (ns), *P<0.05, and **P<0.01 (independent sample t-test). MΦ: macrophage, macrophage; DCs: dendritic cells, dendritic cells.

Figure 9

Figure 10 Differential gene enrichment analysis of dendritic cells from tuberculosis resistant patients

Fig. 11(A) Expression levels of STAT4 mRNA in Mo-DCs of different populations. (B) The expression level and phosphorylation level of STAT4 protein in different populations of Mo-DCs. +: tuberculosis lysate stimulation group; -: blank control group.

Detailed ways

Tuberculosis (TB) is a common and fatal infectious disease caused by Mycobacterium tuberculosis. Tuberculosis usually infects and destroys the lungs and lymphatic system, but other organs such as the brain, central nervous system, circulatory system, urinary system, bones, joints, and even the skin can also be infected. Mycobacterium tuberculosis is mainly human and bovine. Mycobacterium tuberculosis infection has the highest incidence rate. Clinically referred to tuberculosis mostly caused by the above two types. Tuberculosis is mainly transmitted through the respiratory tract, and can also be infected through the digestive tract. A small number of infections are transmitted through skin wounds. Respiratory transmission is the most common and important way. Tuberculosis patients (mainly cavitary tuberculosis) discharge a large number of bacteria-carrying droplets from the respiratory tract and inhale these bacteria-carrying droplets. Microdroplets can cause infection, and microdroplets with a diameter of less than 5μm can reach the alveoli, so they are the most pathogenic. Mycobacteria that reach the alveoli chemoattract and attract macrophages, and are phagocytized by macrophages. Before the establishment of effective cellular immunity, the ability of macrophages to kill it is very limited. Mycobacterium tuberculosis multiplies in the cells, which can cause local inflammation on the one hand, and systemic hematogenous dissemination on the other hand. The root cause of tuberculosis, it generally takes 30-50 days for the body to produce specific cellular immunity to Mycobacterium tuberculosis.

The experimental group of the present invention is divided into four groups: healthy people (HC), tuberculosis resistant patients (Resister), latent tuberculosis infected patients (LTBI), and tuberculosis patients (TB). TB patients, 5 healthy people, 5 latent infected people, and 5 TB patients, that is, 4 groups of 5 people each.

Its inclusion criteria are as follows:

1. Inclusion criteria

(1) Healthy people (HC): no exposure to Mtb; negative T-SPOT test, no other serious medical history.

(2) Tuberculosis resistance (Resister): effective risk factors for high exposure to Mtb; cumulative exposure during exposure; T-SPOT negative for more than two years.

(3) Latent tuberculosis infection (LTBI): have been exposed to Mtb; no TB symptoms; positive T-SPOT test, no history of tuberculosis treatment.

(4) Tuberculosis patients (TB): have been exposed to Mtb; have clinical symptoms of TB; positive sputum smear, positive bacterial isolation and culture, positive nucleic acid test; chest X-ray or CT found lung lesions; tuberculosis in extrapulmonary tissue or lung tissue Pathological changes.

2. Exclusion criteria

(1) Individuals with HIV co-infection, tumors, hepatitis, autoimmune diseases, and other serious diseases (hepatic and renal insufficiency, cardiac insufficiency), and anyone showing uncertain T-SPOT responses.

(2) Those who have recently used related preparations (glucocorticoids, immunosuppressants, immunomodulators) that affect immune function;

(3) According to the investigator's judgment, the subject is not suitable for participating in the trial or has any disease that cannot reliably participate in the whole process of the trial.

Example 1 Flow Cytometry Detects the Distribution of Immune Cells in the Peripheral Blood of the Four Groups of People

1.1 Blood sample collection

Medical staff used purple cap tubes containing anticoagulant EDTA to collect 20ml of fresh peripheral blood from healthy people (HC), tuberculosis resistant patients (Resister), latent tuberculosis infected patients (LTBI) and tuberculosis patients (TB). At room temperature, while processing within 6 hours, the operation process prevents pollution.

1.2 Isolation of peripheral blood mononuclear cells (PBMC)

(1) Use the peripheral blood lymphatic separation fluid to separate the collected blood sample, add 15ml (at least the same volume as the blood sample) peripheral blood lymphatic separation fluid to a 50ml centrifuge tube, mix the 10ml blood sample upside down, carefully add the blood sample to the wall of the lymph On the separation liquid, adjust the parameters of the centrifuge to a temperature of 20°C, an ascending speed (ACC) of 8/decreasing speed (DEC) of 6, a centrifugal force of 600 RCF (g), and centrifuge for 25 minutes.

(2) After centrifugation, the whole system is divided into four layers. From top to bottom, it is a light yellow plasma layer, a ring-shaped milky white mononuclear cell layer, a transparent separation liquid layer, and a red blood cell layer. Use a straw to absorb the second layer of white cells after centrifugation Layer into a new 15ml centrifuge tube, add cleaning solution to 30ml, adjust the centrifuge parameters to a temperature of 20°C, an ascending speed (ACC) of 9/decreasing speed (DEC) of 9, a centrifugal force of 500RCF (g), and centrifuge for 10 minutes.

(3) Recover the above centrifuged supernatant, leave the cell layer, add 2ml erythrocyte lysate, resuspend the cells, lyse for 2-5min, add an equal amount of PBS (or RPMI 1640 medium) to neutralize, at 20°C, ACC9/DEC9 was centrifuged at 500 RCF (g) for 10 min; the recovered supernatant was centrifuged at 20°C for ACC9/DEC9 and 500 RCF (g) for 10 min to recover residual cells.

(4) The supernatant was discarded, the two cells were combined, and 2ml of 1640RPMI medium was added to resuspend to obtain a cell suspension.

1.3 Cryopreservation of peripheral blood mononuclear cells

(1) The obtained peripheral blood mononuclear cell suspension was centrifuged at a centrifugal force of 500 RCF (g) for 10 min at room temperature to collect the cells, and the supernatant was discarded in a biological safety cabinet.

(2) Take a new 15ml centrifuge tube, add 9ml FBS and 1ml DMSO into the tube to make a cell cryopreservation solution, turn it upside down and mix well.

(3) Resuspend the cells with 1ml of the prepared cryopreservation solution, transfer the cells to the cell cryopreservation tube with a pipette gun, then put the cell cryopreservation tube into a gradient freezer box that has been returned to normal temperature, and Add an appropriate amount of isopropanol to the outer tank of the box, put the freezer box in a -80°C refrigerator overnight, and move the cells in the freezer box to liquid nitrogen for storage on the next day.

1.4 Flow cytometry detection of peripheral blood immune cell distribution of four groups of people

1.4.1 Recovery of peripheral blood mononuclear cells

(1) Put pure water into the water bath, adjust the temperature to 37°C in advance, and keep the water temperature at 37°C.

(2) Put 10ml of 1640 complete medium containing 10% FBS into a 15ml centrifuge tube, put it into a carbon dioxide incubator in advance, and incubate the medium to 37°C.

(3) Take out the cryopreservation tube from the liquid nitrogen, put it into a 37°C water bath immediately, shake the cryopreservation tube gently, pay attention to keep the cryopreservation tube upright, and the lower edge of the cryopreservation tube cover must not be submerged in the water surface, so as not to cause cell sample contamination .

(4) After the cell samples in the cryopreservation tube are thawed, transfer them to a biological safety cabinet, use a pipette or a disposable pipette to transfer the cell samples to a 15ml centrifuge tube prepared in advance, centrifuge at 500RCF (g) for 5 minutes to collect the cells, and discard For the supernatant, add 2ml 1640 complete medium with a pipette gun to resuspend and mix evenly. Mix well, then pipette 10 μl of cell suspension and slowly add the cells along the edge of the cell counting plate without generating air bubbles. After the cell suspension is completely submerged in the counting chamber, count the number of cells M in 4 squares on the cell counting plate , total cell number=M/4×10 ⁴ ×20 (dilution factor).

(5) The cells were transferred to a new flow tube for use. Each sample was divided into two tubes, labeled S1 and S2, with 1×10 ⁶ viable cells in each tube.

