CN111000978B - Application of TLT-2 in preparation of medicine for treating tuberculosis - Google Patents

Abstract

The invention relates to the field of biological medicines, in particular to application of TLT-2 in preparation of a medicine for treating tuberculosis, and provides a novel immunotherapy scheme for comprehensive treatment of tuberculosis, the invention researches TLT-2 in active tuberculosis patients to find that the expression level of TLT-2 on mononuclear macrophages of tuberculosis patients is increased, and the TLT-2 on the surface of mouse mononuclear macrophages is blocked by TLT-2 recombinant protein to further research and find that TLT-2 can effectively reduce tubercle bacillus in the lungs of mice and promote anti-tubercular inflammatory reaction, so that TLT-2 is expected to be applied to comprehensive treatment of tuberculosis to prepare a form of the medicine for treating tuberculosis, reduce tubercle bacillus in the lungs of tuberculosis patients, reduce inflammation and finally achieve the aim of cellular immunotherapy.

Description

Application of TLT-2 in preparation of medicine for treating tuberculosis

technical field

The invention relates to the field of biomedicine, in particular to the application of TLT-2 in the preparation of medicines for treating tuberculosis.

Background technique

Tuberculosis is a contagious disease caused by Mycobacterium tuberculosis, which is relatively common in clinical practice. Tuberculosis mostly occurs among young people and rural people. After being infected with tuberculosis, Mycobacterium tuberculosis will invade various body organs of the patient, most of which invade the lungs, so it is also called pulmonary tuberculosis. In addition to lung infections, meningeal, skin, and cervical lymph infections may also occur. The causes of tuberculosis include human-to-human infection, environmental pollution, and the spread of AIDS. The main clinical manifestations are cough, sputum, hemoptysis, malaise, and low-grade fever. Once tuberculosis occurs, it will seriously affect the normal life and work of patients.

Currently, tuberculosis is mainly treated with chemical drugs, but long-term chemotherapy can easily lead to drug resistance of Mycobacterium tuberculosis. With the high rate of drug resistance and the spread of drug-resistant bacteria, the treatment of tuberculosis is becoming more and more difficult. Therefore, there is an urgent need to develop new regimens to treat TB and drug-resistant TB. In recent years, many attempts have been made in the field of biotherapy, such as cellular immunotherapy, cytokines, monoclonal antibodies, etc. Among them, cell therapy is to activate and expand autologous or allogeneic immune effector cells in vitro, and then infuse them back to the patient, so as to kill the target cells and improve the immune function of the patient. Cellular immunotherapy is an important part of the comprehensive treatment of refractory tuberculosis in the future, and the current technology lacks effective methods for cell therapy of tuberculosis.

Contents of the invention

In order to overcome the deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide the application of TLT-2 in the preparation of medicaments for treating tuberculosis.

Myeloid cell triggering receptor-like transcription factor-2 (TREMs like transcript-2, TLT-2) is a member of the myeloid cell triggering receptors (Triggering receptors expressed on myeloid cells, TREMs) immunoglobulin superfamily, is a A novel inflammatory provoking receptor. The present invention finds that the expression of TLT-2 on the mononuclear macrophages of tuberculosis patients is increased through research on tuberculosis patients, and further research finds that TLT-2 on the surface of monocytes and macrophages is blocked by TLT-2 recombinant protein , TLT-2 recombinant protein can effectively reduce the tuberculosis bacteria in the lungs and promote anti-tuberculosis inflammatory response, suggesting that TLT-2 is expected to be used in the comprehensive treatment of tuberculosis.

Therefore, the present invention firstly provides a medicament for treating tuberculosis, said medicament comprising myeloid cell triggering receptor-like transcription factor-2 (also known as TLT-2).

Through research, it is found that: TLT-2 in the human body recognizes and binds to certain components of Mycobacterium tuberculosis, resulting in the inability of macrophages to clear Mycobacterium tuberculosis and inhibiting the anti-tuberculosis inflammatory response. Through exogenous administration of TLT-2 recombinant protein, because it is the same protein as TLT-2 on the surface of macrophages, it can also bind to Mycobacterium tuberculosis, thereby reducing the binding of Mycobacterium tuberculosis to TLT-2 on the surface of macrophages , thereby reducing the TLT-2-mediated inhibitory effect of macrophages.

