Effect Of Weilingxian On Proliferation And Collagen Type Ⅱ,TGF-β1mRNA Expression Of Rabbit Knee Articular Chondrocytes Cultured In Vitro

Objective:Observe the effect of weilingxian on rabbit knee articular chondrocytes proliferation and collagen typeⅡ,TGF-β1mRNA expression,and then explore the effect and possible mechanism of single Chinese medicine on cartilage damage.Methods:Chondrocytes were isolated and harvested from knee articular of New Zealand rabbits,then the phenotype was identified by methods of toluidine blue and immunocytochemical staining;cells of third generation in logarithmic growth phase were randomly divided into two groups,experimental groups were cultured in DMEM with concentration of 0.01,0.05,0.1,0.5,1.0mg/ml Weilingxian,while control group was given normal medium.MTT assay method was adopted to observe proliferation of chondrocytes,immunohistochemistry staining method was employed to detect the expression of collagen typeⅡ,method of RT-PCR was used to evaluate the change of TGF-β1mRNA expression under the intervention of Weilingxian.Results:①Primary cultured chondrocytes was round-shaped,and most adhered after 24h,the appearance was polygonal and irregular-shaped; About 10 days, chondrocytes growed and fused into pieces,the typical appearance was slabstone-like;chondrocytes growed faster after passage, long spindle-shaped chondrocytes appeared after four generations;after six generations,most cells showed long spindle-shaped fibroblast-like appearance,and the rate of growth also slowed down.Extracellular matrix of chondrocytes was stained to be blue by toluidine blue staining,while nucleus was dark blue.The typeⅡcollagen was positive expressed with red-brown particles visible well in the cytoplasm of chondrocytes.②All Weilingxian groups can promote proliferation of chondrocytes,effect of 0.5mg/ml group was significant especially,and the peak of proliferation occured at the 3 day.③The typeⅡcollagen was positive expressed in the cytoplasm of chondrocytes under the intervention of Weilingxian.Chondrocytes in 0.1mg/ml,0.5mg/ml groups were better than other groups on the aspects of typeⅡcollagen positive expression area,absorption value of optical density,rate of positive expression, there were significant differences(P<0.05),while compared with the control group;and there was extreme significant difference between 0.5mg/ml group and the others on the aspects of typeⅡcollagen positive expression area(P<0.01).④0.05,0.1,0.5,1.0mg/ml Weilingxian can promote TGF-β1mRNA expression,and there was no significant difference between four groups (P>0.05),but the 0.5mg/ml group had the best effect.Conclusion: Weilingxian can promote proliferation,collagen typeⅡand TGF-β1mRNA expression of chondrocytes,these may be one of the mechanisms that Weilingxian can work in the treatment of cartilage damage.