1.4.2 Flow cytometry detection to detect the distribution of immune cells in the peripheral blood of the four groups of people

(1) Put pure water in the water bath, adjust the temperature to 56°C in advance, take out a FBS packaged in a 50ml centrifuge tube and thaw it, put it in the water bath after thawing and inactivate it at 56°C for 30 minutes, and then put the inactivated Use a 0.22μm filter to filter good FBS into a new 50ml centrifuge tube for later use.

(2) Take out a bottle of 500ml DPBS buffer solution, add 5ml FBS with a pipette gun to make a cell washing solution containing 1% FBS.

(3) Add 2ml of cell washing solution to the flow tube, vortex and mix with a vortex shaker, then centrifuge at 400g for 5min, discard the supernatant, and resuspend the cell pellet at the bottom of the flow tube with a pipette uniform.

(4) Add 2ml of cell washing solution to the flow tube, vortex and mix with a vortex shaker, then centrifuge at 400g for 5min, discard the supernatant, and resuspend the cell pellet at the bottom of the flow tube with a pipette uniform.

(5) Discard the liquid in the flow tube, add 100 μl of cell washing solution to resuspend the cells, then add 5ul HumanTruStain FcX ^TM , resuspend and mix with a pipette gun, and incubate on ice for 10 minutes in the dark.

(6) Centrifuge all the fluorescent antibodies at 10000g for 5min before use, and then distribute according to the method described in Table 1 and Table 2. Prepare the antibody mixture according to the number of samples. After the preparation, add the prepared in Table 1 to each S1 sample tube. 100μl antibody mixture, add 117.5μl antibody mixture prepared in Table 2 to each S2 sample tube, blow and mix with a pipette gun, and incubate on ice in the dark for 25min;

(7) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend and mix the cell pellet at the bottom of the flow tube with a pipette gun .

Table 1 Configuration method of flow cytometry antibody mixture

Table 2 Configuration method of antibody mixture for flow cytometry

(8) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend and mix the cell pellet at the bottom of the flow tube with a pipette gun .

(9) Add 200 μl of cell wash solution to the flow tube, then add 5 μl of 7AAD dead-life dye, blow and mix with a pipette gun, filter out cell clumps with a flow cap, and then detect with a flow cytometer.

Example 2 Differential Detection of Dendritic Cells from Different Populations

2.1 Establishment of monocyte-derived dendritic cells (Mo-DCs) model

2.1.1 Induction of dendritic cells from peripheral blood monocytes

(1) Collect 20ml of peripheral blood from HC, Resister, LTBI, and TB, and the steps are the same as those described in 1.1 above.

(2) Peripheral blood mononuclear cells were obtained by separating peripheral blood from healthy people (HC), tuberculosis resistant patients (Resister), latent tuberculosis infected patients (LTBI) and tuberculosis patients (TB). The specific operation process was the same as that described in 1.2 above.

(3) Cell counting: Use a pipette to blow and beat the obtained cell suspension evenly, then pipette 50 μl of the cell suspension, transfer it into EP containing 950 μl of PBS for dilution, mix up and down, and pipette 10 μl of the cell suspension Add slowly along the edge of the cell counting plate without generating air bubbles. After the cell suspension is completely submerged in the counting chamber, count the number M of cells in 4 squares on the cell counting plate, the total number of cells = M/4×10 ⁴ × 20 (dilution factor).

(4) Plating: resuspend and mix the cells with 1640 complete medium containing 1% penicillin and streptomycin, adjust the cell density to 2×10 ⁶ /ml, suck into 6-well cell culture plate (2ml/well), 12 Orifice cell culture plate (2ml/well) or 24-well cell culture plate (1ml/well), place the cell culture plate in a carbon dioxide incubator at 37 ° C, 5% CO Under the condition of cultivating _overnight .

(5) Obtaining mononuclear cells: Take out the cell culture plate, suck out the supernatant with a pipette gun and discard, then add 2ml PBS to wash away unattached cells, repeat the washing step twice, and then obtain adherent monocytes .

(6) Induce monocytes to differentiate into dendritic cells: according to the number of wells of the cells, configure a new 1640 complete medium containing 10ng/ml IL4 and 50ng/ml GM-CSF inducer, suck it into a 6-well cell culture plate, Place the cell culture plate in a 12-well cell culture plate (2ml/well) or a 24-well cell culture plate (1ml/well) in a carbon dioxide incubator at 37°C and 5% CO2 for cultivation on the 3rd and 5th day On the 1st day, the 1640 medium containing the inducer was replaced in half, and the immature DC could be obtained on the 6th day. If it is necessary to further obtain mature DC, unadhered cells in the cell culture plate can be collected, that is, immature dendritic cells (imDC), and stimulated with LPS at a final concentration of 100ng/ml for 24h to make them mature.

2.2 Identification of monocyte-derived dendritic cells (Mo-DCs)

2.2.1 Morphological observation of dendritic cells derived from monocytes

During the induction of dendritic cells by monocytes, the cell size and morphological changes were monitored under a high-power microscope.

2.2.2 Flow cytometry detection of the expression of Mo-DCs surface markers

(1) Transfer the induced Mo-DCs from the cell culture plate to the flow tube for later use.

(2) Adjust the temperature of the water bath to 56°C in advance, take out a piece of FBS packed in a 50ml centrifuge tube and thaw it, put it in the water bath after thawing and inactivate it at 56°C for 30 minutes, and then use the inactivated FBS Filter through a 0.22μm filter into a new 50ml centrifuge tube for later use.

(3) Take out a bottle of 500ml DPBS buffer solution, add 5ml FBS with a pipette gun to make a cell washing solution containing 1% FBS; take out the Zombie NIR Fixable Viability Kit reagent, add 500μl DMSO with a pipette gun to prepare the working solution, and let it stand for 2min , after the dye is completely dissolved, use EP tubes for later use.

(4) Add 2ml of cell washing solution to the flow tube, vortex and mix with a vortex shaker, then centrifuge at 400g for 5min, discard the supernatant, and resuspend the cell pellet at the bottom of the flow tube with a pipette uniform.

(5) Repeat the previous step.

(6) Discard the liquid in the flow tube, add 100 μl of cell washing solution to resuspend the cells, then add 5 μl of HumanTruStain FcX ^TM , resuspend and mix with a pipette gun, and incubate on ice for 10 minutes in the dark.

(5) Centrifuge all the fluorescent antibodies at 10000g for 5min before use, then prepare antibody mixture according to the total number of samples according to the method described in Table 3, add 45μl antibody mixture to each sample tube after preparation, and use a pipette After mixing by pipetting, incubate on ice for 25 minutes in the dark;

(6) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend and mix the cell pellet at the bottom of the flow tube with a pipette gun .

(7) Add 1 μl ZOMBIE NIR Fixable Viability Kit working solution to the flow tube, blow and mix with a pipette gun, and incubate on ice in the dark for 10 minutes.

(8) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend and mix the cell pellet at the bottom of the flow tube with a pipette gun .

(9) Repeat the previous step.

(10) Add 200 μl of cell wash solution into the flow tube, filter out cell clumps with a flow cap, and detect the cell suspension with a flow cytometer.

Table 3 Configuration method of flow cytometry antibody mixture

2.3 Identification of the purity of dendritic cells induced by monocytes

(1) Transfer the induced Mo-DCs from the cell culture plate to the flow tube for later use.

(2) Take out a bottle of 500ml DPBS buffer solution, add 5ml FBS with a pipette gun to make a cell washing solution containing 1% FBS.

(3) Add 2ml of cell washing solution to the flow tube, vortex and mix with a vortex shaker, then centrifuge at 400g for 5min, discard the supernatant, and resuspend the cell pellet at the bottom of the flow tube with a pipette uniform.

(4) Repeat the previous step.

(5) Discard the liquid in the flow tube, add 100 μl of cell washing solution to resuspend the cells, then add 5 μl of HumanTruStain FcX ^TM , resuspend and mix with a pipette gun, and incubate on ice for 10 minutes in the dark.

(5) Centrifuge all the fluorescent antibodies at 10,000g for 5 minutes before use, then prepare antibody mixture according to the total number of samples according to the method described in Table 2, add 35μl antibody mixture to each sample tube after preparation, and use a pipette After mixing by pipetting, incubate on ice for 25 minutes in the dark;

(6) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend and mix the cell pellet at the bottom of the flow tube with a pipette gun .

(7) Repeat the previous step.