Furthermore, the above-mentioned drug for treating tuberculosis of the present invention also includes a pharmaceutically acceptable adjuvant for myeloid cell triggering receptor-like transcription factor-2, and the therapeutic effect can be guaranteed by using it in conjunction with the pharmaceutically acceptable adjuvant At the same time, it can also improve the safety, effectiveness and stability of the drug.

The present invention also provides the application of myeloid cell triggering receptor-like transcription factor-2 in the preparation of medicine for treating tuberculosis.

Furthermore, the above-mentioned myeloid cell triggering receptor-like transcription factor-2 of the present invention is a human-derived recombinant myeloid cell triggering receptor-like transcription factor-2 protein, which is more suitable for comprehensive treatment of human tuberculosis; specifically, TLT-2 is It treats tuberculosis by reducing the number of Mycobacterium tuberculosis in the body and promoting the anti-tuberculosis inflammatory response in the body.

Further, the dosage form of the above-mentioned drug of the present invention is an injection dosage form or a dosage form administered through the gastrointestinal tract, so as to increase the scope of application of the drug; specifically, the dosage form administered through the gastrointestinal tract includes commonly used powders, tablets, granules, etc. formulations, capsules, solutions, emulsions, suspensions, etc.; injection dosage forms include commonly used injections (such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections).

Furthermore, when the dosage form of the drug is an injection dosage form, it is sufficient to mix the drug with physiological saline to prepare a solution during use.

Compared with prior art, the beneficial effect of the present invention is:

The present invention studies TLT-2 in patients with active tuberculosis and finds that the expression level of TLT-2 on monocyte-macrophages of tuberculosis patients increases, and the TLT-2 recombinant protein blocks the expression of monocyte-macrophages on the surface of mouse monocyte-macrophages. Further research on TLT-2 found that TLT-2 recombinant protein can effectively reduce the tuberculosis bacteria in the lungs of mice and promote anti-tuberculosis inflammatory response, suggesting that TLT-2 is expected to be applied in the comprehensive treatment of tuberculosis and prepared as a drug for the treatment of tuberculosis. form, to reduce Mycobacterium tuberculosis and reduce inflammation in the lungs of tuberculosis patients. At the same time, because TLT-2 is a human recombinant protein, there is no rejection, and it is more suitable for the comprehensive treatment of tuberculosis.

Description of drawings

Figure 1 is the expression of TLT-2 on monocyte-macrophages of tuberculosis patients (***, p<0.001);

Figure 2 shows the changes in the number of Mycobacterium tuberculosis in the lungs and spleen of mice after the TLT-2 recombinant protein blocked TLT-2 on the surface of mouse mononuclear macrophages (Figure 2A is the number of Mycobacterium tuberculosis in the lungs, Figure 2B is the number of Mycobacterium tuberculosis in the spleen);

Figure 3 is the inflammatory infiltration of mouse lungs after the TLT-2 recombinant protein blocked TLT-2 on the surface of mouse mononuclear macrophages (a, c, e are 10X fields of view, b, d, f are corresponding to a , 20X field of view of c and e, where control is the blank group, BCG+Nacl is the positive control group, and BCG+rmTLT2 is the experimental treatment group).

Detailed ways

Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 TLT-2 expression analysis experiment on monocyte-macrophages of tuberculosis patients

1. Collection of test samples:

Peripheral blood was collected from subjects: patients with active tuberculosis (denoted as ATB) and healthy people (denoted as HC).

2. The detection method specifically includes the following steps:

(1) 3mL of human peripheral venous whole blood and normal saline were mixed and diluted at a ratio of 1:1, and the diluted whole blood was slowly added to the liquid surface of 2ml of Ficoll lymphocyte separation solution.

(2) Centrifuge at 1800 rpm for 30 min to obtain the mononuclear cell layer (buffy coat where PBMCs are located).

