Rat Bone Marrow Mesenchymal Stem Cells And Nucleus Pulposus Cells In Vitro Co-culture Experiments

1 OBJECTIVEUsing stem cell transplantation in the treatment of disc degeneration disease is still limited to the laboratory study. And bone marrow mesenchymal stem cells (BMSCs) can be one of the most potential seed cells. To explore the related mechanisms of BMSCs promoting the expression of the nucleus pulposus cells extracellular matrix and differentiating into nucleus pulposus-like cells in a directly contact micro-environment.2 METHODSSeparate and cultivate mesenchymal stem cells by using density gradient centrifugation and explant-attached method, and identify, train to 3rd generation and freeze-stored. Separate and cultivate the nucleus pulposus cells from disc by using the enzyme digestion method, and identify, train to the fifth generation after morphological degeneration appearance and freeze-stored. The 6th generation of natural degenerated nucleus pulposus cells were isolated, co-cultured with the 4th generation of BMSCs through direct contact in vitro, according to different proportion(75:25,50:50 and 75:25), pure the nucleus pulposus cells group (100:0) and pure BMSCs group (0:100) acted as the positive control and the negative control respectively. The expression of the aggrecan and typeâ…¡collagen were observed by toluidine blue and immunohistochemical separately, both of them were detected through RT-PCR method.3 RESULTSThe results of RT-PCR of collagenâ…¡and aggrecan in the experimental groups (75:25 and 50:50) showed better than the positive control group (100:0), and there were a significant difference of absorbance (A) (P<0.05). The results of immunohistochemical method of collagen II showed more expressions in the experimental group (75:25 and 50:50) than the positive control group (100:0). Proteoglycans of toluidine blue stain reveals cytoplasm metachromaticin the experimental group (75:25 and 50:50) is stronger than the positive control group (100:0). BMSCs turned into nucleus pulposus-like cells appearance and metachromatic granules increased, and the nucleus pulposus cells restore growth activity.4 CONCLUSIONS It demonstrates that the direct contact of MSCs and nucleus pulposus cell is essential for MSCs reversing the nucleus pulposus cells degeneration and MSCs differentiation into nucleus pulposus cells.