(8) Add 200 μl of cell wash solution to the flow tube, then add 5 μl of 7AAD dead-life dye, blow and mix with a pipette gun, filter out cell clumps with a flow cap, and then detect with a flow cytometer.

Table 4 Configuration method of antibody mixture for flow cytometry

2.4 Phagocytosis detection of Mo-DCs in four groups of people

(1) Collect 20ml of peripheral blood from HCs, Resister, LTBI, and TB, and the steps are the same as those described in 1.1 above.

(2) Peripheral blood mononuclear cells were obtained by separating peripheral blood from healthy people (HC), tuberculosis resistant patients (Resister), latent tuberculosis infected patients (LTBI) and tuberculosis patients (TB). The specific operation process was the same as that described in 1.2 above.

(3) The peripheral blood monocytes of the four groups of people were used to induce immature dendritic cells for 6 days, and the specific operation process was the same as that described in 2.1 above.

(4) Count the obtained dendritic cells with a cell counting board (the specific steps are the same as described above), adjust the cell concentration to 1×10 ⁶ /ml, and pipette 100 μl of the cell suspension into the flow tube medium; at the same time, put PRMI 1640 complete medium in a 37°C CO2 incubator to preheat for use; prepare DPBS washing solution containing 1% FBS according to the steps described in the first part.

(5) Take out the Phagocytosis Assay Kit (IgG PE), vortex and mix with a vortex shaker, calculate the amount of reagents (2 μl/tube) according to the number of samples, and draw the required dose of rabbit IgG latex microspheres with a pipette gun, Add it into the preheated PRMI 1640 medium and dilute it 100 times to make a working solution, and mix it with a pipette gun.

(6) Use a pipette gun to draw 200 μl of working solution into the flow tube, pipette and mix well, and place the flow tube in a cell culture incubator at 37° C. and 5% CO ₂ to incubate for 3 hours.

(7) Take out the flow tube, centrifuge at 500g for 5min, discard the supernatant, add 2ml of DPBS washing solution, mix well with a vortex shaker, centrifuge at 500g for 5min, discard the supernatant, and wash again.

(8) Discard the liquid in the flow tube, add 100 μl of cell washing solution to resuspend the cells, then add 5 μl of HumanTruStain FcX ^TM , resuspend and mix with a pipette gun, and incubate on ice for 10 minutes in the dark.

(9) Centrifuge the antibody CD11c-BV605 at 10,000 g for 5 min, pipette 5 μl of antibody into the sample tube, blow and mix with a pipette, and incubate on ice for 25 min in the dark.

(10) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend and mix the cell pellet at the bottom of the flow tube with a pipette gun .

(11) Add 1 μl ZOMBIE NIR Fixable Viability Kit working solution to the flow tube, blow and mix with a pipette gun, and incubate on ice in the dark for 10 minutes.

(12) Add 2ml of cell washing solution into the flow tube, vortex and mix with a vortex shaker, centrifuge at 400g for 5min, discard the supernatant, and resuspend the cell pellet at the bottom of the flow tube with a pipette and mix evenly , and repeat the cleaning.

(13) Add 200 μl of cell washing solution into the flow tube, filter out cell clumps with a flow cap, and detect the cell suspension on a flow cytometer.

2.5 Detection of migration ability of Mo-DCs in four groups of people

(1) Collect 20ml of peripheral blood from HCs, Resister, LTBI, and TB, and the steps are the same as those described in 1.1 above.

(2) Peripheral blood mononuclear cells were obtained by separating peripheral blood from healthy people (HC), tuberculosis resistant patients (Resister), latent tuberculosis infected patients (LTBI) and tuberculosis patients (TB). The specific operation process was the same as that described in 1.2 above.

(3) The peripheral blood monocytes of the four groups of people were used to induce immature dendritic cells for 6 days, and they were stimulated with LPS at a final concentration of 100 ng/ml for 24 hours to make them mature. The specific operation process was the same as that described in 2.1 above.

(4) Transfer the induced dendritic cells to a 15ml centrifuge tube, centrifuge at 500g for 5min, discard the supernatant, resuspend with FBS-free 1640 medium, plate, and starve for 24h.

(5) The dendritic cells starved for 24 hours were counted with a cell counting board (the specific steps were as described above), and the cell concentration was adjusted to 2.5×10 ⁵ /ml.

(6) Add CCL5 into 1640 medium containing 10% FBS with a pipette to make a culture solution containing 100 ng/ml CCL5 for later use.

(7) Use a pipette to add 500 μl of CCL5 culture solution to the lower chamber of the transwell, add 200 μl of cell suspension to the upper chamber, and then place the transwell 24-well plate in a cell culture incubator at 37°C and 5% CO2 for 4 hours.

(8) Take out the upper chamber, wipe it clean with a cotton swab, add 200ul 1×PBS to wash 3 times with a pipette, and then add 500μl 4% tissue fixative to the lower chamber to fix for 20min. Move the upper chamber to a new 24-well culture plate to dry, add 400 μl 0.1% crystal violet to the lower chamber with a pipette to stain for 10 minutes, wash with 1×PBS 3 times until transparent and clear, and let it dry completely Cut off the membrane of the small chamber, place the side with the cells on the glass slide, drop a small amount of PBS on the membrane to flatten the membrane, cover with a cover slip, and then seal the slide.

(9) Turn on the Cytation 5 cell imaging microplate detection system in advance, and set up the detection program after the self-test is completed.

(10) Adjust the objective lens of the cell imaging microplate detection system to ×4, find the position of the chamber membrane in the field of view, then adjust the objective lens to ×20, observe the cells on each chamber membrane after auto-focusing, and randomly select 5 Cells were counted in each field of view, and then the average value was calculated, and the total number of cells on the entire membrane was calculated according to the ratio of the field of view to the chamber membrane, and the migration rate was calculated accordingly.

2.6 Detection of the expression levels of Mo-DCs surface markers in the four groups of people

(1) Collect 20ml of peripheral blood for HCs, Resister, LTBI, and TB, and the steps are the same as those described in 1.1 above.

(2) Separate the peripheral blood of HCs, Resister, LTBI, and TB to obtain peripheral blood mononuclear cells, and the specific operation process is the same as that described in 1.2 above.

(3) Peripheral blood mononuclear cells from the four groups of people were induced to produce immature dendritic cells for 6 days, and then stimulated with LPS at a final concentration of 100 ng/ml for 24 hours to obtain mature dendritic cells. The procedure was the same as that described in 2.1 above.

(4) The expression levels of Mo-DCs surface molecules CD83, HLA-DR, CD80, CD86, and CD40 in the four groups of people were detected by flow cytometry, and the steps were the same as those described in 2.2.2 above.

2.7 Detection of cytokine secretion ability of Mo-DCs stimulated by tuberculosis lysate in four groups of people

2.7.1 Recovery and culture of Mycobacterium tuberculosis

(1) Take out the frozen Mycobacterium tuberculosis H37Rv standard strain from the -80°C refrigerator, and thaw at room temperature.

(2) Use a pipette gun to blow and mix the thawed standard strain of Mycobacterium tuberculosis H37Rv in a biological safety cabinet, draw 100 μl of the bacterial solution and add it along the slope of the neutral Roche medium, and gently rotate the bottle to make the bacterial solution Cover evenly on the slope, mark the strain and inoculation date with a marker pen.

(3) The neutral Roche medium is placed in a constant temperature incubator at 37°C for cultivation, and it can be used after 3-4 weeks of cultivation.

2.7.2 Preparation of tuberculosis lysate

(1) Add 1ml of PBS to the MP tissue homogenate tube, then take out the neutral Roche medium cultured for 3-4 weeks from the 37°C constant temperature incubator, and scrape it with a disposable sterile scraper ring in the biological safety cabinet Take H37RV in the logarithmic growth phase, and transfer the scraped colonies into MP tissue homogenate tubes.

(2) Turn on the MP tissue homogenizer, set the program, fix the homogenization tube and start the homogenization program.

(3) After homogenization, use a 1ml syringe to filter the prepared antigen lysate with a 0.22μm filter into sterile EP tubes, then divide it into multiple sterile EP tubes, and finally use the NanoDrop 2000 The protein concentration was measured with an ultra-micro spectrophotometer, and the protein concentration and sample preparation date were marked with a marker pen, and stored at -20°C for later use.

