CN111808908A - A method for the detection of gaMSCs subsets that promote glioma drug resistance - Google Patents

Abstract

The invention discloses a detection method of glioma-associated mesenchymal stem cells (gammscs) subgroup promoting glioma resistance. The method is characterized in that a human glioma cell line U87 and a glioma primary cell (GBM-1) cultured by a GAMSCs conditioned medium are collected, TMZ is used for processing, and then visual experiment comparison is realized by various detection means, so that the evaluation of the GAMSCs on the drug resistance of the brain glioma in chemotherapy is carried out. The evaluation method can obtain that the gammscs subgroup with low expression of CD90 can promote the expression of FOXS1 in brain glioma cells by secreting IL-6, thereby activating the Epithelial Mesenchymal Transition (EMT) process of tumor cells and forming chemotherapy resistance to temozolomide. The invention further defines the mechanism of promoting the chemotherapy resistance of tumor cells by the brain glioma microenvironment and provides a new idea and a target point for further personalized treatment of the brain glioma.

Description

A method for the detection of gaMSCs subsets that promote glioma drug resistance

technical field

The invention relates to the technical fields of biology and medicine, in particular to a method for detecting a gaMSCs subgroup that promotes drug resistance of brain gliomas.

Background technique

Malignant gliomas are the most common, aggressive, and highly malignant primary brain tumors in adults with a high mortality rate. Despite surgical resection, combined with chemoradiotherapy, median survival is still only 13-16 months, and the five-year survival rate is only 6.7%. Although some novel therapeutic approaches such as immune checkpoint inhibitors, viral therapy, CAR T, dendritic 3-like cell therapy and vaccine therapy have been shown to have some effect on malignant glioma, further research is needed. Currently, temozolomide (TMZ) chemotherapy remains the mainstay of treatment, and temozolomide therapy is the only chemotherapy approved for newly diagnosed malignant gliomas. Temozolomide is an oral alkylating chemotherapy drug that can effectively cross the blood-brain barrier and act on the lesion site, prolonging the survival of postoperative patients. However, most patients will develop resistance to temozolomide over time.

Whether tumor cells are sensitive to chemotherapeutic drugs depends not only on whether their genes are mutated, but also largely affected by the surrounding environment, and the drug resistance mutation of tumor cells themselves is also affected by the surrounding environment to a certain extent. The tumor microenvironment (TMZ) is composed of a variety of extracellular components such as extracellular matrix, various hormones, cytokines, growth factors, endothelial cells, stem cells (including mesenchymal stem cells), immune cells, and fibroblasts. The tumor microenvironment plays an important role in the occurrence and development of tumors. In recent years, related research has gradually increased, and its status has become more and more important. The tumor microenvironment can not only facilitate the occurrence, progression and metastasis of tumors and maintain tumor characterization, but also may play an important role in the formation of chemotherapy resistance.

Mesenchymal stem cells (MCS), also known as pluripotent mesenchymal stem cells, are similar in shape to fibroblasts in vitro, and can proliferate and differentiate into osteoblasts, chondrocytes, and adipocytes under specific stimulation conditions. In tumors, their associated mesenchymal stem cells are an important part of stem cells in the tumor microenvironment. Glioma-associated mesenchymal stem cells (gaMSCs) can transform into pericytes during tumor progression, participate in angiogenesis, and have a negative impact on play a significant role in the progression of gliomas.

However, there is no research on the formation mechanism of gaMSCs resistance to glioma temozolomide at present, and then the present invention provides a method for evaluating the resistance of gaMSCs to glioma chemotherapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to, in view of the above-mentioned deficiencies of the prior art, to propose a detection method for gaMSCs subsets that promote glioma drug resistance, so as to intuitively and truly analyze the influence of gaMSCs on glioma chemoresistance Evaluation.

The present invention provides a method for detecting a gaMSC subset that promotes drug resistance of brain glioma, comprising the following steps:

S1: Collect and isolate gaMSCs, human glioma cell line U87 and glioma primary cells (GBM-1);

S2: Prepare human glioma cell line U87 and glioma primary cells (GBM-1) cultured in DMEM medium and gaMSCs conditioned medium, respectively, to obtain human glioma cell line U87 cultured in DMEM medium ( Control-U87), primary glioma cells (Control-GBM1) cultured in DMEM medium, human glioma cell line U87 (gaMSC-U87) cultured in gaMSCs-conditioned medium, and gliomas cultured in gaMSCs-conditioned medium Primary cells (gaMSC-GBM1);

S3: Control-U87, Control-GBM1, gaMSC-U87 and gaMSC-GBM1 were treated with glioma chemotherapy drugs in vitro to obtain multiple samples to be tested;

S4: The detection, analysis and comparison of the proliferation ability of the sample to be tested with the drug added by CCK-8;

S5: analysis and comparison of cell apoptosis in the sample dosing to be tested by flow analysis technology;

S6: Analyze and compare the migration ability in the drug-dosed sample to be tested by scratch test;

S7: Analysis of the effect of gaMSCs on the gene expression of glioma cells by gene chip;

S8: The expression of FOXS1 was analyzed by PCR and Western-blot;

S9: Detect the changes of epithelial-mesenchymal transition (EMT) markers when FOXS1 is knocked down or overexpressed by lentiviral transfection;

S10: establish an orthotopic nude mouse model by using brain glioma cells and administer glioma chemotherapeutic drugs in vivo, and conduct in vivo drug addition experiments in animals;

S11: The expression changes of Ki-67 in tumor tissues of animals in S10 were detected by immunohistochemistry;

S12: The IL-6 expression and secretion of gaMSCs was analyzed by ELISA;

S13: Analyze and compare the above-mentioned experimental detection results, and conduct drug resistance evaluation.

Further, the glioma chemotherapy drug body is temozolomide (TMZ).

Further, the in vitro concentration of temozolomide is 190-210 uM (umol/L), and the in vivo injection concentration is 45-55 mg/kg.

Further, the glioma primary cells are selected to collect the third-generation passaged cells for experiment and detection.

Further, the gaMSCs need to be sorted for high CD90 expression and low CD90 expression.

