| ObjectiveTo discuss the biological characteristics and its molecular mechanism of hUC-MSCs in tumor microenvironment by constructing the non contact co culture model of hUC-MSCs and MCF-7B breast cancer cells or U251 glioma cells, in order to lay preliminary experimental basis for the clinical application of hUC-MSCs as tumor targeted therapy vector; Injection BMSCs in tumor situ and dynamic observation of its distribution in the tissue by constructing the SD rat glioma model, to provide experimental model for the study of malignant transformation of BMSCs in the microenvironment of brain glioma in vivo.Materimal and Methods1. Experimental animalsMale nude mice,6 weeks old, the weight was range from 15g to 25g; The body weight of Male SD rats was 50±10 g,the age was 3-5 weeks old;Male SD rats,the weight was between 200g to 250g. The above experimental animals were purchased from "Animal Experimental Center Of Chongqing Medical University’2. CellshUC-MSCs were gifted by Chongqing Stem Cell Therapy Engineering Technical Center; Breast cancer cell lines MCF-7B were purchased from Guangzhou Gynefix European Biotechnology Co. Ltd.; Glioma cell lines U251 were purchased from Shanghai cell bank of Chinese Academy of Sciences; Glioma C6 cell lines sourced from Chongqing Medical Institute of Pediatrics Cancer Research; Rat bone marrow derived mesenchymal stem cells were isolated from bone marrow.3. Experimental groupsStudy was divided into three parts: third part had three groups4.Experimental Method The first part and the second part:detection of biologicalcharacteristics of hUC-CMSCs in the tumor microenvironment4.1 Cell morphological characteristics were observed byinvertedmicroscope.4.2 The distribution of cell cycle phase was detected by flow cytometry.4.3 Cell proliferation was analyzed by CCK-8.4.4 Cell karyotype was analyzed.4.5 Tumorigenic ability was evaluated in vivo and subcutaneous tissue was observed by HE staining.4.6 Senescence-assosiated β-galactosidase activity was observed by P-gal staining.4.7 The relative mRNA expression of hTERT,c-Myc and Bcl-xl were detected by RT-qPCR.4.8 The protein expression of P53, c-Myc and Bcl-xl in each group were detected by Western blot.4.9 The protein expression and location of c-Myc and Bcl-xl were observed by immunofluorescence.The third part:the establishment of brain glioma model and the injection and distribution of BMSCCM-Dil in tumor situ4.10 CM-Dil labeling BMSCs were observed by invertedmicroscope.4.11 The establishment of brain glioma model.4.12 The observation of the structure of brain glioma by HE staining.4.13 The distribution of BMSCs in tumor tissue was observed by fluorescence microscope.ResultsThe first part and the second part:analysis of biological characteristics of hUC-MSCs in the tumor microenvironment1. As cultured to 10th generation. hUC-MSCs in the two experimental groups were polymorphism and not uniformity in size.Karyoplasmic ratio slightly increased.The cells arranged in disorder and clustered,showing mild atypia.2. The cells proportion of experiment groups in G1phase was significantly lower than that of blank control group (p<0.05)3. The proliferation rate was significantly increased in the two experimental groups compared with blank control group from day 2 to day 6 (p<0.05)4. hUC-MSCs in experimental groups exhibited a normal karyotype (46,XX),and no significant abnormality of chromosome structure was found in G banding.5.8 weeks later, the nude mice of blank control group and the two experimental groups were dead and not formed package. HE staining showed that the subcutaneous tissues were normal and regularly arranged. Cell cytoplasm stained red and a number of flat nuclei were distributed on the edge of cells.6. As cultured to 6 weeks, β-gal staining showed Senescence-assosiated β-galactosidase activity in the two experimental groups was significantly higher than that in blank control group.7. The relative mRNA levels of c-Myc and Bcl-xl in experimental group1 and 2 were significantly higher than those of blank group (p<0.05). While there was no difference in the mRNA expression of hTERT in the experimental groups compared with the blank group.And contrasting with positive group,the mRNA expression levels in the experimental were extremely lower.8. p53、c-Myc and Bcl-xl protein expressions in the two experimental were significantly higher than the blank (p<0.05)9. c-Myc was located in the nuclei; Bcl-xl was expressed in cytoplasm and nucleus, mainly situated in the outer membrane of the cell.The third part:the establishment of brain glioma model and the injection and distribution of BMSCsCMDil in tumor situ10. BMSCs labeled CM-Dil showed red fluorescence, which was mainly expressed in cell membrane.11.7 d after the establishment of brain glioma model, the brain of SD rats appeared circular abnormal tissue with invasive growth of about 1 cm in diameter on the right side of the cerebral hemisphere.12. HE staining showed that the tumor tissue arranged in dense and was invasive growth,which had unclear boundaries with normal brain tissue.13. In the experimental group,BMSCsCM-Dil implanted into SD rats bearing C6 brain glioma.2 days later, BMSCsCM-Dil was found at the site of needle channel;Then 7 days later, BMSCsCM-Dil was transferred to the edge of the tumor tissue.Conclusion1. The study demonstrate hUC-MSCs were affected by the inflammatory factors in the tumor microenvironment, which were easily enhance the ability of proliferation breakthrough cell cycle restriction by constructing the non contact co-culture model of hUC-MSCs and MCF-7B breast cancer cells or U251 glioma cells. While in the influence of pressure signaling from cancer gene, the activation of p53 protein resulted in the non repairable cells senescence,maintaining the stability of genome stability. hUC-MSCs exposed to the tumor microenvironment might not have the potentials of malignant transformation.2. SD rat C6 brain glioma model was successfully constructed. CM-Dil labeled BMSCs which were implanted into SD rats bearing C6 brain glioma were mainly situated in the edge of the tumor tissue. The study can provide experimental model for the study of malignant transformation of BMSCs in the microenvironment of brain glioma in vivo. |
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