2.7.3 Stimulation of dendritic cells by tuberculosis lysate

(1) Collect 20ml of peripheral blood from HCs, Resister, LTBI, and TB, and the steps are the same as those described in 1.1 above.

(2) Separate the peripheral blood of HCs, Resister, LTBI, and TB to obtain peripheral blood mononuclear cells, and the steps are the same as those described in 1.2 above.

(3) Use a pipette to blow the obtained cell suspension evenly, then pipette 50 μl of the cell suspension, transfer it into an EP tube containing 950 μl of PBS for dilution, mix it upside down, and use a pipette to draw 10 μl of the cell suspension along the Slowly add to the edge of the cell counting plate without generating air bubbles. After the cell suspension is completely submerged in the counting chamber, count the number M of cells in 4 squares on the cell counting plate, the total number of cells = M/4×10 ⁴ × 20 (dilution factor)

(4) Resuspend and mix the cells in 1640 medium containing 1% penicillin and streptomycin and 10% FBS, adjust the cell density to 2×10 ⁶ /ml, suck it into a 6-well cell culture plate, and spread two per person Wells were marked with a marker pen for 0h and 24h, and the cell culture plate was placed in a carbon dioxide incubator for cultivation at 37°C and 5% CO ₂ .

(5) Dendritic cells were induced according to the process of 2.1 for the next experiment.

(6) Use a pipette gun to suck the cell culture medium in the 0h hole into a 15ml centrifuge tube, centrifuge at 500g for 10min, then use a pipette gun to carefully suck out the supernatant and transfer it into the EP tube, be careful not to touch the cell pellet at the bottom, operate After completion, obtain the supernatant and cell pellets, and store them frozen at -80°C; add 40 μg of tuberculosis lysate to the culture well marked 24h, and after 24 hours of stimulation, collect the supernatant and cell pellets according to the above method for 0h, and store them frozen at -80°C .

2.8. Extraction of total cellular RNA

Thaw the cell pellet sample collected in the experimental procedure described in 2.7 on ice, and extract the total RNA of the cells using the Cell/Bacterial Total RNA Extraction Kit of Guangzhou Dongsheng Biotechnology Co., Ltd. The specific operation steps are as follows:

(1) Add β-mercaptoethanol to the solution RD so that the final concentration is 1%, and use it after mixing. Add absolute ethanol to the solution RW at a ratio of 1:4, and use it after mixing.

(2) After each cell pellet sample is placed on ice to melt, add 540 μl of β-mercaptoethanol solution RD to each sample with a pipette, and fully shake and mix with a micro-vortex mixer. If insoluble precipitation occurs, Centrifuge at 12000rpm for 2min at room temperature, and transfer the supernatant to another sterile 1.5ml EP tube.

(3) Use a pipette to add 700 μl RB solution to each sample, mix up and down, transfer the obtained solution and the precipitate into a purification column (purification column + collection tube), mark it, and store it at room temperature with Centrifuge at a speed of 12000rpm for 1min, discard the waste liquid in the collection tube, and put the purification column back into the corresponding collection tube. Due to the limited volume of the purification column, the waste liquid centrifuged into the collection tube can be poured out, and then the liquid mixture of the remaining sample is transferred to the purification column for centrifugation until all the liquid mixture is purified.

(4) Add 350 μl RP solution to each purification column with a pipette, centrifuge at 12000 rpm for 1 min at room temperature, discard the waste liquid in the collection tube, and put the purification column back into the corresponding collection tube.

(5) Use a pipette to add 500 μl of RW solution that has been added with absolute ethanol at a ratio of 1:4 to each purification column, let it stand for 2 minutes, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the For waste, put the purification column back into the corresponding collection tube.

(6) Repeat step (5) again.

(7) Put the purification column back into the corresponding collection tube, continue to centrifuge at 12,000 rpm for 2 minutes at room temperature, pour off the remaining liquid in the collection tube, place each purification column in a fume hood, and place it at room temperature for several minutes , dry the absorbent material thoroughly.

(8) Transfer each purification column into a new RNase-free 1.5ml EP tube, mark it well, add 30μl DEPC water to the adsorption material of each sample with a pipette, let stand at room temperature for 2min, 12000rpm Centrifuge for 2 minutes, discard the purification column, and the centrifuged liquid in the 1.5ml EP tube is the RNA solution.

(9) Use a NanoDrop 2000 ultra-micro spectrophotometer to determine the concentration determination and A260/280 ratio of each RNA sample. RNA samples with an A260/280 ratio of about 2 were selected for subsequent experiments, and all RNA samples were frozen and stored in a -80°C refrigerator for later use.

2.9 Synthesis of cDNA by reverse transcription

All-in-one First-Strand cDNASynthesis SuperMix for qPCR(one-step gDNARemoval)试剂对各个符合标准的RNA样本进行反转录。Using TransGen Biotech's

All-in-one First-Strand cDNASynthesis SuperMix for qPCR (one-step gDNARemoval) reagents perform reverse transcription on each standard RNA sample.

(1) Before performing reverse transcription, the components were separated instantaneously, and the reagents on the tube wall were centrifuged to the bottom of the tube before use. The total RNA reverse transcription system is 20ul, and the configuration method is shown in Table 5. The preparation of the reaction system is carried out on ice.

Table 5 reverse transcription system

(2) After the configuration is completed, mix gently, and set the conditions according to Table 6 on the PCR instrument for reaction.

Table 6 Reverse transcription reaction conditions

(3) After the reaction, the cDNA samples synthesized by reverse transcription were frozen and stored in a -20°C refrigerator for later use.

2.10 RT-qPCR detection of IFN-γ, IL1β, IL6, IL10, IL12, IL23, TGF-β gene expression levels.

2.10.1 Primer design

Search the primer sequences of human GAPDH and IFN-γ, IL1β, IL6, IL10, IL12, IL23, TGF-β on the Primer Bank website, and verify it by Primer-Blast on the NCBI website, provided by Beijing Qingke Biotechnology Co., Ltd. The primers were synthesized, and the primer sequences are shown in Table 7.

Table 7 Primer Sequence

2.10.2 RT-qPCR detection of IFN-γmRNA, IL1βmRNA, IL6mRNA, IL10mRNA, IL12mRNA, IL23mRNA, TGF-βmRNA expression levels

PCR Mix Kit试剂盒检测两组各个时间点IFN-γ、IL1β、IL6、IL10、IL12、IL23、TGF-β基因的表达水平。将cDNA模板进行适当稀释，使GAPDH、FN-γ、IL1β、IL6、IL10、IL12、IL23、TGF-β的扩增循环数(Cycle Threshold，Ct)落在20-30之间为最佳。(1) Using the cDNA obtained in step 2.9 as a template, using GAPDH as an internal reference gene, and using the

The PCR Mix Kit kit was used to detect the expression levels of IFN-γ, IL1β, IL6, IL10, IL12, IL23 and TGF-β genes at each time point in the two groups. Dilute the cDNA template appropriately so that the number of amplification cycles (Cycle Threshold, Ct) of GAPDH, FN-γ, IL1β, IL6, IL10, IL12, IL23, and TGF-β falls between 20-30, which is the best.

(2) According to the reaction system shown in Table 8, each sample was added to the corresponding 96-well PCR reaction plate.

Table 8 RT-QPCR reaction system

(3) After the reaction system is configured on ice, according to the reaction conditions shown in Table 9 in AppliedBiosystems

The reaction was performed on the company's ABI 7500 real-time fluorescent quantitative PCR instrument.

Table 9 RT-QPCR reaction system

2.11 ELISA method to detect the expression level of IFN-γ protein

Thaw the cell culture supernatant obtained in the step 2.7 on ice, and use the ELISA kit of Xinbosheng Biotechnology Co., Ltd. to detect the expression of IFN-γ in the cell culture supernatant of the two groups at each time point in the above step level. The specific operation steps are as follows:

(1) Take out the ELISA kit from the refrigerator 20 minutes in advance to equilibrate to room temperature; take out the collected cell supernatant from the -80 refrigerator in advance and thaw it for later use.

(2) Dilute the 20× concentrated washing solution into 1× working solution with double distilled water according to the required dosage for the current test. Return the unused to 4°C.