Further, the sorting step of described gaMSCs comprises:

S101: The gaMSCs were taken out of the incubator and placed in a clean bench, washed with PBS, digested with Accutase, centrifuged at 1500 rpm for 6 minutes;

S102: Discard the supernatant, add CD90 magnetic bead antibody and sorting Buffer at 1:4, and mix well;

S103: put it in a 4°C refrigerator to avoid light for 15 minutes;

S104: Take out the cells, rinse with sorting buffer, centrifuge at 1500 rpm for 6 minutes;

S105: Discard the supernatant, add 500ul sorting Buffer to resuspend;

S106: drop the cell suspension into the sorting column, and then drop 2 ml of sorting Buffer to wash the column, and the filtered cell suspension is gaMSCs with low CD90 expression (CD90-);

S107: After the above filtration is completed, add 2 ml of DMEM medium containing 20% serum to the sorting column, and then use the handle to quickly push the medium out. At this time, the filtered gaMSCs are CD90 highly expressing gaMSCs (CD90+);

S108: The above CD90- and CD90+ cells were respectively transferred to culture flasks, and cultured in a 37°C, 5% CO2 incubator.

Further, the primer sequences in PCR described in S8 are:

Homo b-actin Forward 5’-AGCGAGCATCCCCCAAAGTT-3’,

Reverse 5'-GGGCACGAAGGCTCATCATT-3',

Homo FOXS1 Forward 5'-CCCAGGTTCCTTGTGGTC-3',

Reverse 5'-CCCAGGTTCCTTGTGGTC-3'.

Further, the PVDF membrane processing method in the Western-blot detection and analysis described in S8 is as follows: soak the PVDF membrane in a blocking solution (TBST containing 5% nonfat milk powder), and place it on a shaker to block for 2 hours; Soak the PVDF membrane in the primary antibody diluted with the primary antibody diluent and incubate overnight at 4°C; wash the PVDF membrane incubated with the primary antibody with TBST 5 times for 5 minutes each time; soak the washed PVDF membrane in the secondary antibody dilution Incubate on a shaker for 2 hours at room temperature in the secondary antibody diluted in PBS; after incubating the secondary antibody, wash 5 times with TBST before use.

Further, the experimental steps of lentivirus transfection described in S9 include:

S201: Prepare a cell suspension with a density of 50,000 cells/ml in DMEM medium containing 10% serum and inoculate it into a 6-well plate, 2 ml per well, and then culture in a 37°C, 5% CO ₂ incubator for 24 hours;

S202: After 24 hours, replace the DMEM medium, 1 ml per well, then add 20ul of 1×10 ⁸ TU/ml virus solution to each well, and continue to culture for 12-16 hours in a 37°C, 5% CO2 incubator;

S203: After 12-16 hours, the medium was replaced and the culture was continued for 72 hours, and the infection effect was observed under a fluorescence microscope;

S204: collecting infected cells and placing them in a medium containing puromycin for screening and purification for later use.

The present invention has the following beneficial effects:

According to the evaluation method of the present invention, it can be concluded that the subgroup of gaMSCs with low CD90 expression can promote the expression of FOXS1 in brain glioma cells by secreting IL-6, thereby activating the epithelial-mesenchymal transition (EMT) process of tumor cells, and forming anti-temozolomide chemotherapy resistance. The present invention further clarifies the mechanism by which the glioma microenvironment promotes the chemoresistance of tumor cells, and provides new ideas and targets for further personalized treatment of gliomas.

Instruction drawings

Fig. 1 is the morphological diagram of the cultured cells of Control-U87 and gaMSC-U87 of the present invention;

Fig. 2 is the schematic diagram of CCK-8 detection result of the present invention;

Fig. 3 is the schematic diagram of scratch experiment detection result of the present invention;

Figure 4 is a schematic diagram of the flow cytometry detection results of the present invention;

5 is a schematic diagram of the results of measuring the expression of FOXS1 in Control-U87 and gaMSC-U87 by the gene chip technology of the present invention;

Fig. 6 is the result schematic diagram of FOXS1 expression of the present invention;

Figure 7 is a schematic diagram of the results of the Western-blot assay of the EMT marker protein of the present invention;

Fig. 8 is the detection schematic diagram of the present invention to verify the synergistic promotion of FOXS1 and EMT;

9 is a schematic diagram of the results of a lentivirus transfection experiment of the present invention;

Figure 10 is a schematic diagram of the comparison results of the in vivo experiments of the present invention;

Figure 11 is a schematic diagram of the comparison results of Ki67 in vivo experiments of the present invention;

Figure 12 is a schematic diagram of the results of IL-6 content comparison and FOXS1 expression comparison of the present invention;

Figure 13 is a schematic diagram of the cell differentiation ability of glioblastoma-related mesenchymal stem cells;

Figure 14 is a schematic diagram of the cell migration ability of glioblastoma-associated mesenchymal stem cells.

Detailed ways

The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, various changes or modifications made to the present invention by those skilled in the art, these equivalent forms also fall within the scope of protection claimed in this application. The proportions in the embodiments of the present invention are all by weight.

Isolation and culture of glioma-associated mesenchymal stem cells (gaMSCs):

(1) Under the condition of obtaining the consent of patients undergoing glioma surgery in Wuhan Union Medical College Hospital and signing the informed consent form, in the operating room of Wuhan Union Union Medical College Hospital, immediately after the glioma specimens were removed, they were placed in a non-sterile room filled with normal saline. Bacteria centrifuge tubes, and immediately transported to the laboratory pre-sterilized sterile ultra-clean bench.

(2) Place the glioma specimen in a petri dish, flush the blood clots with PBS, cut the specimen into 1 cubic millimeter pieces with sterile scissors, and add trypsin to digest.

(3) After 15 minutes, use a cell sieve with a pore size of 70 µm to filter, and centrifuge the filtrate at 1500 rpm for 10 minutes.

(4) Discard the supernatant, add 3 ml of erythrocyte lysis solution, pipette evenly and lyse for 5 minutes, and then centrifuge at 1500 rpm for 5 minutes.

