The Research Of Malignant Transformation Of BMSCs In C6 Glioma Microenvironment

ObjectiveTo discuss the role of NF-κB and STAT3 on the malignant transfromation of BMSCs in C6 glioma microenvironment. And to provide a well model for study the clinical security application of BMSCs and discover new drugs to prevent BMSCs from malignant transformation.Materimal and Methods1. Experimental animalsSD rats, the body weight of rats was between 30g to 50g. The weight of male nude mice was range from 18g to 20g, and the age was range from 5 weeks to 6 weeks. All of the experimental animal were purchased from the Animal Experimental Center of Chongqing Medical University.2. CellsC6 cells lines were obtained from Chongqing Medical Institute of Pediatrics Cancer Research. Rat bone mesenchymal stem cells was isolated from SD rats’femur and tibia, and cultured in vitro.Astrocytes was isolated from new borned SD rats’cerebral cortex and cultured in vitro.3.Experimental groupsThe first part of the experiment was divided four groups:Experimental group, C6 indirect co-cultured with BMSCs; Postive control group,C6 alone cultured; Negtive control group, astrocytes indirect co-cultured with BMSCs; Blank control group, BMSCs alone cultured. The second part was divided into three groups:Experimental group, nude mice injected BMSCs what was cocultured with C6 after 7 days; Positive control group, nude mice injected C6 glioma cells; Blank control group, nude mice injected BMSCs.4. Experimental Method4.1 The morphological changes of the cells was observed by inverted phase contrast microscope.4.2 CD90, CD29 and CD45 expression was dectecd by IF.4.3 NF-κB, STAT3, c-Myc and s-100β expression were detected by RT-qPCR, Western blot and immunofluorescence.4.4 Migration assay was used to dectect cellular migration abilities in each group.4.5 BMSCs after indirect co-culture with C6 were injected in subcutaneous of nude mice.4.6 The lump was detected by HE staining.Results1. BMSCs were negative for CD45, but positive for CD29, CD90 and test group cells showed typical morphology features of tumor cell.2. NF-κB, P-STAT3, c-Myc and s-100β positive expression were situated in the nuclei when co-cultured in vitro, and NF-κB, P-STAT3 and S-100βpositive expression were situated both in cytoplasm and nucleus after injected in subcutaneous of nude mice.3. The expression of NF-κB, STAT3, c-Myc in test group was remarkable higher when compared with blank group and negative group.4. Cellular migration ability in test group was remarkable higher than the blank group.5. BMSCs after indirect co-culture with C6 was developed a lump.6. HE staining showed that a lot of amorphous tumor cells formed the lump, which the lump arranged in disorder, accompanied by necrosis.Conclusion1. In C6 glioma cells microenvironment, the cellular migration ability was improved significantly, and the malignant transformation of BMSCs was related with the activation and overexpression of NF-κB and STAT3.2. The malignant transformed BMSCs could form a lump after injected in subcutaneous of nude mice and the lump were overexpressed NF-κB, STAT3 and s-100β.