(3) Take out the lyophilized standard, centrifuge the lyophilized standard on the tube wall at 1000g for 5min to the bottom of the tube, then pipette 1.4ml standard & specimen universal diluent into the lyophilized standard, let stand After 15 minutes, after it is fully dissolved, gently blow and mix with a pipette. At this time, the concentration of the standard is 1000pg/ml.

(4) Prepare 8 sterile 1.5ml EP tubes, and mark the diluted concentration with a marker pen in order. Use a pipette to add 500μl standard & specimen universal diluent to the 2nd-7th EP tube respectively.

(5) Dilute the dissolved standard product in multiple ratios, draw 1ml from the standard product with a pipette gun and add it to the first EP tube. Pipette 500 μl into the second EP tube, and so on for doubling dilution until the sixth EP tube, the corresponding concentration of each EP tube is 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5 pg/ml, 31.25pg/ml, 15.6pg/ml, 0pg/ml.

(6) Take out the strips required for the test from the sealed bag that has been equilibrated to room temperature, put the unused strips and desiccant back into the aluminum foil bag, press the self-sealing strip, seal the bag, and put it back at 4°C.

(7) Add 100 μl of Standard & Specimen Universal Diluent to the blank well, and add specimens or different concentration standards to the remaining corresponding wells.

100 μl each standard product, and 2 replicates were set in each well. Seal the reaction well with sealing tape. Before the reaction, gently tap the frame of the microplate to mix well, then place the plate in a 37°C incubator and incubate for 90 minutes in the dark.

(8) Prepare biotinylated antibody working solution 20 minutes in advance: calculate the required amount of biotinylated antibody according to the number of test wells, and then dilute 30× concentrated biotinylated antibody into 1× working solution with biotinylated antibody diluent .

(9) After the incubation time is over, shake off the liquid in the wells, pat dry on clean absorbent paper, add 350 μl of washing solution to each well with a multi-channel pipette, let it stand for 30 seconds, shake off the liquid, and dry it on thick absorbent paper. Pat dry. Repeat plate washing 5 times.

(10) Add 100 μl of biotinylated antibody diluent to the blank well with a pipette gun, and add 100 μl of biotinylated antibody working solution to the remaining wells with a multichannel pipette. Seal the reaction wells with new sealing tape, then place the plate in a 37°C incubator and incubate for 60 minutes in the dark.

(11) Prepare the enzyme conjugate working solution 20 minutes in advance: Calculate the required enzyme conjugate dosage according to the number of test wells, then dilute the 30× concentrated enzyme conjugate into 1× working solution with the enzyme conjugate diluent, and place it at room temperature in the dark spare.

(12) After the incubation time is up, wash the plate 5 times according to the plate washing method described in step (9).

(13) Use a pipette gun to add 100 μl enzyme conjugate dilution solution to the blank well, and add 100 μl enzyme conjugate working solution to the remaining wells with a multichannel pipette. Seal the reaction wells with new sealing tape, then place the plate in a 37°C incubator and incubate for 30 minutes in the dark.

(14) Turn on the power of the microplate reader, preheat the instrument, and set the detection program in advance.

(15) After the incubation time is up, wash the plate 5 times according to the plate washing method described in step (9).

(16) Add 100 μl chromogenic substrate (TMB) to each well with a multichannel pipette, and then place the plate in a 37° C. incubator and incubate for 15 minutes in the dark.

(17) After the incubation time is up, add 100 μl of reaction termination solution to each well with a multichannel pipette to terminate the reaction, mix well and measure the OD450 value within 3 minutes. Draw the standard curve with the average value of OD450 of each concentration of the standard as the vertical axis and the corresponding concentration of the standard as the horizontal axis.

(18) Calculate the concentration of IFN-γ in the supernatant of each cell culture according to the formula of the standard curve, and perform subsequent result analysis.

(19) After the experiment is completed, put the unused reagents back into the refrigerator according to the specified storage temperature.

Example 3 Detection of intracellular Mtb survival in macrophages after co-incubation of Mo-DCs from different populations with macrophages infected with Mycobacterium tuberculosis

3.1 Recovery and passage of U937 cells

(1) Turn on the switch of the electric constant temperature water bath and preheat it to 37°C in advance.

(2) Take out the frozen U937 cells from the liquid nitrogen tank, and after the liquid nitrogen on the surface of the cell cryopreservation tube evaporates, put it into a 37°C electric constant temperature water bath to thaw.

(3) Use 75% alcohol to clean the countertop of the biosafety cabinet, blow and mix the cell suspension with a pipette, transfer it to a sterile 15ml centrifuge tube, add 10ml RPMI 1640 complete medium, and store at room temperature Centrifuge at 800 rpm for 5 min, and discard the supernatant carefully.

(4) Use a pipette to absorb an appropriate amount of RPMI 1640 complete medium to resuspend the cell pellet, mix by pipetting, and transfer to a cell culture bottle.

(5) Carefully observe the morphology of the cells under a microscope, and after confirming that the cells are in good condition, place the cell culture flask in

Culture in a 37°C, 5% CO ₂ cell incubator.

(6) Take out the cell culture bottle from the 5% CO2 cell incubator, observe the growth state of the cells under a microscope, when the cells grow well, the outline is clear, and the density reaches about 2×10 ⁶ cells/ml, put them in the biological safety cabinet Transfer the cell suspension to a sterile 50ml centrifuge tube, centrifuge at 800rpm for 5min at room temperature, and discard the supernatant carefully.

(7) Use a pipette to absorb an appropriate amount of RPMI 1640 complete medium to resuspend the cell pellet, pipette and mix well, and transfer the cell suspension to a new cell culture flask at a ratio of 1:2-1:3 for passage. The medium was changed every 2-3 days, and cells with vigorous growth and uniform size were selected for subsequent experiments.

3.2 Induce U937 cells to differentiate into macrophages

(1) When the number of cultured cells meets the requirements of the experiment, observe the cells under a microscope to confirm that the cells are growing well, centrifuge at 800 rpm for 5 minutes at room temperature, carefully discard the supernatant, and add an appropriate amount of fresh RPMI 1640 for complete culture Resuspend the cell pellet.

(2) Adjust the cell density to 2×10 ⁵ /ml by cell counting, and add phorbol to the cell suspension

ester, so that the final concentration was 10ng/ml, the cell suspension was thoroughly mixed, and 1ml was inoculated in a 24-cell culture plate for culture.

(4) Place the cell culture plate in a 37°C, 5% CO2 cell incubator to induce differentiation overnight, and after 16 hours, it can transform into adherent macrophages, which can be used for subsequent experiments.

3.3 Co-incubation of Mo-DCs from different populations with macrophages infected with Mycobacterium tuberculosis

(1) Collect 20ml of peripheral blood for HCs, Resister, LTBI, and TB, and the steps are the same as those described in 1.1 above.

(2) Separate the peripheral blood of HCs, Resister, LTBI, and TB to obtain peripheral blood mononuclear cells. The specific operation process is described in 1.2 above.

(3) Use a pipette to blow the obtained cell suspension evenly, then pipette 50 μl of the cell suspension, transfer it into an EP tube containing 950 μl of PBS for dilution, mix it upside down, and use a pipette to draw 10 μl of the cell suspension along the Slowly add to the edge of the cell counting plate without generating air bubbles. After the cell suspension is completely submerged in the counting chamber, count the number M of cells in 4 squares on the cell counting plate, the total number of cells = M/4×10 ⁴ × 20 (dilution factor)

(4) Resuspend and mix the cells with RPMI 1640 medium containing 10% FBS, adjust the cell density to 2×10 ⁶ /ml, inhale 2ml into a 6-well cell culture plate, lay two wells for each person, and mark with a marker After 2 hours and 24 hours, the cell culture plate was placed in a carbon dioxide incubator for cultivation at 37°C and 5% CO ₂ .

(5) Dendritic cells were induced according to the process of 2.1 for the next experiment.

(6) U937 was induced to differentiate into macrophages according to the method described in 3.2.