(5) Discard the supernatant, wash the cells with DMEM medium, and carefully transfer the cell suspension to a 15ml centrifuge tube pre-added with 4ml of lymphocyte separation medium to stratify the liquid, then adjust the acceleration and deceleration to 0 at 1000rpm and centrifuge for 20 minutes .

(6) After centrifugation, transfer the intermediate buffy coat layer to PBS and wash twice, then add the cells to DMEM medium containing 20% fetal bovine serum, 1% penicillin and streptomycin, and place them at 37°C for 5 minutes. The cells were cultured in a % CO ₂ incubator, and the medium was changed and passaged according to the growth conditions.

Isolation and culture of primary glioma cells (GBM-1):

(1) Place the glioma specimen in a petri dish, add MEM-α medium after flushing the blood clot with PBS, cut the specimen into small pieces of about 1 cubic millimeter after cutting off the electrocoagulation necrosis with sterile scissors.

(2) After homogenizing the small pieces, add trypsin and digest at 37°C for 30 minutes

(3) Lyse the digested cell suspension with erythrocyte lysate for 10 minutes,

(4) Wash the excess lysate with PBS, and then centrifuge at 1500 rpm for 5 minutes.

(5) Discard the supernatant, wash the cells with Neuroblast medium (containing N2, B27, hEGF, FGF and heparin), then transfer them to a culture flask with extracellular matrix, and culture the cells at 37°C, 5% CO ₂ The cells were cultured in an incubator, and the medium was changed or passaged about every 5 days according to the growth rate of the cells, and the third-generation cells were collected for experiments and detection.

Subculture of human glioma cell line U87:

(1) Take out the cells from the incubator, place the cells in a clean bench, pour off the medium, and add PBS to rinse once.

(2) Pour off the PBS, add 1 ml of trypsin, and place it at 37°C for digestion for 1-2 minutes.

(3) Transfer the digested cells to a 15 ml centrifuge tube, add 1 ml of DMEM medium containing 10% serum to neutralize trypsin, and then centrifuge at 1000 rpm for 5 minutes.

(4) Discard the supernatant and resuspend the cells in 4 ml of DMEM medium containing 10% serum.

(5) Transfer the cell suspension to a T25 culture flask and place it in a cell incubator at 37°C with 5% CO ₂ .

Glioma-related mesenchymal stem cell sorting:

(1) The glioma-related mesenchymal stem cells were taken out of the incubator and placed in a clean bench, washed with PBS, digested with Accutase, and centrifuged at 1500 rpm for 6 minutes.

(2) Discard the supernatant, add CD90 magnetic bead antibody and sorting Buffer at 1:4, and mix well.

(3) Put the reaction in a 4°C refrigerator to avoid light for 15 minutes.

(4) The cells were taken out, washed with sorting buffer, and centrifuged at 1500 rpm for 6 minutes.

(5) Discard the supernatant, add 500ul sorting Buffer and resuspend.

(6) The cell suspension is dropped into the sorting column, and then 2 ml of sorting Buffer is dropped to wash the column, and the filtered cell suspension is the glioma-related mesenchymal stem cells with low CD90 expression.

(7) After the above filtration is completed, add 2 ml of DMEM medium containing 20% serum to the sorting column, and then use the handle to quickly push out the medium. At this time, the filtered glioma-related cells with high CD90 expression mesenchymal stem cells.

(8) The above cells were respectively transferred to culture flasks and cultured in a 37°C, 5% CO ₂ incubator.

Preparation of conditioned medium for glioma-related mesenchymal stem cells:

(1) Take out the glioma-associated mesenchymal stem cells with a density of about 50% from the incubator and place them in a clean bench.

(2) Pour off the medium, rinse 2-3 times with PBS, add 4 ml of serum-free DMEM medium, and place in a 37°C, 5% CO ₂ incubator for 3 days.

(3) After 3 days, take out the culture bottle, transfer the culture solution to a 15ml centrifuge tube, centrifuge at 1000g for 10 minutes.

(4) Collect the supernatant and place it in a -20°C refrigerator for later use.

CCK-8 proliferation assay:

(1) Digest and centrifuge U87 cells, prepare a cell suspension of 50,000 cells/ml in DMEM medium containing 10% serum, and inoculate in a 96-well plate with 100 ul per well.

(2) After the cells were completely adherent, washed three times with PBS, and added serum-free DMEM medium and glioma-related mesenchymal stem cell conditioned medium respectively.

(3) Add the corresponding concentration of temozolomide and culture in an incubator at 37°C and 5% CO ₂ .

(4) After culturing to the time required for measurement, take out the 96-well plate, add 10ul of CCK-8 reagent to each well, and place it in an incubator to continue culturing for 2 hours.

(5) Detect the OD value of each well at a wavelength of 450 nm in a microplate reader after 2 hours.

Scratch test:

(1) Inoculate U87 cells in a 6-well plate and culture in an incubator. When the cells reach about 90% density, take them out and place them in a clean bench.

(2) Draw a straight line through the diameter in the middle of each hole with a 10ul pipette tip.

(3) After rinsing with PBS, take pictures under a microscope, which is a 0-hour picture.

(4) Rinse with PBS, add DMEM medium and glioma-related mesenchymal stem cell conditioned medium, and add 200uM (umol/L) temozolomide, and place in an incubator for 24 hours.

(5) Take out the photo after 24 hours, which is the 24-hour picture.

Flow Cytometry:

(1) U87 cells cultured under different conditions were digested with EDTA-free trypsin, the cell suspension was collected, centrifuged at 1200 rpm for 5 minutes.

(2) Discard the supernatant and wash the cells with PBS 2-3 times.

(3) Add 5uL of 7-AAD staining solution to each 50uL of Binding Buffer, mix well and set aside.

(4) Add 55ul of the above prepared solution to each tube, and after mixing, incubate at room temperature for 5-15 minutes in the dark.

(5) Then add 450uL Binding Buffer and mix well.

(6) Add 1uL Annexin V-APC, mix well, and incubate at room temperature for 5-15 minutes in the dark.

