CN111808908A - Detection method of gaMSCs subgroup promoting 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

Detection method of gaMSCs subgroup promoting glioma drug resistance

Technical Field

The invention relates to the technical field of biology and medicine, in particular to a detection method of a gammscs subgroup promoting brain glioma drug resistance.

Background

Malignant glioma is the most common highly malignant primary brain tumor with extremely strong invasiveness in adults, and has extremely high fatality rate. Despite surgical resection, assisted by chemotherapy, median survival is only 13-16 months, with a five-year survival rate of only 6.7%. Although several novel therapeutic approaches such as immune checkpoint inhibitors, viral therapies, CAR T, dendritic 3 cell therapies and vaccine treatments have been shown to have some effect on glioblastomas, further research is still needed. Temozolomide (TMZ) chemotherapy is still the mainstay of treatment today and temozolomide therapy is the only chemotherapy approved for newly diagnosed glioblastomas. Temozolomide is an oral alkylating chemotherapeutic medicine, can effectively penetrate through a blood brain barrier to act on a diseased part, and prolongs the life cycle of a postoperative patient. However, most patients develop resistance to temozolomide over extended periods of time.

Whether the tumor cells are sensitive to the treatment of the chemotherapeutic drugs depends on whether the genes are mutated or not, and the tumor cells are influenced by the surrounding environment to a great extent, and the drug resistance variation of the tumor cells is also influenced 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, fibroblasts, and the like. The tumor microenvironment plays an important role in the occurrence and development of tumors, and related researches are gradually increased in recent years, and the position of the microenvironment is more and more important. The tumor microenvironment can not only facilitate the generation, the development and the metastasis of the contained various types of cells and various extracellular components and maintain the representation of the tumor, but also has important effect on the formation of chemotherapy drug resistance.

Mesenchymal stem cells (MCS), also known as multipotent mesenchymal stem cells, have in vitro morphology similar to fibroblasts and can proliferate and differentiate into various cell lines of osteoblasts, chondrocytes and adipocytes under specific stimulation conditions. In tumors, the related mesenchymal stem cells are an important part of stem cells in the tumor microenvironment, and the brain glioma-related mesenchymal stem cells (gasmscs) can be transformed into pericytes during tumor progression, participate in angiogenesis and play an obvious role in the progression of glioma.

However, at present, the research on the drug resistance forming mechanism of the gammscs to the glioma temozolomide is not available, and then the invention provides a method for evaluating the drug resistance of the gammscs to the glioma chemotherapy.

Disclosure of Invention

The invention aims to provide a detection method of a gaMSCs subgroup promoting glioma drug resistance aiming at the defects of the prior art, so as to visually and truly analyze and evaluate the influence of the gaMSCs on the drug resistance of glioma chemotherapy.

The invention provides a detection method of a GAMSC subgroup promoting brain glioma drug resistance, which comprises the following steps:

s1: collecting and separating to obtain gammscs, a human glioma cell line U87 and a glioma primary cell (GBM-1);

s2: respectively preparing a human glioma cell line U87 and a glioma primary cell (GBM-1) which are cultured by a DMEM medium and a gamSCs conditioned medium, thus obtaining a human glioma cell line U87 (Control-U87) which is cultured by the DMEM medium, a glioma primary cell (Control-GBM 1) which is cultured by the DMEM medium, a human glioma cell line U87 (gamSC-U87) which is cultured by the gamSCs conditioned medium and a glioma primary cell (gamSC-GBM 1) which is cultured by the gamSCs conditioned medium;

s3: carrying out glioma chemotherapy drug in-vitro dosing treatment on Control-U87, Control-GBM1, gasMC-U87 and gasMC-GBM 1 to obtain a plurality of samples to be detected;

s4: detecting, analyzing and comparing the proliferation capacity of the dosed sample to be detected through CCK-8;

s5: detecting the analysis and comparison of the apoptosis condition in the drug-added sample to be detected by a flow analysis technology;

s6: analyzing and comparing the migration capacity of the sample to be tested after adding the medicine through a scratch test;

s7: analyzing the influence of the gammscs on the gene expression of the glioma cells by a gene chip;

s8: analyzing the expression condition of FOXS1 by PCR and Western-blot;

s9: detecting changes in epithelial-mesenchymal transition (EMT) markers when knockdown or overexpression of FOXS1 was obtained by lentivirus transfection;

s10: establishing a nude mouse in-situ model through brain glioma cells, and in-vivo administering a glioma chemotherapeutic drug object to perform an in-vivo drug adding experiment on animals;

s11: detecting Ki-67 expression changes in tumor tissues in the animals by immunohistochemistry in S10;

s12: IL-6 expression and secretion of the gamSCs were analyzed by ELISA;

s13: and analyzing and comparing the detection results of the experiments to evaluate the drug resistance.

Further, the glioma chemotherapeutic drug object is Temozolomide (TMZ).

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

Further, the glioma primary cells are selected and collected with third generation passage cells for experiment and detection.

Further, the gaMSCs were sorted for high expression of CD90 and low expression of CD 90.

Further, the step of sorting the gaMSCs comprises:

s101: taking the gamSCs out of the incubator, placing the gamSCs in a super clean bench, washing the gamSCs by PBS, digesting the gamSCs by Accutase at 1500rpm, and centrifuging the gamSCs for 6 minutes;

s102: discarding the supernatant, adding the CD90 magnetic bead antibody and the sorting Buffer at a ratio of 1:4, and flushing and mixing uniformly;

s103: placing the mixture in a refrigerator at 4 ℃ to avoid light for reaction for 15 minutes;

s104: taking out cells, sorting and washing by Buffer, centrifuging at 1500rpm for 6 minutes;

s105: discarding the supernatant, adding 500ul of sorting Buffer for resuspension;

s106: dripping the cell suspension into a sorting column, then dripping 2ml of sorting Buffer to wash the column, and obtaining the low-expression GaMSCs (CD 90-) of CD90 in the filtered cell suspension;

s107: after the filtration, 2ml of DMEM medium containing 20% serum is added into the separation column, then the culture medium is rapidly pushed out by a handle, and the filtered medium is the GaMSCs (CD 90 +) with high CD90 expression;

s108: the CD 90-and CD90+ cells were transferred to flasks and cultured in a 5% CO2 incubator at 37 ℃.

