Mice Mesenchymal Stem Cells Differentiate Into Chondrocytes With Rhzizoma Drynariaeor Radix Rehmanniae Preparata.

Object:To investigate the possibility and mathod that mesenchymal stem cells (MSCs) of adult mice to differentiate into chondrocytes in vitro induced by Rhzizoma drynariae or Radix rehmanniae preparata.Methods:Rhzizoma drynariae or Radix rehmanniae preparata were extracted with water. The extraction was filtered when it was hot, then it was evaporated to dry. Dissolve and dilute the residue to about 10ug/ml with 70% ethanol and keep solution in 4℃.Bone mesenchymal stem cells were obtained from infancy SD mice, separated by gradient centrifugation method, and purified by monolayer culture. The cells which were not to the bottles cliff were wiped off by exchanging culture liquid. When the proportion of cells is more than 80 percent of the culture bottles bottom area, the cells were assimilated by enzyme, and cultivated to propagate.When the third generation cells were attained, they were put into the 96-hole cultivate plate, making 5000 cells in one hole. After cells sticked to the bottle bottom cliff, the various density extraction of Radix rehmanniae and Rhzizoma drynariae were added into the culture liquid respectively. At last, the quantity of the alive cells were measured by MTT test after cultured 1 week.When the third generation cells were attained, they were inoculated on glass patch. The extraction of Radix rehmanniae and Rhzizoma drynariae were added into the culture liquid respectively, after cells sticked to the bottle bottom cliff. Then the protein expressions of collagen typeⅡweredetected by immunohistochemical staining, after cultured about 2 weeks.Compared the figure of differentiated cells cultivated by Radix rehmanniae and Rhzizoma drynariae with elastic chondrocyte. Compared them by the quantificational analyse of immunohistochemical staining of typeⅡcollagen and the statistic analyse.Compared the figure of differentiated cells cultivated with adding nothing. There were difference in the quantificational analyse of immunohistochemical staining of typeⅡcollagen and the statistic analyse.Results :Induced by Rhzizoma drynariae, the fibroblast-like MSCs turned to be triangular or polygonal in shape, some turned to be round.,most cells grow well. Cells were positive for immunohistochemical staining of collagen typeⅡrespectively after 2 weeks. Lots of cells stained, the staining in cytoplasm, and the staining very week in the cell clearance .Affected by Radix rehmanniae preparata, the fibroblast-like MSCs retained triangular or polygonal in shape. Cells propagated rapidly, but cells were positive for immunohistochemical staining of collagen typeⅡafter 2 weeks.The quantificational analyse of immunohistochemical staining of typeⅡcollagen and the statistic analyse: There were obvious difference between in the cells cultivated by adding traditional medicine Rhzizoma drynariae and that cultivated by adding nothing; There were not obvious difference in the cells cultivated by adding traditional medicine Radix rehmanniae preparata and that cultivated by adding nothing.Conclusion:Radix rehmanniae preparata can not induce the expression of typeⅡcollagen. Radix rehmanniae preparata cannot induce bone marrow derived mesenchymal stem cells into chondrocytes. The cells showed negative staining of collagen typeⅡ.Rhzizoma drynariae can induce the expression of collagen typeⅡ, Rhzizoma drynariae can induce bone marrow derive mesenchymal stem cells into chondrocytes.The optimal dosage of that Rhzizoma drynariae induced bone marrow mesenchymal stem cells into chondrocyte was 0.02ug/5000cells.The traditional medicine Rhzizoma drynariae can be one inducer that induced bone marrow mesenchymal stem cells into chondrocytes.