(7) Take out the neutral Roche culture medium of Mycobacterium tuberculosis cultured for 3-4 weeks from the 37°C constant temperature incubator, and use a disposable sterile scraper loop to scrape the bacteria in the logarithmic growth phase in the biological safety cabinet. H37RV, move the scraped colonies into a sterile grinding bottle, add 3-5 drops of 5% Tween-80 with a pipette gun, cover the lid, vortex with a vortex shaker for 30s, disperse the strains, and finish the grinding After standing still for 15 minutes to allow the aerosol to settle, then open the grinding bottle and add 2ml of RPMI1640 culture medium with a pipette gun, and mix well to obtain the bacterial suspension of Mycobacterium tuberculosis.

(8) Turn on the UV spectrophotometer, pipette 900 μl of RPMI 1640 medium into the cuvette, and zero the spectrophotometer to make the initial OD600=0.00. Take a 15ml centrifuge tube, add 3ml RPMI 1640 with a pipette gun, then add 500μl bacterial suspension, mix well, draw 900μl into the cuvette to measure the OD600 value, adjust the concentration of the bacterial solution according to the OD600 value determined this time, until OD600=1.0, the corresponding bacterial solution concentration is 1×10 ⁸ /ml.

(9) Infect macrophages with MOI=10:1, calculate the required number of Mycobacterium tuberculosis according to the number of test wells, add the corresponding volume of bacterial suspension to RPMI 1640 medium to prepare a culture solution containing bacteria for future use.

(10) Discard the old culture medium of macrophages with a pipette, add 1ml PBS to each well to wash twice, and wash away PMA.

(11) Add 1 ml of RPMI 1640 medium containing bacteria with a pipette, and then put the 24-well cell culture plate into the cell culture incubator for infection for 2 hours.

(12) Use a pipette to suck the induced dendritic cells from different populations into a 15ml centrifuge tube, centrifuge at 500g for 5min, discard the medium, add 1ml of PBS to resuspend the cell pellet, centrifuge at 500g for 5min, repeat twice to wash off Antibiotics, and finally resuspended with 1ml RPMI 1640 medium for later use.

(13) Pipette 500 μl of 5% SDS into 50 ml of PBS to prepare 0.05% SDS for later use.

(14) Take out all cells 2 hours after infection, discard the culture medium containing bacteria in all wells, add 1ml PBS to each well to wash twice, and wash away extracellular bacteria. Add 1 ml of RPMI 1640 medium to the control well, add 1 ml of RPMI 1640 medium containing dendritic cells to each of the 2h and 24h test wells, and then put the 24-well cell culture plate into the cell culture incubator for incubation.

(15) After incubation for 2 hours, the cell culture plate was taken out from the cell culture incubator, the culture medium of the control well and the test well for 2 hours was discarded, and then 1 ml of 0.05% SDS 1 ml was added with a pipette to lyse the cells for 5 min.

(16) Take out the sterile EP tubes, each hole corresponds to 3 EP tubes, mark the stock solution, 10 ^-1 , and 10 ^-2 in turn with a marker pen, and add 900 μl PBS to the second and third EP tubes for later use.

(17) After the cells are lysed, use a pipette to pipette repeatedly, then draw the obtained cell lysate into the first EP tube, invert and mix well, then draw 100 μl from the first EP tube and add it to the second EP tube , and so on to perform multiple dilutions until the third EP tube, and the concentration of the bacterial solution corresponding to each EP tube is the stock solution, 10 ^-1 , and 10 ^-2 in turn. Place the cell culture plate into the cell culture incubator to incubate and continue the incubation.

(18) Take out the 7H10 bacterial culture dish prepared in advance, mark the bacterial species, concentration and date with a marker pen, then use a pipette to draw 50 μl of cell lysate from the EP tube of the corresponding concentration and transfer it into the bacterial culture dish, and use The coating rod spreads the cell lysate evenly, and coats the plate sequentially from low concentration to high concentration.

(19) Seal the coated bacterial culture dish with a parafilm, and then place it in a bacterial incubator at 37°C for cultivation.

(20) After incubation for 24 hours, the cell culture plate was taken out from the cell culture incubator, the culture medium of the control well and the test well for 24 hours was sucked away, and then 1ml of 0.05% SDS 1ml was added with a pipette to lyse the cells for 5min, and then follow the above (17 ) (18) (19) steps described in multiple dilutions, plating and culture.

(21) Cultivate Mycobacterium tuberculosis for 3-4 weeks, and count CFU when the colony grows to be visible to the naked eye and uniform in size, for further statistical analysis.

3.4 Experimental conclusion

(1) Myeloid DC cells are highly expressed in Resister, suggesting that they play an important role in the formation of tuberculosis resistance (see Figure 2).

(2) Dendritic cells can be successfully induced by human peripheral blood mononuclear cells (see FIG. 3 ).

(3) Among the four groups of people, the dendritic cells of tuberculosis resisters had stronger phagocytic ability (see Figure 4).

(4) Among the four groups of people, the dendritic cells of tuberculosis-resistant people had stronger migration ability (see Figure 5).

(5) Among the four groups of people, the high expression of HLA-DR surface markers on dendritic cells of tuberculosis-resistant individuals indicates that they have stronger antigen-presenting ability; the high expression of CD83 surface markers on dendritic cells of tuberculosis-resistant individuals indicates that Its maturity is higher (see Figure 6).

(6) Among the four groups of people, the dendritic cells of tuberculosis-resistant individuals had a stronger ability to secrete IFN-γ (see Figure 7).

(7) The dendritic cells of tuberculosis resisters can enhance the ability of macrophages to kill Mycobacterium tuberculosis (see Figure 8).

Example 4 Differential Gene Enrichment Analysis of Dendritic Cells from Tuberculosis Resistant Patients and Analysis of Enhanced IFN-γ Secretion Pathway

Based on the above experimental conclusions, the high expression of myeloid DC cells in vivo, stronger phagocytic ability, migration ability, high expression of CD83 surface markers and stronger ability to secrete IFN-γ in tuberculosis resistant patients, and thus the resistance to tuberculosis Differential gene enrichment analysis was carried out on the dendritic cells of tuberculosis resistant patients, and the molecular mechanism of IFN-γ secretion by dendritic cells of tuberculosis resistant patients was further explored.

4.1 Tuberculosis lysate stimulates myeloid dendritic cells in tuberculosis resistant patients

(1) Collect 20ml of peripheral blood from 3 tuberculosis resistant patients, and separate the peripheral blood to obtain peripheral blood mononuclear cells. The specific operation process is the same as that described in 1.1 and 1.2 above.

(2) Count with a cell counting board, adjust the cell density to 2×10 ⁶ /ml, use a 6-well cell culture plate to plate, inhale 2ml of cell suspension in each well, and lay 2 wells for each person, which are respectively the test well and the control well hole.

(3) According to the procedure described in 2.1, monocytes were induced to produce dendritic cells.

(4) Add 40 μg of tuberculosis lysate to the test wells with a pipette gun, and leave the control wells untreated, then move the cell culture plate to a cell culture incubator for 24 hours.

(5) After 24 hours, use a pipette gun to suck the cell culture medium into 1.5ml EP tubes, centrifuge at 500g for 10min, then use a pipette gun to carefully suck out the supernatant and discard it. NResister1, NResister2, NResister3, the test wells are named PResister1, PResister2, PResister3 respectively, mark the corresponding sample names with a marker pen, and store frozen at -80°C.

4.2 Extraction of total cellular RNA

(1) Thaw the cell pellet collected in 4.1 above on ice for later use.

(2) According to the operation process in the second part, the total cellular RNA was extracted using the cell/bacterial total RNA extraction kit of Guangzhou Dongsheng Biotechnology Co., Ltd.

(3) The concentration determination and A260/280 ratio of each RNA sample were measured using a NanoDrop 2000c ultra-micro-volume ultraviolet spectrophotometer. RNA samples with an A260/280 ratio of around 2 were selected for sequencing, and all RNA samples were frozen and stored in a -80°C refrigerator.

4.3 On-machine sequencing

The extracted RNA samples were sent to Beijing Berry Hekang Biotechnology Co., Ltd. for transcriptome sequencing (mRNAsequencing, mRNA-seq), using the Illumina sequencing platform to sequence the cDNA formed by the reverse transcription of the mRNA, and to study the gene structure and Gene expression abundance reveals mechanisms of Mycobacterium tuberculosis clearance by dendritic cells of tuberculosis resisters. The specific experimental process is as follows:

4.3.1 RNA detection

The RNA concentration was initially quantified using a NanoDrop 2000 spectrophotometer, and then the Agilent 4200 assay was used to interpret the quality of the RNA sample and accurately quantify the concentration. Select samples with no RNA degradation or slight degradation, no obvious DNA and small fragment contamination, and the total amount and concentration meet the standards (total amount ≥ 2 μg; concentration ≥ 40 ng/μl) for subsequent library construction.