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’-CCCAGGGTTCCTTGTGGTC-3’，

Reverse 5’-CCCAGGGTTCCTTGTGGTC-3’。

further, the method for treating the PVDF membrane in the Western-blot detection and analysis in S8 comprises the following steps: soaking the PVDF membrane in a sealing solution (TBST containing 5% skimmed milk powder), and sealing on a shaking table for 2 hours; after the sealing is finished, soaking the PVDF membrane in primary antibody diluted by primary antibody diluent and incubating overnight at 4 ℃; washing the incubated PVDF membrane with TBST for 5 times, 5 minutes each time; soaking the washed PVDF membrane in a secondary antibody diluted by a secondary antibody diluent, and incubating for 2 hours on a shaking table at room temperature; after incubation, the secondary antibody was washed 5 times with TBST and used.

Further, the lentivirus transfection experiment step in S9 includes:

s201: cell suspension with 50000 cells/ml density was prepared in DMEM medium containing 10% serum and inoculated into 6-well plates at 2 ml/well, then at 37 ℃ with 5% CO₂Culturing in an incubator for 24 hours;

s202: after 24 hours, the DMEM medium was changed to 1ml per well, and then 20ul of 1X 10 medium was added to each well⁸Continuously culturing the virus solution with TU/ml in a 5% CO2 incubator at 37 ℃ for 12-16 hours;

s203: after 12-16 hours, the culture medium is replaced to continue culturing for 72 hours, and the infection effect is observed under a fluorescence microscope;

s204: and collecting infected cells, and screening and purifying the infected cells in a culture medium containing puromycin for later use.

The invention has the following beneficial effects:

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 method further defines the mechanism of promoting tumor cell chemotherapy drug resistance by the brain glioma microenvironment, and provides a new thought and target for further personalized treatment of the brain glioma.

Drawings

FIG. 1 is a morphological diagram of the cultured cells of Control-U87 and gasSC-U87 according to the present invention;

FIG. 2 is a schematic diagram showing the detection result of CCK-8 according to the present invention;

FIG. 3 is a schematic diagram of the scratch test result of the present invention;

FIG. 4 is a schematic diagram of the results of flow cytometry analysis of the present invention;

FIG. 5 is a schematic diagram showing 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 a graph showing the results of expression of FOXS1 according to the invention;

FIG. 7 is a schematic diagram showing the result of determining EMT marker protein by Western-blot;

FIG. 8 is a schematic diagram of the test of the present invention to verify the synergy of FOXS1 with EMT;

FIG. 9 is a schematic representation of the results of a lentivirus transfection experiment of the present invention;

FIG. 10 is a diagram showing the results of in vivo experiments according to the present invention;

FIG. 11 is a diagram showing the result of alignment of Ki67 in vivo experiments according to the present invention;

FIG. 12 is a graph showing the content alignment of IL-6 and the expression alignment of FOXS1 according to the present invention;

fig. 13 is a schematic diagram of the cell differentiation capacity of glioma-associated mesenchymal stem cells;

fig. 14 is a schematic view of cell migration ability of glioma-associated mesenchymal stem cells.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, various changes or modifications of the present invention may be made by those skilled in the art, and equivalents may fall within the scope of the claims of the present application. The proportions in the examples of the invention are by weight.

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

(1) under the condition of obtaining the consent of patients who are in Wuhan cooperative hospital for glioma surgery and signing informed consent, after glioma specimens are excised, the glioma specimens are immediately placed in a sterile centrifuge tube filled with normal saline in the Wuhan cooperative hospital operating room and immediately transferred to a sterile super clean bench sterilized in advance in a laboratory.

(2) Placing the glioma specimen in a culture dish, flushing out blood clots by using PBS, shearing the specimen into small pieces with the size of 1 cubic millimeter by using sterile scissors, and adding pancreatin for digestion.

(3) After 15 minutes, the mixture was filtered using a cell sieve with a pore size of 70 μm, and the filtrate was centrifuged at 1500rpm for 10 minutes.

(4) The supernatant was discarded, 3ml of erythrocyte lysate was added, the mixture was shaken well and lysed for 5 minutes, and then centrifuged at 1500rpm for 5 minutes.

(5) The supernatant was discarded, the cells were suspended in DMEM medium, and the cell suspension was carefully transferred to a 15ml centrifuge tube containing 4ml of the lymphocyte separation medium to allow the cells to separate into layers, followed by centrifugation at 1000rpm with 0 adjustment for 20 minutes.

(6) After centrifugation, the intermediate buffalo membrane cell layer was washed 2 times with PBS, and the cells were added to DMEM medium containing 20% fetal bovine serum, 1% penicillin and streptomycin and placed at 37 ℃ with 5% CO₂Culturing in the cell culture box, and changing the culture medium and carrying out passage according to the growth condition.

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

(1) placing the glioma specimen in a culture dish, flushing blood clots by PBS, adding MEM-alpha culture solution, shearing the electrocoagulation necrosis by sterile scissors, and shearing the specimen into small blocks of about 1 cubic millimeter.

(2) Homogenizing the small pieces, adding pancreatin, and digesting at 37 deg.C for 30 min

(3) The digested cell suspension was lysed using red blood cell lysate for 10 minutes,

(4) excess lysate was washed with PBS and centrifuged at 1500rpm for 5 minutes.