(7) No dye solution was added to the negative control, and only one dye solution was added to the single positive control.

(8) On-board detection.

Control-U87 and gaMSC-U87 were obtained by culturing U87 cells with DMEM medium and glioma-related mesenchymal stem cell conditioned medium, respectively. The morphology of the cells in the two groups was observed, and the cells in both groups were attached to the wall and grew in a spindle shape with no shape. difference (Figure 1). CCK-8 was used to measure the difference of cell proliferation between the two groups under the condition of different concentrations of temozolomide, and it was found that the proliferation ability of gaMSC-U87 was stronger than that of Control-U87; further, the difference of cell proliferation ability of the two groups at different times was measured under the condition of 200 μM temozolomide, the same gaMSC-U87 -U87 was more proliferative than Control-U87 (Figure 2). The migration ability of the two groups of cells was determined by scratch experiments under the condition of 200 μM temozolomide, and it was found that the migration of gaMSC-U87 was better than that of Control-U87 (Fig. 3). Flow cytometry was used to measure cell apoptosis, and the results showed that regardless of the presence of temozolomide, the apoptosis rate of gaMSC-U87 was lower than that of Control-U87 (Figure 4). For further description of the cell differentiation and cell migration of tumor-associated mesenchymal stem cells, please refer to Figure 13 (D, E, F are schematic diagrams of the differentiation of adipocytes, osteocytes and chondrocytes, respectively) and Figure 14.

Among them: Figure 1. Morphology of U87 cells in different culture conditions. Control-U87 on the left and gaMSC-U87 on the right. ×40, scale bar=200 μm; Figure 2. Proliferation of U87 cells under different conditions. A. The proliferation of two groups of cells under different concentrations of temozolomide. B. The proliferation of two groups of cells at different culture time when the concentration of temozolomide was fixed at 200µM. μM=μmol/L, n=3, **P<0.01, ****P<0.0001; Figure 3. 24-hour scratch migration of Control-U87 and gaMSC-U87 cells. Migration on the left, quantitative statistical analysis on the right. n=3, *P<0.05; Figure 4. Apoptosis of Control-U87 and gaMSC-87 cells. The left side is the flow-through apoptosis situation, and the right side is the quantitative statistical analysis. -: without temozolomide, +: with temozolomide, n=3, *P<0.05, ****P<0.0001.

Real-time PCR:

(1) Take the cells out of the incubator and place them in a biological safety cabinet. After washing with PBS, add 1 ml of Trizol, and then transfer the cells to a 1.5 ml EP tube for lysis for 10 minutes.

(2) Add 200ul of chloroform, mix and react for 5 minutes, then centrifuge at 4°C and 12000rpm for 15 minutes.

(3) Transfer the upper layer to a 1.5ml EP tube, add 400ul isopropanol, mix well and let stand for 10min, then centrifuge at 4°C, 12000rpm for 10min.

(4) Discard the supernatant, add 1 ml of 75% ethanol, vortex to mix, and centrifuge for 5 minutes at 4°C, 10,000 rpm.

(5) Repeat step (4) once.

(6) Discard the supernatant, place it in the air to dry for 10 minutes, and then add 20 ul of DEPC water to dissolve it.

(7) Take 2 ul of the above-dissolved RNA to measure the OD260/OD280 value with a microspectrophotometer, and the value must be 1.8-2.0 to meet the requirements of subsequent experiments. Total RNA was stored at -80°C for later use.

(8) Reverse transcription into cDNA.

(9) PCR detection, curve drawing, data analysis.

Primer sequence: Homo b-actin Forward 5'-AGCGAGCATCCCCCAAAGTT-3'

Reverse 5'-GGGCACGAAGGCTCATCATT-3'

Homo FOXS1 Forward 5’-CCCAGGTTCCTTGTGGTC-3’

Reverse 5’-CCCAGGTTCCTTGTGGTC-3’

The two groups of cells were cultured under the condition of 200 μM temozolomide, and gene chip technology was used to determine the difference in gene expression between the two groups of cells. It was found that compared with Control-U87, FOXS1 was most significantly up-regulated in gaMSC-U87 (Figure 5). The expression of FOXS1 in the two groups of cells was further verified by PCR (Fig. 6A) and WB (Fig. 6B), and it was found that the expression of FOXS1 in gaMSC-U87 was significantly higher than that in Control-U87.

Among them: Figure 5. Heat map of gene expression differences between Control-U87 and gaMSC-U87 cells. After culturing for 3 days under the condition of 200 μM temozolomide, the samples were detected by gene chip and analyzed statistically. n=3, P<0.05; Figure 6. Differences in FOXS1 expression between Control-U87 and gaMSC-U87 cells. A. PCR technology to detect the difference of FOXS1 expression in two groups of cells. B. WB technique detected the difference of FOXS1 expression in two groups of cells. n=3,***P<0.001.

Western Blot:

(1) Pre-culture the cells in a 6-well plate. When the cells are full, remove the cells, discard the culture medium, and add pre-cooled PBS at about 4°C to wash for 3 times.

(2) Add 100ul of pre-configured PMSF-containing lysis solution to each well, and lyse on ice for 30 minutes.

(3) After the lysis is completed, the cells are scraped off with a cell scraper, transferred to a 1.5 ml EP tube (operated on ice), and then centrifuged at 12,000 rpm at 4°C for 5 minutes.

(4) After centrifugation, take the supernatant and aliquot and store it at -20°C for later use.

(5) Dilute the BSA standard to prepare standard proteins with concentrations of 1, 0.8, 0.6, 0.4, and 0.2.

(6) Add each concentration standard into the 96-well plate, two parallel wells of each concentration, 20ul per well, and 20ul of PBS for the blank control well, add the protein stock solution to be tested and the protein solution diluted 10 times and 100 times with PBS for each 3 parallel wells at each concentration, and 20ul per well was also added to the 96-well plate.

(7) Mix solution A and solution B in the BCA kit at a ratio of 50:1, then add 200ul to each well, and react in the dark for 30 minutes.

(8) After the reaction was completed, the OD568 was measured by a microplate reader, and the protein concentration was calculated.