(3) Aspirate buffy coat cells, seal with 1% BSA buffer, and perform flow staining to mark TLT-2 (CD11b-PacificBlue, TLT-2-PE) expressed on monocytes and macrophages, staining at 4 degrees for 30 minutes , fixed with 1% paraformaldehyde and tested on the machine, the expression of TLT-2 was counted as the percentage of TLT2 ⁺ CD11b ⁺ Cells).

3. Experimental results:

As shown in the results in Figure 1, the expression of TLT-2 was increased in monocyte-macrophages of tuberculosis patients compared with healthy controls. It is suggested that TLT-2 on mononuclear macrophages may play an important role in tuberculosis infection.

Example 2 TLT-2 experiment of TLT-2 recombinant protein blocking the surface of mouse monocyte-macrophage

1. Experiment preparation: mouse TLT-2 recombinant protein; Mycobacterium bovis BCG; 7H10 medium; normal saline; C57BL mice.

2. Experimental steps:

(1) Collect the BCG cultured in liquid by centrifugation, and grind the BCG with a tissue grinding rod to prepare a single bacterial suspension of BCG;

(2) Calculate the bacterial concentration by detecting the turbidity of a single bacterial suspension of BCG, and then intravenously infect mice with a dose of BCG 1×10 ⁶ /mouse;

(3) Infected mice were injected intraperitoneally with 1 ug of TLT-2 recombinant protein (denoted as BCG+rmTLT2 group), and the control group was injected with NaCl;

(4) 7 days after infection, re-inject 1ug/TLT-2 recombinant protein to enhance the effect.

(5) Judgment of curative effect: The experiment was terminated on 21 days, the mice were dissected, the lungs and spleen were taken, and the organs were ground to obtain the tissue grinding fluid. The tissue grinding fluid was lysed with 0.01% Triton100 at a ratio of 1:10, and 100uL was taken. The lysate was plated on a 7H10 plate, and after 21 days of culture at 37 degrees, the individual bacterial colonies growing on the plate were counted. Taking the Nacl treatment group as a control, the number of Mycobacterium tuberculosis in the lungs and spleens of the mice treated with the TLT-2 recombinant protein was detected ( FIG. 2 ). Partial lung tissue blocks were taken, fixed with 10% formalin, and stained with HE on the pathological sections to observe the inflammatory infiltration of the lungs of the mice ( FIG. 3 ).

3. Experimental results:

As shown in the results in Figure 2, compared with the Nacl treatment group, the TLT-2 recombinant protein blocked TLT-2 on the surface of mouse mononuclear macrophages, and the amount of Mycobacterium tuberculosis in the lungs and spleen of the mice was significantly reduced, indicating that blocking The immune mechanism of bacterial clearance in mice was strengthened after cutting off TLT-2 on the surface of mouse mononuclear macrophages.

As shown in the results in Figure 3, compared with the Nacl treatment group, TLT-2 recombinant protein blocked TLT-2 on the surface of mouse monocyte-macrophage cells, and the inflammatory response in the lungs of mice was enhanced, indicating that blocking TLT-2 The inflammatory effect of the anti-TB immunity in the mice was enhanced.

It can be seen from the above results that the TLT-2 recombinant protein can effectively reduce the tuberculosis bacteria in the lungs of mice, promote the anti-tuberculosis inflammatory response, and is beneficial to the comprehensive treatment of tuberculosis, suggesting that the TLT-2 recombinant protein is expected to be used in the comprehensive treatment of tuberculosis .

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (5)

1. Application of medullary cell triggering receptor-like transcription factor-2 in preparing medicine for treating tuberculosis is provided.

2. The use of claim 1, wherein the myeloid cell-triggering receptor-like transcription factor-2 is a human recombinant myeloid cell-triggering receptor-like transcription factor-2 protein.

3. The use according to claim 1, wherein the treatment of tuberculosis is a reduction in the number of mycobacterium tuberculosis in the body.

4. The use according to claim 1, wherein the treatment of tuberculosis is the promotion of an anti-tubercular inflammatory response in vivo.

5. The use according to claim 1, wherein the medicament is in the form of an injection or a gastrointestinal tract.

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