(5) The supernatant was discarded, and the cells were resuspended in Neuroplast broth (containing N2, B27, hEGF, FGF and heparin) and transferred to extracellular matrix-plated flasks at 37 ℃ with 5% CO₂According to the growth rate of the cells, the liquid is changed or the cells are passaged once every 5 days, and third generation cells are collected for experiment and detection.

Subculture of human glioma cell line U87:

(1) the cells were removed from the incubator and placed in a clean bench, the medium was decanted, and washed 1 time with PBS.

(2) The PBS was poured off, 1ml of pancreatin was added, and the mixture was digested at 37 ℃ for 1 to 2 minutes.

(3) The digested cells were transferred to a 15ml centrifuge tube, and 1ml of DMEM medium containing 10% serum was added to neutralize the pancreatin, followed by centrifugation at 1000rpm for 5 minutes.

(4) The supernatant was discarded and 4ml of DMEM containing 10% serum was used to resuspend the cells.

(5) The cell suspension was transferred to a T25 flask and placed at 37 ℃ in 5% CO₂Cultured in a cell culture box.

Sorting the brain glioma-related mesenchymal stem cells:

(1) taking out the brain glioma-related mesenchymal stem cells from the incubator, placing the brain glioma-related mesenchymal stem cells in an ultra-clean bench, washing the cells with PBS, digesting the cells with Accutase at 1500rpm, and centrifuging the cells for 6 minutes.

(2) The supernatant was discarded, and the CD90 magnetic bead antibody and sorting Buffer were added at 1:4, followed by mixing.

(3) The reaction was carried out at 4 ℃ in a refrigerator under dark conditions for 15 minutes.

(4) The cells were removed, washed with a sorting Buffer, 1500rpm, and centrifuged for 6 minutes.

(5) The supernatant was discarded and 500ul of sorting Buffer was added for resuspension.

(6) And dripping the cell suspension into a sorting column, and then dripping 2ml of sorting Buffer to wash the column, wherein the filtered cell suspension is the brain glioma-related mesenchymal stem cell with low expression of CD 90.

(7) After the filtration is finished, 2ml of DMEM culture medium containing 20% serum is added into the sorting column, then the culture medium is quickly pushed out by a handle, and the filtered mesenchymal stem cells with high expression of CD90 brain glioma are obtained.

(8) The cells were transferred to culture flasks, respectively, at 37 ℃ with 5% CO₂Culturing in an incubator.

Preparing a conditioned culture solution of the brain glioma-related mesenchymal stem cells:

(1) taking out the brain glioma-associated mesenchymal stem cells with the density of about 50% from the culture box, and placing the cells in a super clean bench.

(2) Pouring out the culture medium, washing with PBS for 2-3 times, adding serum-free DMEM medium 4ml, standing at 37 deg.C and 5% CO₂Cultured in an incubator for 3 days.

(3) After 3 days, the flask was removed, and the culture broth was transferred to a 15ml centrifuge tube, 1000g, and centrifuged for 10 minutes.

(4) The supernatant was collected and placed in a refrigerator at-20 ℃ for future use.

CCK-8 proliferation assay:

(1) u87 cells were digested and centrifuged, and 50000 cells/ml cell suspension was prepared in DMEM medium containing 10% serum and seeded in 96-well plates at 100ul per well.

(2) And after the cells are completely attached to the wall, washing the cells for three times by PBS, and respectively adding a serum-free DMEM culture medium and a brain glioma-related mesenchymal stem cell conditioned medium.

(3) Adding temozolomide with corresponding concentration, and standing at 37 deg.C and 5% CO₂Cultured in an incubator.

(4) After the culture is carried out for the time required to be measured, the 96-well plate is taken out, 10ul of CCK-8 reagent is added into each well, and then the culture is carried out in an incubator for 2 hours.

(5) After 2 hours, the OD of each well at a wavelength of 450nm was measured in a microplate reader.

Scratch test:

(1) the U87 cells were seeded in 6-well plates, cultured in an incubator, and when the cells reached about 90% density, they were removed and placed in a clean bench.

(2) A straight line through the diameter was drawn in the middle of each hole with a 10ul lance tip.

(3) And (5) washing by PBS, and then taking a picture under a microscope to obtain a 0-hour picture.

(4) And (3) washing with PBS, adding a DMEM culture medium and a brain glioma-associated mesenchymal stem cell conditioned medium respectively, adding 200uM (umol/L) temozolomide, and placing in an incubator for 24 hours.

(5) And taking out the picture after 24 hours to obtain a 24-hour picture.

Flow cytometry:

(1) u87 cells cultured under different conditions were digested with trypsin without EDTA, and the cell suspension was collected, centrifuged at 1200rpm for 5 minutes.

(2) The supernatant was discarded and the cells were washed 2-3 times with PBS.

(3) Adding 5uL 7-AAD dye solution into every 50uL Binding Buffer, and uniformly mixing for later use.

(4) 55ul of the prepared solution is added into each tube, mixed evenly and incubated for 5-15 minutes at room temperature in a dark place.

(5) 450uL of Binding Buffer was then added and mixed well.

(6) Adding 1uL Annexin V-APC, mixing, and incubating at room temperature in dark for 5-15 min.

(7) Negative control is not added with staining solution, and single positive control is added with 1 staining solution respectively.

(8) And (6) performing detection on the machine.