4.3.2 Library construction

After the RNA sample is qualified, the library can be constructed. The library construction process is as follows:

(1) Use magnetic beads with Oligo(dT) to enrich eukaryotic mRNA;

(2) breaking mRNA into short fragments;

(3) using the fragmented mRNA as a template, using six base random primers to synthesize the first strand of cDNA;

(4) Add buffer, dNTPs, and enzymes to synthesize the second cDNA strand (dTTP, dATP, dGTP, and dCTP are added for common library construction, and dUTP, dATP, dGTP, and dCTP are added for strand-specific library construction);

(5) Purify the obtained double-stranded cDNA, repair the end, add A tail and connect the sequencing adapter, and then select the fragment size;

(6) PCR amplification enrichment cDNA library.

4.3.3 Library Quality Inspection

After the library was constructed, Qubit 3.0 was used for preliminary quantification, and then qPCR was used to accurately quantify the effective concentration of the library.

4.3.4 On-machine sequencing

After passing the library inspection, the IlluminaNovaSeq 6000 sequencing platform was used for PE150 sequencing.

4.4 Bioinformatics analysis

The raw data (Raw reads) obtained by sequencing contains a small number of reads with adapters, repetitions, and low quality. These Reads will affect the subsequent comparison and analysis. We first fine-filter the Raw reads to obtain effective and high-quality reads. Effective data (Clean reads), and then based on the effective data for differential gene expression analysis, mining differentially expressed genes before and after treatment. Then link differentially expressed genes with biological phenomena and their underlying mechanisms, and perform functional enrichment analysis (GO enrichment analysis and KEGG enrichment analysis) on differentially expressed genes to discover signals that play a key role in biological processes The molecular mechanism of pathways and related biological processes, the specific analysis process is shown in Figure 9.

4.5 Detection of target protein expression level by real-time fluorescent quantitative PCR

4.5.1 Primer design

The primer sequences of human GAPDH and STAT4 were searched on the Primer Bank website, verified by Primer-Blast on the NCBI website, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 10.

Table 10 Primer Sequence

4.6.2 Detection of STAT4 mRNA expression level by real-time fluorescent quantitative PCR

Use the remaining cDNA in the second part to perform real-time fluorescent quantitative PCR, and the specific operation process is the same as that described in 2.10 above.

4.6 Extraction of total protein of Mo-DCs from different populations and determination of protein concentration by BCA method

4.7.1 Tuberculosis lysate stimulates dendritic cells of Mo-DCs from different populations

(1) Collect 20ml of peripheral blood from HC, Resister, LTBI, and TB, and separate the peripheral blood to obtain peripheral blood mononuclear cells. The specific operation process is the same as the first part.

(2) Count with a cell counting board, adjust the cell density to 2×10 ⁶ /ml, use a 6-well cell culture plate to plate, inhale 2ml of cell suspension in each well, and lay 2 wells for each person, which are respectively the test well and the control well hole.

(3) Induce monocytes to generate dendritic cells according to the process described in the second part.

(4) Add 40 μg of tuberculosis lysate to the test wells with a pipette gun, and leave the control wells untreated, then move the cell culture plate to a cell culture incubator for 24 hours.

(5) Move the 500mM sodium metavanadate stock solution to room temperature to melt, adjust the metal bath to 100°C, then move the melted sodium metavanadate to the metal bath for activation for 10 minutes, and then cool to room temperature.

(6) Use a pipette to draw 500 μl of sodium metavanadate and 500 μl of 30% H ₂ O ₂ and mix them in a 1.5 ml EP tube to obtain a final concentration of 250 mM sodium pervanadate solution.

(7) Preheat the RPMI 1640 medium in a 37°C cell culture incubator, then take 500ml of the preheated medium and pack it into a 50ml centrifuge tube, use a pipette to draw 1ml of 250mM sodium pervanadate solution and add 50ml of RPMI The 1640 medium is made into a medium containing 5mM sodium pervanadate, and it is prepared for immediate use.

(7) Take out the cell culture plate after 24 hours, add 2ml of medium containing sodium pervanadate into each well, and then move to the cell culture incubator for 10 minutes of incubation.

(8) Use a pipette to transfer the cell culture medium to a 15ml centrifuge tube, centrifuge at 500g for 10min, discard the supernatant, then add 2ml of PBS to resuspend the cells, centrifuge at 500g for 10min, discard the supernatant to obtain a cell pellet.

4.7.2 Extraction of total protein and determination of protein concentration by BCA method

(1) According to the method shown in Table 11, prepare the cell lysate required for each sample, mix well and place on ice for later use.

Table 11 Configuration method of cell lysate

(2) Add 120 μl of prepared cell lysate to each 1.5ml EP tube containing cell pellets, blow and mix well with a pipette, place on ice, and lyse the protein for 30 minutes.

(3) After the protein is fully lysed, place the 1.5ml EP tube in a refrigerated microcentrifuge, centrifuge at 12000rpm for 10min at 4°C, and transfer the supernatant to a new sterile 1.5ml EP tube , mark well, and take 12 μl of each sample and dilute 5 times with PBS buffer solution for later use.

(4) Dilute the standard substance: use PBS as the diluent to dilute the BSA standard substance according to Table 3.

Table 12 BSA standard concentration preparation table

(5) Configure the BCA working solution: calculate the total amount of the BCA working solution required according to the total number of standards of each concentration and the samples to be tested. Prepare BCA reagent and Cu reagent at a ratio of 50:1 to make BCA working solution, mix thoroughly and store at room temperature for later use.

(6) Take 25 μl of the standard substance and the diluted sample to be tested and add them to the corresponding sample wells of the 96-well plate, and set 2 duplicate wells for each sample.

(7) After adding the standard and each sample to be tested into the corresponding wells, add 200 μl of BCA working solution to each well with a multi-channel pipette, cover the 96-well plate, and place it at 37 ° C for constant temperature incubation Incubate in the box for 30min.

(8) Turn on the multi-functional microplate reader in advance and set up the detection program.

(9) Measure the absorbance at 562nm of each well with a multi-functional microplate reader, take the average value of the absorbance of each concentration of the standard as the ordinate, and the corresponding concentration of the standard as the abscissa, draw a standard curve, and draw the standard curve formula.

(10) According to the standard curve formula, calculate the concentration of the sample to be tested after each dilution, and then calculate each

The protein concentration of the sample stock solution.

(11) Find the sample with the lowest concentration, calculate the protein amount of the sample according to the volume, and dilute the remaining samples with PBS buffer to adjust the concentration and volume of all samples to be consistent with the sample with the lowest concentration.

(12) According to the current volume of each sample, add an appropriate amount of 5× protein loading buffer to each sample to make the final concentration 1×.

(13) Heat all the samples with a constant temperature metal bath at 95°C for 10 minutes.

(14) After heating, centrifuge at 12,000 rpm for 1 min at room temperature, and store in a -80°C refrigerator for later use.

4.7 Western blot detection of target protein expression and phosphorylation level

(1) Carefully clean the glass plates, 15-hole combs and brackets used for glue making with detergent and deionized water, and dry them naturally for later use.

(2) Fix the glass plate on the bracket so that the bottom of the glass plate is parallel to the sponge pad and fit closely to ensure that the bottom will not leak glue.

(3) Use the 10% One-Step PAGE Gel Fast Preparation Kit from Nanjing Novozyme Co., Ltd. to prepare the upper layer gel and the lower layer gel respectively according to Table 13 and 14 (Note: APS is added at the end of the glue filling).

Table 13 upper glue formula

Table 14 Underlayer Adhesive Formula

(4) Add APS to the lower layer of glue and mix well, then pour it into the glue glass plate, so that the liquid level is about 1.5cm away from the edge of the short glass plate.