(9) Mix the extracted protein with 5X protein loading buffer and boil in boiling water for 10 minutes.

(10) Prepare 5% stacking gel and 12% separating gel.

(11) Add the protein and MAKER to the sample hole with a loading gun, electrophoresis at a constant voltage of 80V until the bromophenol blue is at the junction of the stacking gel and the separating gel, and then continue electrophoresis at 120V until the bromophenol blue reaches the bottom of the gel.

(12) After the electrophoresis is completed, cut off the gel band containing the target protein, cut the PVDF membrane of the corresponding size, clamp it in the order of blackboard-filter paper-gel-FVDF membrane-filter paper-whiteboard, put it into the membrane transfer tank, The blackboard is transferred to the negative electrode.

(13) After the membrane transfer is completed, soak the PVDF membrane in blocking solution (TBST containing 5% skimmed milk powder), and place it on a shaker to block for 2 hours.

(14) After the blocking is completed, soak the PVDF membrane in the primary antibody diluted with the primary antibody diluent and incubate at 4°C overnight.

(15) Wash the PVDF membrane incubated with the primary antibody 5 times with TBST, 5 minutes each time.

(16) Immerse the washed PVDF membrane in the secondary antibody diluted with the secondary antibody diluent, and incubate on a shaker for 2 hours at room temperature.

(17) After incubating the secondary antibody, wash 5 times with TBST.

(18) Mix the ECL enhancement solution and the peroxidase solution at a ratio of 1:1 and drop them on the PVDF membrane. After the band is obvious, the filter paper absorbs the excess liquid, covers the plastic wrap, presses the X film, and then drips it in order to develop The film is developed, fixed and processed with a fixer solution, dried, scanned and analyzed.

Lentiviral transfection experiments:

(1) Prepare a cell suspension with a density of 50,000 cells/ml in DMEM medium containing 10% serum and inoculate it into a 6-well plate, 2 ml per well, and then culture in a 37°C, 5% CO ₂ incubator for 24 hours.

(2) After 24 hours, replace the DMEM medium, 1 ml per well, then add 20ul of 1×10 ⁸ TU/ml virus solution to each well, and continue to culture for 12-16 hours in a 37°C, 5% CO ₂ incubator.

(3) After 12-16 hours, the medium was replaced and the culture was continued for 72 hours, and the infection effect was observed under a fluorescence microscope.

(4) Collect infected cells and place them in a medium containing puromycin for screening and purification for later use.

Control-U87, gaMSC-U87, Control-GBM1 (glioma primary cells GBM-1 treated with DMEM medium), gaMSC-GBM1 (glioma-associated mesenchymal stem cells cultured under the condition of 200uM temozolomide) Glioma primary cells GBM-1) treated with solution, EMT marker proteins (E-Cadherin, N-Cadherin) were determined by WB, and the results showed that compared with Control-U87 and Control-GBM1, gaMSC-U87 and gaMSC-GBM1 EMT The processes were both facilitated (Fig. 7). In order to verify that the up-regulation of FOXS1 promotes the EMT process, U87 and GBM-1 cells were knocked down or overexpressed by lentivirus, and then measured by WB technology. , Control-GBM1), the EMT process was promoted in the overexpression group (OE-U87, OE-GBM1), whereas the knockdown group (KD-U87, KD-GBM1) was the opposite, and the negative control group (NC-U87, NC-GBM1) No change (Figure 8).

The FOXS1-overexpressing or knockdown U87 stable cells constructed by lentivirus transfection were cultured under the condition of 200 μM temozolomide, and then the scratch experiments, CCK-8 experiments and flow cytometric apoptosis experiments were performed. The results showed that compared with Control-U87, OE-U87 had increased migration ability and proliferation ability, and decreased apoptosis rate; KD-U87 showed the opposite trend (Figure 9).

Among them: Figure 7. Expression of EMT marker proteins in U87 cells and GBM-1 cells under different conditions. A. The expression of E-Ccdheron and N-Cadherin protein was detected by WB technique, B. Statistical analysis of WB results of U87 cells in two groups. C. Statistical analysis of GBM-1 in two groups. n=3, **P<0.01; Figure 8. The expression of FOXS1 and EMT marker proteins in U87 cells and GBM-1 stable cells with knockdown or overexpression, A. WB technology detects FOXS1, E-Ccdheron and N-Cadherin proteins Expression, statistical analysis of WB results of B.U87 cells. C. Statistical analysis of WB results of GBM-1 cells, n=3, **P<0.01, ***P<0.001, ****P<0.0001; Figure 9. U87 stable cell line with knockdown or overexpression of FOXS1 resistance situation. A. Scratch migration and statistical analysis of cells in different groups under the condition of 200 μM temozolomide for 24 hours, B. The proliferation of cells in different groups under the condition of 200 μM temozolomide for 48 hours, C. The apoptosis of cells in different groups under the condition of 200 μM temozolomide, n=3 , *P<0.05, **P<0.01, ***P<0.001.

Animal experiment:

(1) 4-week-old nude mice were anesthetized with 4% chloral hydrate and fixed on the brain stereotaxic apparatus.

(2) Disinfect the scalp of mice with active iodine, cut the scalp, and fully expose the bregma.

(3) 1mm in front of the bregma, open 2mm aside and drill a hole with a 1ml syringe needle, then use a microsyringe along this hole, insert the needle 3.5mm, and inject 10ul of the U87 cell suspension under different conditions into different mice respectively. Inject 1ul per minute, and leave the needle for 5 minutes after injection.

(4) After the needle was pulled out, the bone hole was closed with bone wax, the scalp was sterilized and sutured, and the animals were raised and observed in an SPF environment.

(5) After 3 weeks, mice were given intraperitoneal injection of temozolomide (50 mg/kg) for 5 consecutive days, and then continued to observe.

(6) Mice were sacrificed 35 days after surgery, and tumors were removed for further experiments.