Culturing U87 cells with DMEM culture medium and brain glioma-related mesenchymal stem cell conditioned medium to obtain Control-U87 and gamMSC-U87, observing the morphology of the two groups of cells, wherein the two groups of cells grow adherent to each other, are fusiform and have no morphological difference (figure 1). CCK-8 determines the difference of cell proliferation of two groups under the condition of temozolomide with different concentrations, and finds that the proliferation capacity of the gasSC-U87 is stronger than that of Control-U87; furthermore, the difference in proliferation potency of the two groups was determined at different times under 200. mu.M temozolomide condition, and the proliferation potency of gamSC-U87 was also stronger than that of Control-U87 (FIG. 2). Two groups of cells were tested for migration ability by scratch assay under 200. mu.M temozolomide conditions and it was found that gamSC-U87 migration was superior to Control-U87 (FIG. 3). The apoptosis condition is determined by flow cytometry, and the result shows that the apoptosis rate of the gassc-U87 is less than that of Control-U87 (figure 4) no matter whether temozolomide exists or not; the present application also refers to the attached drawings of the prior patent applications of the inventor for further description of cell differentiation and cell migration of the glioma-associated mesenchymal stem cells, please refer to fig. 13 (D, E, F is a schematic diagram of differentiation of adipocytes, osteocytes and chondrocytes, respectively) and fig. 14.

Wherein: FIG. 1 morphology of U87 cells in different culture conditions. The left side is Control-U87 and the right side is gasSC-U87. X 40, scale bar =200 μm; FIG. 2 proliferation of U87 cells under different conditions. A. Proliferation of two groups of cells in different concentrations of temozolomide. B. And (5) fixing the proliferation condition of two groups of cells under different culture times when the concentration of temozolomide is 200 mu M. μ M = μmol/L, n =3, # P <0.01, # P < 0.0001; FIG. 3 shows the 24-hour scratch migration of Control-U87 and gasSC-U87 cells. Migration on the left and quantitative statistical analysis on the right. n =3, < 0.05; FIG. 4 apoptosis of Control-U87 and gasSC-87 cells. Flow-through apoptosis was shown on the left and quantitative statistical analysis on the right. -: no temozolomide, + is added: plus temozolomide, n =3, P <0.05, P < 0.0001.

Real-time fluorescent quantitative PCR:

(1) the cells were removed from the incubator and placed in a biosafety cabinet, washed with PBS and then added with 1ml Trizol, and the cells were blown out of the wall and transferred to a 1.5ml EP tube for lysis for 10 minutes.

(2) 200ul of chloroform was added thereto, and the mixture was mixed well and reacted for 5 minutes, followed by centrifugation at 12000rpm at 4 ℃ for 15 minutes.

(3) The upper layer was transferred to a 1.5ml EP tube, 400ul isopropanol was added, mixed well and left for 10min, and then centrifuged at 12000rpm at 4 ℃ for 10 min.

(4) The supernatant was discarded, 1ml of 75% ethanol was added, and the mixture was vortexed, mixed at 4 ℃ and 10000rpm, and centrifuged for 5 minutes.

(5) Repeating the step (4) for 1 time.

(6) The supernatant was discarded, dried in air for 10 minutes, and then dissolved by adding 20ul DEPC water.

(7) Taking the dissolved RNA2ul, and measuring the OD260/OD280 value by using a micro spectrophotometer, wherein the value is required to be 1.8-2.0 to meet the requirements of subsequent experiments. The total RNA is stored at-80 ℃ for later use.

(8) Reverse transcription into cDNA.

(9) And (4) PCR detection, curve drawing and data analysis.

The primer sequence is as follows: homo b-action Forward 5'-AGCGAGCATCCCCCAAAGTT-3'

Reverse 5’-GGGCACGAAGGCTCATCATT-3’

Homo FOXS1 Forward 5’-CCCAGGGTTCCTTGTGGTC-3’

Reverse 5’-CCCAGGGTTCCTTGTGGTC-3’

Two groups of cells are cultured under the condition of 200 mu M temozolomide, and the gene expression difference of the two groups of cells is measured by a gene chip technology, and the FOXS1 in the gaMSC-U87 is most obviously upregulated relative to Control-U87 (figure 5). The expression of FOXS1 in both groups of cells was further verified by PCR (FIG. 6A) and WB (FIG. 6B) techniques, and it was found that FOXS1 expression in gasSC-U87 was significantly higher than in Control-U87.

Wherein: FIG. 5 is a heatmap of the differences in gene expression between Control-U87 and gamSC-U87 cells. After 3 days of culture at 200. mu.M temozolomide concentration, the cells were examined and statistically analyzed using a gene chip. n =3, P < 0.05; FIG. 6 FOXS1 expression differences between Control-U87 and gamSC-U87 cells. PCR technology detects the difference of FOXS1 expression between two groups of cells. Wb technology detects differences in FOXS1 expression between two groups of cells. n =3, P < 0.001.

Western Blot:

(1) Culturing cells with a 6-well plate in advance, taking out the cells after the cells grow full, abandoning the culture solution, and adding PBS (phosphate buffer solution) with precooling temperature of about 4 ℃ for washing 3 times.

(2) 100ul of a pre-prepared lysis solution containing PMSF was added to each well and lysed on ice for 30 minutes.

(3) After completion of lysis, the cells were scraped off with a cell scraper, transferred to a 1.5ml EP tube (ice-on operation), and then centrifuged at 12000rpm at 4 ℃ for 5 minutes.

(4) After centrifugation, the supernatant is taken and packaged separately and can be stored at-20 ℃ for later use.

(5) The BSA standard was diluted to prepare standard proteins at concentrations of 1, 0.8, 0.6, 0.4, and 0.2.

(6) Adding each concentration standard substance into a 96-well plate, adding 20ul PBS into each hole with two parallel holes with each concentration, adding 20ul PBS into a blank control hole, adding the protein stock solution to be detected and the protein solution diluted by 10 times and 100 times by PBS into 3 parallel holes with each concentration, and adding 20ul PBS into the 96-well plate into each hole.

(7) And (3) mixing the solution A and the solution B in the BCA kit according to the ratio of 50: 1, mixing uniformly, adding 200ul of the mixture into each hole, and reacting for 30 minutes in a dark place.

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

(9) The extracted protein was mixed with 5 x protein loading buffer and cooked in boiling water for 10 min.

(10) 5% concentrated gum and 12% separation gum were prepared.

