牛膝总皂苷含药关节液对骨关节炎体外软骨细胞增殖及凋亡的实验研究

The Experimental Research of Medicated Joint Fluid of Total Saponins of Achyranthes from Experimental Rabbit Model of OA for Cartilage Cell Proliferation and Apoptosis

目的:观察牛膝总皂苷(TSA)含药关节液对兔骨关节炎(OA)体外软骨细胞增殖及凋亡的影响。方法:将30只兔分为TSA组、硫酸氨基葡萄糖组、空白对照组,每组10只,分别予TSA、硫酸氨基葡萄糖、蒸馏水灌胃7d后制备含药关节液;进行OA造模,鉴定OA造模成功后体外提取关节软骨细胞进行培养、鉴定。将P3代软骨细胞分为TSA组、硫酸氨基葡萄糖组、空白对照组、经典软骨增殖组,分别用10%TSA含药关节液+10%FBS+DMEM∶F12(1∶1)培养液、10%硫酸氨基葡萄糖含药关节液+10%FBS+DMEM∶F12(1∶1)培养液、10%空白含药关节液+10%FBS+DMEM∶F12(1∶1)培养液、10%FBS+DMEM∶F12(1∶1)培养液进行培养,对培养后的软骨细胞进行细胞形态学观察,用CCK-8法检测细胞增殖情况,免疫荧光染色检测Ⅱ型胶原蛋白表达,Annexin V-PE/7-AAD双染流式细胞仪检测细胞凋亡率。结果:OA软骨细胞体外分离成功,经甲苯胺蓝染色鉴定细胞为软骨细胞来源;CCK-8法观察四组细胞生长曲线,绘制的生长曲线符合Logistic生长曲线规律,TSA组在第2～7天细胞增殖速度明显高于其它3组,差异有统计学意义(P<0.05);连续培养7d后,与空白对照组比较,TSA组Ⅱ型胶原蛋白表达显著,差异有统计学意义(P<0.01),硫酸氨基葡萄糖组、经典软骨增殖组Ⅱ型胶原蛋白表达明显,差异有统计学意义(P<0.05);流式细胞仪检测显示,与空白对照组(总凋亡率为17.61%±1.19%)比较,经典软骨增殖组(总凋亡率为16.50%±1.42%)细胞凋亡减少不明显,差异无统计学意义(P>0.05)。与空白对照组比较,TSA组(总凋亡率为5.15%±1.96%)、硫酸氨基葡萄糖组(总凋亡率为7.76%±1.72%)细胞凋亡减少明显,差异有统计学意义(P<0.01)。结论:TSA含药关节液能有效提高细胞活力、促进软骨细胞增殖、提高Ⅱ型胶原蛋白表达,且降低软骨细胞早期、晚期凋亡率,对证实TSA是牛膝作用于软骨细胞的主要物质基础具有重要意义。

Objective:To observe the effect of total saponins of achyranthes(TSA)containing pharmaceutical articular fluid on the proliferation and apoptosis of chondrocytes in vitro in the OA model of experimental rabbits.Methods:30 experimental rabbits were divided into TSA group,glucosamine sulfate group and blank control group,10 of which were treated with TSA glucoside,glucosamine sulfate and distilled water respectively for 7 d.After the successful modeling of OA model,articular chondrocytes were extracted in vitro for culture and identification.P3 generation chondrocytes were divided into four groups:TSA group,glucosamine sulfate group,blank control group and classic chondrogenic prolifer-ation group were cultured with 10% TSA drug-containing joint fluid+10%FBS+DMEM∶F12(1∶1)medium,10% glucosamine sulfate drug-containing joint fluid+10%FBS+DMEM∶F12(1∶1)medium,10% blank drug-containing joint fluid+10%FBS+DMEM∶F12(1∶1)medium,10%FBS+DMEM∶F12(1∶1)medium.To cultivate the cartilage cell to cell morphological observation,after using CCK-8 cases,immunofluorescence staining method to detect cell proliferation test typeⅡcollagen expression,Annexin V-PE/7-AAD double flow cytometry to detect cell apoptosis rate.Results:OA chondrocytes were isolated successfully in vitro,and the cells were identified as chondrocytes by toluidine blue staining.The growth curves of the cells in the four groups were observed by the cck-8 method,and the growth curves were consistent with the Logistic growth curves.The proliferation rate of cells in the TSA group was significantly higher than that in the other three groups on the 2 nd to 7 th day(P<0.05).Continuous culture after 7 d,compared with blank control group,the TSA Ⅱtype collagen expression significantly(P<0.01),glucosamine sulfate group,classic cartilage proliferation typeⅡcollagen expression obviously(P<0.05).Flow cytometry showed that compared with the blank control group(total apoptosis rate:17.61%+1.19%),apoptosis was not significantly reduced in the classic chondrogenic proliferation group(total apoptosis rate:16.50%+1.42%)(P>0.05).Compared with the blank control group,TSA group(total apoptosis rate:5.15%+1.96%)and glucosamine sulfate group(total apoptosis rate:7.76%+1.72%)showed significant decrease in apoptosis(P<0.01).Conclusion:The TSA medicated joint fluid can effectively improve the cell vitality,promote cartilage cell proliferation,improve typeⅡcollagen protein expression,and decrease the rate of early and late apoptosis of cartilage cells,to confirm the TSA is applied to chondrocytes cultured the main material foundation has an extremely important theoretical significance,its mechanism needs further exploration.