The mouse tumor formation experiments were performed with U87 cells under different conditions. The results showed that the survival time of the mice in the gaMSC-U87 group was lower than that of the mice in the Control-U87 group (Figure 10B). Immunohistochemical analysis showed that compared with the Control-U87 group, the gaMSC-U87 group had larger tumors (Fig. 10C) and expressed more Ki67 (Fig. 11A); Mice in the OE-U87 group had larger tumors (Fig. 10D) and expressed more Ki67 (Fig. 11B), and the opposite was true in the KD-U87 group.

Among them: Figure 10. Mouse tumorigenesis and survival of U87 cells under different conditions. A. Tumor formation of U87 cells under different conditions, DMEM-U87 is the tumor of mice inoculated with U87 cells treated with DMEM but not treated with temozolomide, -: not treated with temozolomide, +: treated with temozolomide, B. Survival analysis of mice inoculated with Control-U87 and gaMSC-U87 cells, C. Quantification of tumor size in mice inoculated with Control-U87 and gaMSC-U87 cells, D. Control-U87, NC-U87, KD-U87 , and quantitative analysis of tumor size in mice inoculated with OE-U87 cells, n=3, *P<0.05, **P<0.01, ***P<0.001; Figure 11. The expression difference of Ki67 in different cell groups. A. Immunohistochemical results and quantitative analysis of Ki67 in mouse tumor tissues inoculated with Control-U87 and gaMSC-U87 cells, B. Mouse tumor tissues inoculated with Control-U87, NC-U87, KD-U87, and OE-U87 cells Immunohistochemical results and quantitative analysis, ×400, scalebar=250 μm, n=3, *P<0.05, **P<0.01, ***P<0.001.

Immunohistochemical experiments:

(1) Dehydrate the tissue in 75%, 85%, 90%, and 95% alcohol in sequence for 4 hours, 2 hours, 1.5 hours, and 1 hour; and then dehydrate in absolute ethanol twice for half an hour each time.

(2) The transparent agent was used for transparent treatment, and then the tissue was immersed in wax (60°C) 3 times for 1 hour each time.

(3) Embedding after dipping in wax, then slicing and baking.

(4) Dewaxing the sections and heating them in an electric ceramic furnace for antigen retrieval.

(5) Drop 3% hydrogen peroxide solution on the slices, place them at room temperature for 15 minutes, and then wash with PBS three times for 3 minutes each time.

(6) Dry the slide with absorbent paper, draw a circle around the tissue with an immunohistochemical pen, and drop the diluted goat serum to seal for 30 minutes.

(7) After sealing, blot up the excess liquid, draw circles along the tissue with an oil-based pen, add 1:100 diluted Ki67 antibody dropwise, and incubate overnight in a humidified box at 4°C.

(8) Wash the picks incubated with the primary antibody three times with PBS for 3 minutes each time, absorb the excess water, add the secondary antibody and incubate at room temperature for 20 minutes.

(9) After incubating the secondary antibody, rinse with PBS for 4 times, 3 minutes each time, add DAB chromogenic solution to develop color, observe under a microscope, and rinse the slide when the color is appropriate.

(10) Counterstained with Harris hematoxylin for 30s-1min, washed with water, differentiated with 1% hydrochloric acid alcohol, and then washed with PBS to return to blue.

(11) Rinse the slides, dehydrate and air-dry them, and then cover the slides and air-dry them before taking pictures under a microscope for analysis.

ELISA experiment:

(1) Take the ELISA kit out of the refrigerator and place it at room temperature, wait until the temperature is equilibrated to room temperature before proceeding to the next step.

(2) Dilute the IL-6 standard with standard/specimen universal diluent to 200, 100, 50, 25, 12.5, 6.25, 3.125, 0 pg/ml.

(3) Add different concentrations of standards and samples to the plate, 100ul per well, and add standard/specimen general dilution to blank wells, then seal the plate with tape, and incubate at 36°C for 90 minutes in the dark.

(4) After the incubation, pour out the liquid and add 350ul washing solution to each well to wash for 30 seconds, then pour it out, and wash the plate 5 times in this way.

(5) After washing the plate, pat the plate dry on a thick stack of absorbent paper, add 100ul of pre-prepared biotinylated antibody working solution to each well, add biotinylated antibody diluent to blank wells, and incubate at 36°C in the dark 60 minutes.

(6) After incubation, wash the plate 5 times, pat the plate dry, add 100ul of pre-configured enzyme conjugate working solution to each well, add enzyme conjugate dilution solution to blank wells, and incubate at 36°C for 30 minutes in the dark.

(7) After the incubation, wash the plate 5 times, pat the plate dry, add 100ul of chromogenic substrate to each well, and incubate at 36°C for 15 minutes in the dark.

(8) After incubation, add 100ul of reaction stop solution to each well and immediately measure OD450 in a microplate reader and save the analysis results.

ELISA was used to detect DMEM medium (Control) and glioma-related mesenchymal stem cell conditioned medium (gaMSC-CM), and it was found that IL-6 content in gaMSC-CM was significantly higher than that in Control group. Glioma-associated mesenchymal stem cell conditioned medium (CD90+gaMSC-CM) and glioma-associated mesenchymal stem cell conditioned medium with low CD90 expression (CD90-gaMSC-CM), the results showed that CD90-gaMSC- The IL-6 content in CM was significantly higher than that in CD90+gaMSC-CM (Fig. 12A). Further use DMEM medium (Control group), DMEM medium with IL-6 (IL-6 group), and DMEM medium with IL-6 and IL-6 neutralizing antibody (IL-6 + anti IL- Group 6) U87 and GBM-1 were cultured under the condition of 200 μM temozolomide, and then the expression of FOXS1 was detected by WB. The anti-IL-6 group could reverse the up-regulation of FOXS1 expression (Fig. 12B).

Among them: Figure 12. The effect of IL-6 on cellular FOXS1 expression. A. The content of IL-6 in different culture medium determined by ELISA experiment, B. The expression of FOXS1 was determined by WB. n=3, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001.