(11) Adding protein and MAKER into the sample loading hole by using a sample loading gun, performing constant-pressure electrophoresis at 80V until the bromophenol blue is positioned at the junction of the concentrated gel and the separation gel, and continuing electrophoresis at 120V until the bromophenol blue reaches the bottom of the gel.

(12) After the electrophoresis is finished, cutting off a gel strip containing the target protein, cutting a PVDF membrane with a corresponding size, sequentially clamping the gel strip, the PVDF membrane, the filter paper and the whiteboard according to the sequence of blackboard-filter paper-gel-FVDF membrane-filter paper-whiteboard, putting the clamped gel strip into a membrane rotating groove, and rotating the membrane by the blackboard corresponding to a negative electrode.

(13) After the membrane transfer was completed, the PVDF membrane was soaked in a blocking solution (TBST containing 5% skim milk powder) and placed on a shaker for 2 hours.

(14) After blocking was complete, the PVDF membrane was soaked in primary antibody diluted with primary antibody diluent and incubated overnight at 4 ℃.

(15) The incubated primary resistant PVDF membrane was washed 5 times with TBST for 5 minutes each.

(16) The washed PVDF membrane is soaked in the secondary antibody diluted by the secondary antibody diluent again, and incubated for 2 hours on a shaking table at room temperature.

(17) After incubation, the secondary antibody was washed 5 times with TBST.

(18) ECL enhancement solution and peroxidase solution 1: 1, uniformly mixing, dripping on a PVDF film, after a band is obvious, sucking redundant liquid by filter paper, covering a preservative film, pressing an X film, then dripping developing solution in sequence for development, fixing by the fixing solution, washing the film, airing, scanning and analyzing.

Lentivirus transfection experiments:

(1) cell suspension with 50000 cells/ml density was prepared in DMEM medium containing 10% serum and inoculated into 6-well plates at 2 ml/well, then at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 24 hours.

(2) After 24 hours, the DMEM medium was changed to 1ml per well, and then 20ul of 1X 10 medium was added to each well⁸Viral solution TU/ml, 37 ℃, 5% CO₂The cultivation in the incubator is continued for 12-16 hours.

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

(4) And collecting infected cells, and screening and purifying the infected cells in a culture medium containing puromycin for later use.

Control-U87, gamMSC-U87, Control-GBM1 (DMEM medium-treated glioma primary cells GBM-1), gamMSC-GBM 1 (glioma primary cells GBM-1 treated with conditioned medium of brain glioma-associated mesenchymal stem cells) cultured under 200uM temozolomide conditions, and WB assay for EMT marker protein (E-Cadherin, N-Cadherin) showed that EMT progression was promoted relative to Control-U87 and Control-GBM1, gamMSC-U87 and gamMSC-GBM 1 (FIG. 7). To verify that upregulation of FOXS1 promoted EMT progression, U87 and GBM-1 cells were either knocked down or overexpressed FOXS1 using lentiviruses and then assayed using the WB technique, resulting in the same trend for both cells, i.e., EMT progression was promoted in the overexpression group (OE-U87, OE-GBM 1) versus the Control group (Control-U87, Control-GBM 1), and in contrast in the knock down group (KD-U87, KD-GBM 1), the negative Control group (NC-U87, NC-GBM 1) did not change (fig. 8).

The cells of the FOXS1 overexpression or knockdown U87 stable strain constructed by lentivirus transfection are cultured under the condition of 200 mu M temozolomide, and then a scratch experiment, a CCK-8 experiment and a flow cell apoptosis experiment are carried out. The results show that compared with Control-U87, the migration capacity and proliferation capacity of OE-U87 are increased, and the apoptosis rate is reduced; KD-U87 showed the opposite trend (FIG. 9).

Wherein: FIG. 7 shows the EMT marker protein expression of U87 cells and GBM-1 cells under different conditions. And B, performing WB (broad band) technology to detect the expression conditions of E-Ccddheron and N-Cadherin proteins, and performing WB result statistical analysis on two groups of U87 cells. C. Two groups of GBM-1 statistical analyses. n =3,. P < 0.01; FIG. 8 shows the expression of FOXS1 and EMT marker protein in the stable strain of knocked-down or over-expressed U87 cells and GBM-1 cells, the expression of FOXS1, E-Ccddheron and N-Cadherin proteins detected by the A.WB technology, and the WB result statistical analysis of the B.U87 cells. Gbm-1 cells WB results statistical analysis, n =3, # P <0.01, # P <0.001, # P < 0.0001; FIG. 9 drug resistance of U87 cell stable strain knocked down or over expressing FOXS 1. A. 24-hour scratch migration and statistical analysis under 200 μ M temozolomide condition in different groups of cells, 48-hour proliferation under 200 μ M temozolomide condition in different groups of cells, and flow apoptosis under 200 μ M temozolomide condition in different groups of cells, n =3, P <0.05, P <0.01, P < 0.001.

Animal experiments:

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

(2) The mouse scalp was disinfected with active iodine, and the scalp was cut open to fully expose bregma.

(3) A1 ml syringe needle is used for drilling a hole 1mm before bregma and 2mm aside, then a micro syringe is used for inserting a needle 3.5mm along the hole, 10ul of U87 cell suspension under different conditions is respectively injected into different mice, 1ul is injected every minute, and the needle is left for 5 minutes after the injection.

(4) Sealing the bone hole with bone wax after removing the needle, sterilizing and sewing scalp, and breeding and observing in SPF environment.

(5) Mice were given an intraperitoneal injection of temozolomide (50 mg/kg) for 5 consecutive days after 3 weeks, and then observations were continued.

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

Mouse tumorigenesis experiments were performed with U87 cells under different conditions, and the results showed that the survival of the gasSC-U87 group mice was lower than that of the Control-U87 group mice (FIG. 10B); after removal of the mouse tumors, tumor sizes were compared and Ki67 immunohistochemical analysis was performed, showing that the tumors were larger in the gassc-U87 group (FIG. 10C) and expressed more Ki67 (FIG. 11A) than in the Control-U87 group; compared with Control-U87 group mice, OE-U87 group mice had larger tumors (FIG. 10D), expressed more Ki67 (FIG. 11B), and KD-U87 group was opposite.

