| ObjectiveInvestigate the relation between bone marrow mesenchymal stem cells(mesenchymal stem cells, MSCs) tumorigenesis and tyrosine kinasesignaling molecules in the tumor simulated microenvironment, and thusprovided certain pre-theoretical foundation and experimental basis for thefuture safe and effective use of MSCs.Methods and materials1. Cell culture：Took Wistar rat to dissociate femur and tibia, and thenIsolated, cultured and amplified MSCs in vitro by adhesion methods;Took New-borned Wistar rat to dissociate Cerebral cortex, and thenisolated and cultured and amplified Astrocytes in vitro by adhesionmethods; C6glioma cells lines were obtained from Tumor ResearchCenter of the Institute of Pediatrics in Chongqing children hostipal.Allthe three cell lines were cultured using the same medium as MSCs.2. Establishment of grouping: PART1：The screening experiment isdivided into three groups:Experimental group(co-cultured with C6), Control group (co-cultured with astrocytes) Blank control group(MSCs alone)；PART2：The verificationexperiment is divided intotwo groups: Experimental group(treated with the same amount of IL-6in the tumor microenvironment), Control group (treated with normalIL-6)；3. Experimental methods: PART1：(1)observed morphological changesof MSCs by Olympus phase-contrast microscope（;2）P53and MDM2ofMSCs in each group were measured by PCR, Immunofluorescence；（3）HGF，IL-6and BFGF expression in each group were detected byELISA;（4） the STAT3protein expression of MSCs treated withcorresponding ELISA amount tyrosine kinase signaling molecule weredetected by immunofluorescence. PART2：（1）MSCs proliferationchanges between each group were detected by cell growth curve;（2）MSCs proliferation changes between each group were detected by cellgrowth curve;(3)P53and MDM2of MSCs in each group weremeasured by PCR and Western blotting；(4)The ability of each groupMSCs growing into tumor were detected by nude mice transplant test.RESULTSPART1： The screening anyalysis of related factors induced MSCstumorgenesis1. The4th generation of purified MSCs were fibroblast-like and uniformed.The purified astrocytes were star-shaped, slender and has a branch. C6 glioma cells were slender, closely arranged, the nucleus mass ratio.MSCs cell morphology in the experimental group after1week becamemore obvious:obvious cellular atypia, nuclear-mass ratio increases. Cellmorphology of the control group did not change significantly, stillshowed a fibroblast-like arrangement.2. the experimental group of p53mRNA relative expression was0.55±0.10, compared with the control group,the expression was decreasedstatistically significant (P <0.01); MDM2mRNA expression level ofexperimental group was1.56±0.21and1.56times the control group,increased significantly(P <0.01) P53mRNA expression level of theControl group was1.10±0.16times as compared to the blank controlgroup, the two had no statistical difference (p>0.05); And MDM2mRNA in the two control groups, was not statistically different either(p>0.05). MDM2protein in experimental group increased90%±7%ascompared to control group (P<0.01)；The level of mutant P53proteinincreased to87%±5%in experimental group as compared to controlgroup (P<0.01);3. HGF and il-6significantly increased significantly （p<0.01）whileBFGF had no significant difference in Experimental group ascompared to Control group and Blank control groupl.（p>0.05）HGF〠il-6and BFGF had no significant difference between Controlgroup and Blank control group（p>0.05）. 4. P-STAT3expression increased significantly in IL-6overdose group ascompared to IL-6normaldose group.(P<0.01).PART2：IL-6tumor effect on mesenchymal stem cells1. Growth curve showed the experimental group MSCs reduced contactinhibition after two months;2. Two months later, the relative p53mRNA expression level ofexperimental group is0.17±0.07compared with the control group,expression level between the two groups was statistically different(P<0.01); MDM2mRNA expression in the experimental group was2.64±0.5times as compared with the control group, and was significantlyincreased (P <0.01).3. Western blot results of p53and mdm2protein:After two months, p53protein in the control group was0.33±0.11, while p53protein in theexperimental group was0.10±0.03, the p53protein in the experimentalgroup was significantly lower than control group (P <0.05). MDM2proteins increased from0.23±0.09to0.73±0.14form the controlgroup to the experimental group, were significantly different (P<0.05).These indicated the reduction of p53protein and MDM2amplification occurred in the experimental group cells.4. Three months after transplant, we could see tumor grow in nude mice ofthe experimental group, while not see in the control group. Take theinoculation site in the experimental group for HE staining, we could see a lot of amorphous tumor cells, the cells arranged in disorder,accompanied by necrosis. While the HE staining of the control groupresults showed that the inoculation site still retained the staining ofnormal muscle structure.Conclusions1. Overexpression of kinase signaling molecules in vitro could promoteMSCs to get the biological characteristics of tumor cells in the vitrosimulated tumor microenvironment.2. Overexpression of IL-6could promote MSCs malignanttransformation in the tumor microenvironment and the malignanttransformed MSCs could form tumors after implanted in nude mice. |