Evaluation:

EMT is a biological process whereby epithelial cells interact with the basement membrane through their basal surface and undergo multiple biochemical changes that transform them into a mesenchymal phenotype, including enhanced migratory capacity, invasiveness, resistance to apoptosis resistance, etc. The main feature of EMT is a decrease in E-Cadherin levels accompanied by an increase in N-Cadherin levels, resulting in changes in cellular cadherin-based adhesion, which play a role in regulating development and organogenesis. The emergence of EMT during tumor progression allows non-invasive and non-metastatic benign tumor cells to acquire the ability to infiltrate surrounding tissues and ultimately metastasize to distant sites. In gliomas, EMT can transform glioma cells into cells with weaker adhesion and dysregulated cytoskeleton, resulting in greater motility and chemoresistance. The resistance of gliomas to chemotherapy is closely related to the activation of EMT. Promoting the activation of EMT in gliomas can increase the expression of MGMT and then increase the resistance of glioma cells to TMZ. TMZ is a prodrug of alkylating agents that delivers methyl groups to the purine bases of DNA (O6-guanine; N7-guanine and N3-adenine), while MGMT removes the alkyl group from the O6 position of guanine Therefore, high levels of MGMT in cancer cells can produce a resistant phenotype by impairing the therapeutic effect of TMZ.

When the method of the present invention uses temozolomide to kill glioma cells in vitro, U87 cells cultured with glioma-related mesenchymal stem cell conditioned medium (gaMSC-CM) have stronger proliferation and migration ability and less cell apoptosis death and increased resistance of glioma cells to temozolomide. Further gene chip analysis found that the expression of FOXS1 in U87 cells cultured with gaMSC-CM was the most obvious. FOXS1 is a member of the FOX protein family, which are evolutionarily conserved transcriptional regulators. Members of this family play important roles in many biological processes, such as proliferation, migration, apoptosis, and invasion. FOX family proteins are expressed in many tumors, and different proteins in the family play different roles in different tissue cells, some promote tumor progression, and some inhibit tumor occurrence. For example, FOXO3a can inhibit EMT of prostate cancer cells by affecting the transduction of WNT/β-catenin signaling pathway, while FOXM can regulate the urokinase plasminogen activator system to promote pancreatic cancer EMT. The expression of FOXS1 in gastric cancer tissue is significantly higher than that in precancerous tissue, and the expression in gastric cancer cells is also significantly higher than that in gastric epithelial cells. FOXS1 can promote cell EMT and affect the survival rate of gastric cancer patients. High FOXS1 expression indicates poor prognosis of gastric cancer patients. Meanwhile, gaMSC-CM could enhance the expression of N-cadherin and down-regulate the expression of E-cadherin in U87 and GBM-1 cells. Furthermore, a stable transfected cell line with FOXS1 overexpression and knockdown was established, and it was found that FOXS1 upregulation could promote cell EMT, increase cell proliferation and migration, and reduce cell apoptosis, while FOXS1 knockdown was the opposite. In vivo experiments, it was found that mice inoculated with U87 cells cultured with gaMSC-CM had a lower survival period than the control group, formed larger tumors, and expressed more Ki67. At the same time, U87 cells overexpressing FOXS1 also formed larger tumors in the mouse brain and expressed more Ki67, whereas knocking down FOXS1 showed the opposite result.

IL-6 is a soluble glycosylated polypeptide chain and one of the major cytokines in the tumor microenvironment. The tumor microenvironment can secrete high levels of IL-6, which can promote tumorigenesis and increase tumor resistance to therapy by promoting tumor proliferation, migration, and inhibiting apoptosis. Therefore, gaMSC-CM was measured and found to contain a large amount of IL-6. Glioma-related mesenchymal stem cells can be divided into two subtypes with high expression of CD90 and low expression of CD90, and IL-6 is mainly secreted by the subtype with low expression of CD90. Therefore, adding IL-6 to the culture medium can effectively promote the expression of FOXS1 in glioma cells, and adding IL-6 neutralizing antibody can reverse this effect.

To sum up, the present analysis shows that glioma-related mesenchymal stem cells can promote the expression of FOXS1 in glioma cells, and then promote the chemoresistance of gliomas to temozolomide by promoting EMT. The up-regulation of FOXS1 is mainly due to the stimulation of IL-6 secreted by glioma-associated mesenchymal stem cells with low CD90 expression. The method of the present invention provides new ideas and targets for reducing or reversing the chemoresistance of glioma.

The material sources employed in the specific embodiments of the present invention are provided below:

(1) DMEM medium, MEM-α medium, PBS (HyClone, USA)

(2) Fetal Bovine Serum (BI, Israel)

(3) Pancreatin (Biyuntian, China)

(4) Accutase (Stem Cell, Canada)

(5) T25 flask, centrifuge tube, petri dish, culture plate, cell sieve (Corning, USA)

(6) Penicillin/Streptomycin (GibcoBRL, USA)

(7) Lymphocyte Separation Buffer, RBC Lysis Buffer (Thermo Fisher, China)

(8) CCK-8 kit (Dojindo Laboratories, Japan)

(9) ELISA kit (Xinbosheng, China)

(10) CD90 Magnetic Bead Antibody, Magnetic Bead Separation Column (Milrenyi, Germany)

(11) FOXS1 antibody (Thermo, USA)

(12) N-Cadherin, E-Cadherin, IL-6 neutralizing antibody (Abcam, China)

(13) Ki67 antibody (Proteintech, China)

(14) Recombinant human IL-6 (PEPROTECH, USA)

(15) β-actin antibody (Boster Biologicals, China)

(16) BALB/c-nu nude mice (Viton Lever, China)

(17) U87 cells (American Type Culture Collection, USA)

(18) Lentivirus (Genkey Gene, China)

The embodiments of the present invention have been exemplarily described above, but the content is only a preferred embodiment of the present invention, and should not be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements according to the scope of the application of the present invention shall fall within the scope of the patent of the present invention.