Wherein: FIG. 10. mouse tumorigenesis and survival of U87 cells under different conditions. A. Under different conditions for tumor formation of U87 cells, DMEM-U87 is a tumor of mice inoculated with DMEM-treated U87 cells, but not treated with temozolomide, -: no temozolomide treatment, +: survival analysis of mice vaccinated with temozolomide, b.control-U87 and gaMSC-U87 cells, quantification of tumor size of mice vaccinated with c.control-U87 and gaMSC-U87 cells, quantification of tumor size of mice vaccinated with d.control-U87, NC-U87, KD-U87, and OE-U87 cells, n =3, <0.05, <0.01, < 0.001; picture 11. expression differences of Ki67 in different cell groups. Tumor tissues Ki67 immunohistochemistry and quantitation in mice inoculated with control-U87 and galmsc-U87 cells, b.control-U87, NC-U87, KD-U87, and OE-U87 cells, × 400, scalebar =250 μm, n =3, × P <0.05, × P <0.01, × P < 0.001.

Immunohistochemical experiments:

(1) dehydrating the tissue in 75%, 85%, 90% and 95% alcohol for 4 hours, 2 hours, 1.5 hours and 1 hour in sequence; then dehydrated in absolute ethyl alcohol for 2 times, each time for half an hour.

(2) The clearing agent was applied to the tissue and the tissue was then waxed (60 ℃)3 times for 1 hour each time.

(3) After being soaked in wax, the mixture is embedded, and then sliced and baked.

(4) The sections were dewaxed and then heated in an electric ceramic oven for antigen retrieval.

(5) A3% hydrogen peroxide solution was dropped on the sections, left at room temperature for 15 minutes, and then washed 3 times with PBS for 3 minutes each.

(6) The slide was blotted dry with absorbent paper, an immunohistochemical pen was circled along the tissue, and diluted goat serum was added dropwise and sealed for 30 minutes.

(7) After sealing, sucking the redundant liquid, drawing a circle along the tissue by using an oily pen, and dropwise adding 1: ki67 antibody was incubated overnight at 100 dilution in a wet box at 4 ℃.

(8) The incubated primary antibody was washed with PBS 3 times for 3 minutes each, excess water was blotted off, and secondary antibody was added and incubated for 20 minutes at room temperature.

(9) After the second antibody is incubated, the second antibody is washed for 4 times by PBS, 3 minutes each time, DAB color developing solution is added for color development, observation is carried out under a mirror, and the slide is washed when the color development is proper.

(10) Counterstaining with Harris hematoxylin for 30s-1min, washing with water, differentiating with 1% hydrochloric acid alcohol, and washing with PBS to turn blue.

(11) And washing the slide, dehydrating and air-drying, sealing and air-drying, and taking a picture under a microscope for analysis.

ELISA experiments:

(1) and taking the ELISA kit out of the refrigerator, placing the kit at room temperature, and carrying out the next step when the temperature is balanced to the room temperature.

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

(3) Adding standard substances and samples to be detected with different concentrations into the plate, wherein each well is 100ul, adding standard substance/specimen universal diluent into a blank well, sealing with a plate sealing adhesive tape, and incubating for 90 minutes at 36 ℃ in a dark place.

(4) After incubation was complete, the liquid was poured out and washed with 350ul of wash solution per well for 30 seconds, then poured off and the plate washed 5 times.

(5) After washing the plate, the plate was patted dry on thick absorbent paper, 100ul of the prepared biotinylated antibody working solution was added to each well, and the diluted biotinylated antibody was added to the blank well, followed by incubation for 60 minutes at 36 ℃ in the dark.

(6) After incubation, the plate was washed 5 times, patted dry, 100ul of pre-prepared working solution of enzyme conjugate was added to each well, dilution of enzyme conjugate was added to blank wells, and incubation was carried out for 30 minutes at 36 ℃ in the dark.

(7) After incubation, the plates were washed 5 times, patted dry, 100ul chromogenic substrate was added per well, and incubated for 15 minutes at 36 ℃ in the dark.

(8) After incubation was complete, 100ul of reaction stop solution was added to each well and OD450 was measured immediately in a microplate reader and the assay results were saved.

ELISA experiments are used for detecting a DMEM culture medium (Control) and a glioma-related mesenchymal stem cell conditioned medium (gasMC-CM), the IL-6 content in the gasMC-CM is found to be obviously higher than that in the Control group, and the CD90 high-expression glioma-related mesenchymal stem cell conditioned medium (CD 90+ gasMC-CM) and the CD90 low-expression glioma-related mesenchymal stem cell conditioned medium (CD 90-gasMC-CM) are further detected, and the result shows that the IL-6 content in the CD 90-gasMC-CM is obviously higher than that in the CD90+ gasMC-CM (FIG. 12A). Further, U87 and GBM-1 were cultured in 200. mu.M temozolomide in DMEM medium (Control group), IL-6-supplemented DMEM medium (IL-6 group), and IL-6-neutralizing antibody (IL-6 + anti IL-6 group), and WB examined the expression of FOXS1, showing that FOXS1 was significantly upregulated in IL-6 group compared to Control group, while FOXS1 was reversibly upregulated in IL-6 + anti IL-6 group supplemented with neutralizing antibody (FIG. 12B).

Wherein: FIG. 12 Effect of IL-6 on expression of FOXS1 in cells. The content of IL-6 in different culture solutions determined by ELISA experiment, and the expression content of FOXS1 determined by B.WB. n =3, P <0.05, P <0.01, P < 0.001P < 0.0001.

