The Study On Avoiding BMSCs Being Malignant Transformation In The Tumor Microenvironment Simulated By C6 Glioma Treated By TPCA1

Objective:To establish the models of bone marrow mesenchymal stem cells(BMSCs)which treated by TPCA1 co-cultured indirectly with C6 glioma cells,We investigate the way to avoid the malignant tansformation of BMSCs in the tumor microenvironment simulated by C6 glioma cells,Which provides reference for the safety application of BMSCs as the clinical carrier for tumor targeted therapy.Methods 1 Experiment group This study is divided into two parts: 1.1 The first part is grouped as follow Blank control group—BMSCs cultured separately Positive control group—C6 glioma cells cultured separately Experiment group—BMSCs co-cultured with C6 glioma cells indirectly:divided into seven sub group 3.2 The second part is grouped as follow Blank control group—BMSCs cultured separately Positive control group—C6 glioma cells cultured separately Experiment group—BMSCs co-cultured with C6 glioma cells indirectly:divided into three sub group Group 1:co-cultured without treatment Group 2:co-cultured +DMSO Group 3:co-cultured +200nmol/L TPCA1(300noml/L)2 Experiment method 2.1 Apply the way of cell co-cultured indirectly 2.2 Observe the morphological changes of BMSCs by microscope 2.3 Detect the cytotoxicity of TPCA1 to BMSCs by CCK8 2.4 Identify the BMSCs by flow cytometry(FCM)2.5 Detect the m RNA expression of STAT3?NF-?B?C-myc by Real Time fluorescent Quantitative PCR 2.6 Detect the protein expression of STAT3?NF-?B?C-myc by Westtern Blot 2.7 Detect the protein expression of P-STAT3 ? NF-?B ? C-myc by Immunofluorescence Assay 2.8 Detect the ability of migration and invasion by migration test and invasion testExperiment result The first part 1.1 BMSCs extracted primarily was identified by FCM,the positive expression rate of CD90 was 99.8%,the the positive expression rate of CD29 was 92.5%,CD34 and CD45 was notexpressed.1.2 Observe the cellular morphology by the microscope.The BMSCs cultured separately arranged regularly,C6 glioma cells arranged irregularly.The BMSCs co-cultured with C6 glioma cells indirectly arranged more irregularly than cultured separately.The BMSCs co-cultured with C6 glioma cells indirectly and treated by TPCA1 increased more slowly significantly with the increase of drug concentration,the cellular morphology was more regular than the co-cultured with C6 glioma cells.1.3 We found that the increase of BMSCs got slow gradually with the increase of drug concentration.1.4 We found the m RNA expression of STAT3?NF-?B?C-myc decreased gradually with the increase of drug concentration.The second part 2.1 We found that the cells of negative group were arranged regularly,the cells of positive group were arranged irregularly,the BMSCs co-cultured indirectly with C6 glioma cells treated by TPCA1 were more regular than without treatment by the microscope.2.2 The migration ang invasion of BMSCs co-cultured indirectly with C6 glioma cells treated by TPCA1 were significantly lower than without treatment(p<0.05).2.3 The m RNA and protein expression of STAT3?NF-?B?C-myc were significantly lower withouttreatment(p<0.05).Conclusion TPCA1 could inhibit the cell proliferation of the BMSCs co-cultured indirectly with C6 glioma cells,inhibit the m RNA expression of STAT3?NF-?B?C-myc with a concentration dependence,and the best inhibitory concentration was 200nmol/L.The biological characteristics of the BMSCs co-cultured indirectly with C6 glioma cells had changed,the m RNA and protein of STAT3?NF-?Bwere overexpressed,the m RNA expression downstream target gene C-myc was significantly increased.The malignant change of the BMSCs co-cultured indirectly with C6 glioma cells treated by TPCA1 was inhibited,which shown that TPCA1 could play a role in avoiding the BMSCs being malignant transformation in the tumor microenvironment simulated by C6 glioma.