Claims (9)

1. a detection method of the gaMSCs subgroup of promoting brain glioma drug resistance, is characterized in that, comprises the following steps: S1: Collect and isolate glioma-associated mesenchymal stem cells (gaMSCs), human glioma cell line U87 and glioma primary cells (GBM-1); S2: Prepare human glioma cell line U87 and glioma primary cells (GBM-1) cultured in DMEM medium and glioma-associated mesenchymal stem cell (gaMSCs) conditioned medium, respectively, to obtain DMEM culture Human glioma cell line U87 (Control-U87) cultured in medium, primary glioma cells (Control-GBM1) cultured in DMEM medium, human glial cultured in conditioned medium of glioma-related mesenchymal stem cells Tumor cell line U87 (gaMSC-U87) and glioma primary cells (gaMSC-GBM1) cultured in conditioned medium of glioma-related mesenchymal stem cells; S3: Control-U87, Control-GBM1, gaMSC-U87 and gaMSC-GBM1 were treated with glioma chemotherapy drugs in vitro to obtain multiple samples to be tested; S4: The detection, analysis and comparison of the proliferation ability of the sample to be tested with the drug added by CCK-8; S5: analysis and comparison of cell apoptosis in the sample dosing to be tested by flow analysis technology; S6: Analyze and compare the migration ability in the drug-dosed sample to be tested by scratch test; S7: Analysis of the effect of gaMSCs on the gene expression of glioma cells by gene chip; S8: The expression of FOXS1 was analyzed by PCR and Western-blot; S9: Detect the changes of epithelial-mesenchymal transition (EMT) markers when FOXS1 is knocked down or overexpressed by lentiviral transfection; S10: establish an orthotopic nude mouse model by using brain glioma cells and administer glioma chemotherapeutic drugs in vivo, and conduct in vivo drug addition experiments in animals; S11: The expression changes of Ki-67 in tumor tissues of animals in S10 were detected by immunohistochemistry; S12: The IL-6 expression and secretion of gaMSCs was analyzed by ELISA; S13: Analyze and compare the above-mentioned experimental detection results, and conduct drug resistance evaluation. 2 . The method for detecting gaMSCs subsets that promote drug resistance of gliomas according to claim 1 , wherein the glioma chemotherapeutic drug body is temozolomide (TMZ). 3 . 3. The method for detecting gaMSCs subsets that promote glioma drug resistance according to claim 2, wherein the in vitro concentration of temozolomide is 190-210 uM (umol/L), and the in vivo injection The concentration is 45-55 mg/kg. 4 . The method for detecting a gaMSCs subgroup that promotes drug resistance of gliomas as claimed in claim 1 , wherein the glioma primary cells are selected to collect the third-generation passaged cells for experiment and detection. 5 . 5. The method for detecting a subgroup of gaMSCs that promotes drug resistance of gliomas as claimed in claim 1, wherein the glioma-related mesenchymal stem cells need to carry out high expression of CD90 and low expression of CD90 sorting. 6. The method for detecting a subgroup of gaMSCs that promotes glioma drug resistance as claimed in claim 5, wherein the sorting step of the glioma-related mesenchymal stem cells comprises: S101: Take out the glioma-related mesenchymal stem cells from the incubator and place them in a clean bench, rinse with PBS, digest with Accutase, and centrifuge at 1500 rpm for 6 minutes; S102: Discard the supernatant, add CD90 magnetic bead antibody and sorting Buffer at 1:4, and mix well; S103: put it in a 4°C refrigerator to avoid light for 15 minutes; S104: Take out the cells, rinse with sorting buffer, centrifuge at 1500 rpm for 6 minutes; S105: Discard the supernatant, add 500ul sorting Buffer to resuspend; S106: drop the cell suspension into the sorting column, and then drop 2 ml of sorting Buffer into the column to wash the column, and the filtered cell suspension is glioma-related mesenchymal stem cells with low CD90 expression (CD90 ^- ); S107: After the above filtration is completed, add 2 ml of DMEM medium containing 20% serum to the sorting column, and then use the handle to quickly push out the medium. At this time, the filtered glioma-related mesenchymal cells with high CD90 expression Stem cells (CD90 ⁺ ); S108: The above ^CD90- and CD90 ⁺ cells were transferred to culture flasks, respectively, and cultured in a 37°C, 5% CO ₂ incubator. 7. the detection method of a kind of gaMSCs subgroup of promoting brain glioma drug resistance as claimed in claim 1, is characterized in that: the primer sequence in PCR described in S8 is: Homo b-actin Forward 5’-AGCGAGCATCCCCCAAAGTT-3’, Reverse 5'-GGGCACGAAGGCTCATCATT-3', Homo FOXS1 Forward 5'-CCCAGGTTCCTTGTGGTC-3', Reverse 5'-CCCAGGTTCCTTGTGGTC-3'. 8. the detection method of a kind of gaMSCs subgroup of promoting brain glioma drug resistance as claimed in claim 1, is characterized in that: the PVDF membrane processing method in Western-blot detection analysis described in S8 is: PVDF membrane Immerse in blocking solution (TBST containing 5% nonfat dry milk) and place on a shaker to block for 2 hours; after blocking, soak PVDF membrane in primary antibody diluted with primary antibody diluent and incubate overnight at 4°C; wash with TBST Incubate the PVDF membrane with the primary antibody 5 times for 5 minutes each time; soak the washed PVDF membrane in the secondary antibody diluted with the secondary antibody diluent, and incubate it on a shaker for 2 hours at room temperature; after incubating the secondary antibody, use After washing 5 times with TBST, it was set aside. 9. the detection method of a kind of gaMSCs subgroup of promoting brain glioma drug resistance as claimed in claim 1, is characterized in that: described in S9, the lentivirus transfection experimental step comprises: S201: Prepare a cell suspension with a density of 50,000 cells/ml in DMEM medium containing 10% serum and inoculate it into a 6-well plate, 2 ml per well, and then culture in a 37°C, 5% CO2 incubator for 24 hours; S202: After 24 hours, replace the DMEM medium, 1ml per well, then add 20ul of 1×108TU/ml virus solution to each well, and continue to culture for 12-16 hours in a 37°C, 5% _CO2 incubator; S203: After 12-16 hours, the medium was replaced and the culture was continued for 72 hours, and the infection effect was observed under a fluorescence microscope; S204: collecting infected cells and placing them in a medium containing puromycin for screening and purification for later use.

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