Evaluation:

EMT is a biological process whereby epithelial cells, through their interaction with the basement membrane, undergo a variety of biochemical changes that transform them to mesenchymal phenotype, including enhanced migratory capacity, invasiveness, resistance to apoptosis, etc. The main feature of EMT is the reduction of the level of E-Cadherin (E-Cadherin) and the simultaneous increase of the level of N-Cadherin (N-Cadherin), causing alterations in the adhesion of cells based on Cadherin, which plays a key role in regulating development and organogenesis. The appearance of EMT during tumor progression allows non-invasive and non-metastatic benign tumor cells to acquire the ability to infiltrate the surrounding tissue and eventually metastasize to distant sites. Glioma cells can be transformed into cells with weaker adhesion and disordered cytoskeleton through EMT, so that stronger motility and chemotherapy resistance can be obtained. The chemotherapy resistance of glioma is closely related to the activation of EMT, and the activation of EMT in glioma is promoted to increase the expression of MGMT so as to increase the resistance of glioma cells to TMZ. TMZ is a prodrug of alkylating agents, which can deliver a methyl group to the purine base of DNA (O6-guanine; N7-guanine and N3-adenine), while MGMT can remove the alkyl group from the O6 position of guanine, so that high levels of MGMT in cancer cells can produce a resistant phenotype by impairing the therapeutic effect of TMZ.

When temozolomide is used for killing glioma cells in vitro, the U87 cells cultured and treated by a conditioned medium (gasSC-CM) of the brain glioma-related mesenchymal stem cells have stronger proliferation and migration capacity and less apoptosis, and the resistance of glioma cells to temozolomide medicaments is increased. Further, it was found by gene chip analysis that U87 cells cultured in gasSC-CM showed the most significant up-regulation of FOXS1 expression. FOXS1 belongs to the FOX family of proteins, which are evolutionarily conserved transcriptional regulators, and which play important roles in many biological processes, such as proliferation, migration, apoptosis, invasion, etc. The FOX family protein is expressed in a plurality of 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 inhibits EMT in prostate cancer cells by affecting the transduction of WNT/β -catenin signaling pathway, while FOXM modulates urokinase plasminogen activator system to promote EMT in pancreatic cancer. The expression of FOXS1 in gastric cancer tissues is obviously higher than that of precancerous tissues, the expression in gastric cancer cells is also obviously higher than that of gastric epithelial cells, FOXS1 can promote cell EMT and influence the survival rate of gastric cancer patients, and the high expression of FOXS1 indicates that the prognosis of the gastric cancer patients is poor. Meanwhile, the gasSC-CM can enhance the expression of U87 and GBM-1 cell N-cadherin and can down-regulate the expression of E-cadherin. Further, a stable transgenic cell line with over-expressed and knocked-down FOXS1 was established, and it was found that up-regulation of FOXS1 promotes cell EMT, increases cell proliferation and migration, and reduces apoptosis, and the opposite is true when FOXS1 is knocked-down. In vivo experiments, it was found that mice inoculated with U87 cells cultured with gasSC-CM had a lower survival time than the control group, developed larger tumors, and expressed more Ki 67. Meanwhile, U87 cells over-expressing FOXS1 also formed larger tumors in mouse brain, expressing more Ki67, and knocking down FOXS1 showed the opposite result.

IL-6 is a soluble glycosylated polypeptide chain, one of the major cytokines in the tumor microenvironment. The tumor microenvironment can secrete high-level IL-6, and can promote tumorigenesis by promoting tumor proliferation and migration, inhibiting apoptosis and the like, and increase the drug resistance of tumors to treatment. Therefore, GAMSC-CM was assayed and found to contain a large amount of IL-6. The brain glioma-associated mesenchymal stem cells can be divided into two subtypes of high expression of CD90 and low expression of CD90, and IL-6 is mainly secreted by a subtype of low expression of CD 90. Therefore, the addition of IL-6 to the culture medium was effective in promoting the expression of glioma cells FOXS1, while the addition of IL-6 neutralizing antibodies reversed this effect.

In conclusion, the brain glioma-related mesenchymal stem cells obtained by the analysis of the invention can promote the expression of glioma cells FOXS1, and further promote the chemotherapy resistance of glioma to temozolomide by promoting EMT. While the up-regulation of FOXS1 was mainly due to stimulation by IL-6 secreted by brain glioma-associated mesenchymal stem cells with low expression of CD 90. The method of the invention provides a new idea and target for reducing or reversing chemotherapy resistance of glioma.

The following provides sources of materials used in specific embodiments of the invention:

(1) DMEM medium, MEM-alpha 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, erythrocyte lysate (Saimeifei, China)

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

(9) ELISA kit (Xinbo Sheng, China)

(10) CD90 magnetic bead antibody, magnetic bead sorting column (Milrenyi, Germany)

(11) FOXS1 antibody (Thermo, USA)

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

(13) Ki67 antibody (Proteitech, China)

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

(15) Beta-actin antibody (doctor De biology, China)

(16) BALB/c-nu nude mouse (Weitonglihua, China)

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

(18) Lentivirus (Jikai gene, China)

