| ObjectiveTo investigate the methods of isolation , cultivation and induction to differentiate into chondrocyte in vitro by monolayer culture of rat bone marrow mesenchymal stem cells (MSCs). Try to set up a simplest and feasible method to induce mesenchymal stem cells to express chondrocyte phenotype. Tlien the MSCs from rats bone marrow of different age were induced by this method. Hie differentiation rate of the MSCs derived from different age rats was statistical analys-ized.Materials and methodsThe mesenchymal stem cells were obtained from both femur of the rats of different age, and then centrifugated with Percoll solution. The cells were resus-pended with low - glucose Dulbeccos modified Eagles medium containing 15% FBS and cultured . After 4 hours, the culture medium was replaced, thus the suspending cells were purified. After that,the culture medium was replaced every 2 or 3 days. We drew the growth lines of 3rd passage cells from 4 to 6weeks old or 12 months old rats. We used third passage cells for chondrogenic differentiation with serum - free High - glucose Dulbecco's modified Eagles medium only containing transforming growth factor - beta 1. After two weeks induced, the cells were detected for type — II collagen by immunohistochemical analysis and in - situ hybridization . At last the differentiation rates of rats of 4 to 6weeks old or 12 months old were campared by statistical analysis.Results(1) The primary cultured MSCs would get 80 ~ 90% confluence within 2 weeks generally. The offspring cells grew more rapidly and would get 80 ~ 90% confluence within 5 to 7 days. (2)The third passage cells from different age rats both regenerated quickly. There would be incubation time for 1 ~ 2 days, then the cells would exponentially grow for 3 ~ 4 days. At last these cells' quantity would reach a platform. The doubling generating times were both short and only 3 - 4 days. There was no discrepancy in the growth pattern of the third passage MSCs between different age rats (3) After induced for 2 weeks , parts of the cells became flat, spacious in shape or had several spurius. (4) Immunohistochemical analysis for collagen type - II shown that there was significant difference between the induced cells and the un - induced cells derived from all rats. Collagen type - II was detected in some cells of the induced group and there were brown particles in the endochylema of these cells. The un — induced cells were all negtive. (5) In - situ hybridization for collagen type - II mRNA manifested that there was marked distinction between the induced cells and the un - induced cells derived from all rats. Collagen type - II mRNA was detected in some cells of the induced group and there were brown particles in the kytoplasm of these cells. The un - induced cells were all negtive. (6) There was no difference in the def-ferentiation rate between diverse age rats by statistical analysis. .Conclution(1) High purity MSCs could be obtained by density gradient centrifugation with Percoll fluid. The MSCs accrementited active in vitro. (2)The third passage MSCs from different age rats both regenerated quickly, and there was no difference between them. (3 ) The MSCs differentiated into chondyocyte after they were cultured with HG - DMEM only added transforming growth factor - beta 1. TGF - β1 could induce MSCs to chondrogenesis by oneself (4) Under our experiment conditions, there was no distinction in the differentiation rate of the third...
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