(5) After adding APS to the lower layer of glue, mix thoroughly immediately, without waiting for the lower layer of gel to solidify, gently inject the mixed solution into the glass plate with a pipette, and gently insert the comb.

(6) Stand at room temperature for 15 minutes. After the gel is solidified, pull out the comb vertically upwards gently.

(7) Remove the glass plate filled with glue from the bracket, install it on the electrophoresis device, lock it with a buckle to prevent liquid leakage, put it into the electrophoresis tank, and add 1× electrophoresis buffer to it, and its height must be at least more than Top of the two glass plates.

(8) After the protein samples are arranged in order, slowly load the sample and 5 μl protein marker in a volume of 25 μl (the remaining volume is supplemented with 1×Loading buffer), and add 25 μl 1×Loading buffer to other blank wells.

(9) After loading the sample, correctly connect the positive and negative poles of the power supply and the electrophoresis tank, adjust the voltage to 80V, and after electrophoresis for 30 minutes, adjust the voltage to 120V and then perform electrophoresis for about 1 hour. During the electrophoresis process, pay attention to observe the separation of the markers, and stop the electrophoresis in due course according to the size of the protein to be tested.

(10) After the electrophoresis is finished, disconnect the power supply, remove the glass plate from the electrophoresis device, pry the two layers of glass plates apart, cut off the stacking gel above, and then soak the separating gel in 1× transfer buffer.

(11) Use scissors to cut off a PVDF membrane of appropriate size, and then use tweezers to clamp the PVDF membrane and place it in methanol for activation for 15s.

(12) Soak the clamps, sponge pad, and filter paper used for membrane transfer with 1× transfer buffer, and then place the sponge pad, filter paper, separation gel, PVDF membrane, filter paper, and sponge pad in sequence from the negative electrode to the positive electrode. Take care to avoid the formation of air bubbles during placement.

(13) After confirming that the orientation is correct, close and fasten the clips, place them in the electroporation cell, and add 1× transfer buffer until the top of the clips is completely submerged. Correctly connect the positive and negative poles of the power supply and the electrotransfer tank, place the entire electrotransfer device in an ice water bath, adjust the current to 200mA, and transfer the membrane for 3.5h.

(14) After electroporation, disconnect the power supply, take out the clamp, carefully take out the PVDF membrane with tweezers, cut the PVDF membrane according to the size of the target protein, mark it, and seal it with blocking solution on a horizontal shaker for 2 hours at room temperature.

(15) Dilute each primary antibody in appropriate proportions with blocking solution (p-STAT41:500, STAT41:500, β-actin 1:5000), and place the PVDF membrane in a heat-sealable hybridization bag with tweezers , add an appropriate amount of diluted primary antibody solution, seal with a plastic sealing machine after exhausting air bubbles, and incubate overnight at 4°C on a horizontal shaker.

(16) Take out the PVDF membrane from the hybridization bag with tweezers, recover the primary antibody, and wash 4 times with 1×TBST buffer solution on a horizontal shaker, 20 min each time.

(17) Dilute the secondary antibody with the same species source as the primary antibody with blocking solution at a ratio of 1:10000, put the PVDF membrane in a heat-sealable hybridization bag with tweezers, add an appropriate amount of diluted secondary antibody solution, and drain Seal with a plastic sealing machine after air bubbles, and incubate at room temperature for 1 h on a horizontal shaker.

(18) Take the PVDF membrane out of the hybridization bag with tweezers, and wash it with 1×TBST buffer solution on a horizontal shaker for 4 times, 20 min each time.

(19) According to the number of PVDF membranes, use a pipette to pipette an appropriate amount of ECL supersensitive luminescent solution A and ECL supersensitive luminescent solution B, mix them evenly at a ratio of 1:1, and use a pipette to evenly add the mixed solution dropwise on the surface of the PVDF membrane.

(20) When developing, the membrane can be put into a gel imaging chromogenic instrument to take pictures; it can also be placed in a dark box for darkroom compression.

(21) After the development is completed, the PVDF membrane can be placed in the primary antibody and secondary antibody removal solution and washed on a horizontal shaker for 20 minutes to elute the antibody on the PVDF membrane, then re-block with the blocking solution, and continue to incubate with other primary antibodies , and the rest of the steps are as described above.

4.8 Analysis of Experimental Results

Transcriptome analysis was performed on myeloid dendritic cells of tuberculosis resistant patients, and the KEGG database was used to annotate the information of the up-regulated differential genes at the level of biological pathways and perform Pathway enrichment analysis. The analysis results (Figure 10) showed that the differential genes were mainly concentrated in Cytokine-cytokine receptor interaction signaling pathway, chemokine signaling pathway, Toll-like receptor signaling pathway, complement system signaling pathway, etc. Among them, the cytokine-cytokine receptor interaction signaling pathway is the most enriched, and this pathway The enrichment of IFN-γ, combined with the above results, shows that IFN-γ plays a crucial role in the process of clearing Mtb in tuberculosis resisters.

Transcriptome results showed that IFN-γ was one of the core genes for the clearance of Mtb by dendritic cells of tuberculosis resisters, and further explored the molecular mechanism of IFN-γ secretion by dendritic cells of tuberculosis resisters. Firstly, the expression level of STAT4mRNA in Mo-DCs of different populations was studied by real-time fluorescent quantitative PCR, and the results (Figure 11A) showed that the expression level of STAT4mRNA in dendritic cells of tuberculosis resistance was higher than that of the other three groups of people; and then explored by Western blot experiment The expression level and phosphorylation level of STAT4 protein in different populations were analyzed, and the results (Figure 11B) showed that after being stimulated with tuberculosis lysate for 24 hours, the expression level and phosphorylation level of STAT4 protein in the dendritic cells of tuberculosis resistant patients were different from those in the four groups. Average is the highest. It shows that tuberculosis resistance promotes the secretion of IFN-γ through STAT4 signaling pathway.

Claims (10)

1. Use of a biomolecule in the preparation of a formulation for killing mycobacterium tuberculosis, characterized in that the biomolecule is a nucleic acid, a protein and/or a polypeptide of STAT4 molecule.

2. Use of a biomolecule according to claim 1 for the preparation of a formulation for killing mycobacterium tuberculosis, wherein the polypeptide further comprises a polypeptide constructed from a nucleic acid or protein composition comprising a STAT4 molecule nucleic acid or a composition comprising a STAT4 molecule protein.

3. Use of a biomolecule according to claim 1 for the preparation of a formulation for killing mycobacterium tuberculosis, the formulation being a pharmaceutical formulation.

4. Use of a biomolecule according to claim 1 for the preparation of a formulation for killing mycobacterium tuberculosis, wherein the STAT4 biomolecule may also improve or enhance at least one property selected from the group consisting of:

A. enhancing phagocytic capacity of dendritic cells;

B. enhancing the migration capacity of dendritic cells;

C. improving the HLA-DR surface marker expression of the dendritic cells;

D. improving the expression of a CD83 surface marker of the dendritic cells;

E. enhancing IFN-gamma secretion ability;

F. enhancing the capability of macrophages to kill mycobacterium tuberculosis.

5. Use of a biomolecule in the preparation of a tuberculosis immunotherapeutic preparation.

6. Use of a biomolecule according to claim 5 in the preparation of a formulation for tuberculosis immunotherapy, said immunotherapy comprising targeting antibodies, tuberculosis vaccines, secondary cell therapies, immune checkpoint inhibitors, cytokines and immune adjuvants.

7. Use of a biological molecule for the preparation of an immunodiagnostic reagent for detecting a susceptibility to mycobacterium tuberculosis in a population, characterized in that the immunodiagnosis comprises a diagnosis of the expression level of STAT4 molecules in a subject.

8. Use of a biomolecule in the construction of a cell model against mycobacterium tuberculosis.

9. The use according to claim 8, wherein the cells are dendritic cells.

10. A dendritic cell line having a function of killing mycobacterium tuberculosis, said dendritic cell having a higher level of STAT4 expression and/or phosphorylation than that of STAT4 in a normal dendritic cell, and said dendritic cell line having at least one of the following functions:

G. the phagocytic capacity is stronger;

H. the migration capability is stronger;

HLA-DR surface marker high expression;

J.CD83 surface marker high expression;

IFN-gamma secretion ability is stronger;

l. has the ability to kill Mycobacterium tuberculosis.

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