The embodiments of the present invention have been described above by way of example, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within 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 a gammscs subgroup with drug resistance to brain glioma is characterized by comprising the following steps:

s1: collecting and separating to obtain brain glioma-related mesenchymal stem cells (gammscs), a human glioma cell line U87 and glioma primary cells (GBM-1);

s2: respectively preparing a human glioma cell line U87 and a glioma primary cell (GBM-1) which are cultured by DMEM culture medium and brain glioma-related mesenchymal stem cell (galMSCs) conditioned medium, and obtaining a human glioma cell line U87 (Control-U87) cultured by DMEM culture medium, a glioma primary cell (Control-GBM 1) cultured by DMEM culture medium, a human glioma cell line U87 (galMSC-U87) cultured by brain glioma-related mesenchymal stem cell conditioned medium and a glioma primary cell (galMSCs-GBM 1) cultured by brain glioma-related mesenchymal stem cell conditioned medium;

s3: carrying out glioma chemotherapy drug in-vitro dosing treatment on Control-U87, Control-GBM1, gasMC-U87 and gasMC-GBM 1 to obtain a plurality of samples to be detected;

s4: detecting, analyzing and comparing the proliferation capacity of the dosed sample to be detected through CCK-8;

s5: detecting the analysis and comparison of the apoptosis condition in the drug-added sample to be detected by a flow analysis technology;

s6: analyzing and comparing the migration capacity of the sample to be tested after adding the medicine through a scratch test;

s7: analyzing the influence of the gammscs on the gene expression of the glioma cells by a gene chip;

s8: analyzing the expression condition of FOXS1 by PCR and Western-blot;

s9: detecting changes in epithelial-mesenchymal transition (EMT) markers when knockdown or overexpression of FOXS1 was obtained by lentivirus transfection;

s10: establishing a nude mouse in-situ model through brain glioma cells, and in-vivo administering a glioma chemotherapeutic drug object to perform an in-vivo drug adding experiment on animals;

s11: detecting Ki-67 expression changes in tumor tissues in the animals by immunohistochemistry in S10;

s12: IL-6 expression and secretion of the gamSCs were analyzed by ELISA;

s13: and analyzing and comparing the detection results of the experiments to evaluate the drug resistance.

2. The method of claim 1 for detecting a subpopulation of brain glioma resistant gaMSCs, wherein: the glioma chemotherapeutic drug object is Temozolomide (TMZ).

3. The method of claim 2, wherein the subset of brain glioma-resistant gaMSCs is selected from the group consisting of: the in vitro sample adding concentration of the temozolomide is 190-210uM (umol/L), and the in vivo injection concentration is 45-55 mg/kg.

4. The method of claim 1 for detecting a subpopulation of brain glioma resistant gaMSCs, wherein: and selecting and collecting third generation passage cells for experiment and detection of the glioma primary cells.

5. The method of claim 1 for detecting a subpopulation of brain glioma resistant gaMSCs, wherein: the brain glioma-associated mesenchymal stem cells need to be sorted by high expression of CD90 and low expression of CD 90.

6. The method of claim 5, wherein the subset of brain glioma-resistant gaMSCs is selected from the group consisting of: the sorting step of the brain glioma-related mesenchymal stem cells comprises the following steps:

s101: taking out the brain glioma-related mesenchymal stem cells from the incubator, placing the brain glioma-related mesenchymal stem cells in an ultra-clean bench, washing the cells with PBS, digesting the cells with Accutase at 1500rpm, and centrifuging the cells for 6 minutes;

s102: discarding the supernatant, adding the CD90 magnetic bead antibody and the sorting Buffer at a ratio of 1:4, and flushing and mixing uniformly;

s103: placing the mixture in a refrigerator at 4 ℃ to avoid light for reaction for 15 minutes;

s104: taking out cells, sorting and washing by Buffer, centrifuging at 1500rpm for 6 minutes;

s105: discarding the supernatant, adding 500ul of sorting Buffer for resuspension;

s106: dripping the cell suspension into a sorting column, then dripping 2ml of sorting Buffer to wash the column, and obtaining the mesenchymal stem cells related to the brain glioma with low expression of CD90 (CD 90) from the filtered cell suspension^-）；

S107: after the filtration, 2ml of DMEM medium containing 20% serum is added into the sorting column, then the culture medium is rapidly pushed out by using a handle, and the filtered mesenchymal stem cells (CD 90) related to the CD90 high-expression glioma are obtained⁺）；

S108: respectively combining the above-mentioned CDs 90^-And CD90⁺The cells were transferred to a culture flask at 37 ℃ with 5% CO₂Culturing in an incubator.

7. The method of claim 1 for detecting a subpopulation of brain glioma resistant gaMSCs, wherein: the primer sequences in the PCR described in S8 are:

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

Reverse 5’-GGGCACGAAGGCTCATCATT-3’，

Homo FOXS1 Forward 5’-CCCAGGGTTCCTTGTGGTC-3’，

Reverse 5’-CCCAGGGTTCCTTGTGGTC-3’。

8. the method of claim 1 for detecting a subpopulation of brain glioma resistant gaMSCs, wherein: the PVDF membrane treatment method in the Western-blot detection and analysis in S8 comprises the following steps: soaking the PVDF membrane in a sealing solution (TBST containing 5% skimmed milk powder), and sealing on a shaking table for 2 hours; after the sealing is finished, soaking the PVDF membrane in primary antibody diluted by primary antibody diluent and incubating overnight at 4 ℃; washing the incubated PVDF membrane with TBST for 5 times, 5 minutes each time; soaking the washed PVDF membrane in a secondary antibody diluted by a secondary antibody diluent, and incubating for 2 hours on a shaking table at room temperature; after incubation, the secondary antibody was washed 5 times with TBST and used.

9. The method of claim 1 for detecting a subpopulation of brain glioma resistant gaMSCs, wherein: the lentivirus transfection experiment steps in S9 include:

s201: preparing cell suspension with density of 50000 cells/ml by using DMEM medium containing 10% serum, inoculating the cell suspension into a 6-well plate, wherein each well contains 2ml cells, and culturing the cell suspension in a 5% CO2 incubator at 37 ℃ for 24 hours;

s202: after 24 hours, the DMEM medium was changed to 1ml per well, and 20ul of 1X 108TU/ml virus solution was added to each well at 37 ℃ with 5% CO₂Continuously culturing for 12-16 hours in the incubator;

s203: after 12-16 hours, the culture medium is replaced to continue culturing for 72 hours, and the infection effect is observed under a fluorescence microscope;

s204: and collecting infected cells, and screening and purifying the infected cells in a culture medium containing puromycin for later use.

Images