牛膝总皂苷体外干预人膝骨关节炎软骨细胞Ⅱ型胶原表达及最佳浓度的实验研究  

In vitro intervention and optimal concentration of total saponins from Achyranthis Bidentatae Radix on the expression of type II collagen in chondrocytes of human knee osteoarthritis

作　　者：陈浩雄 霍少川 马笃军[2] 徐启良 黄学成 林新珍 徐宁达 CHEN Haoxiong;HUO Shaochuan;MA Dujun;XU Qiliang;HUANG Xuecheng;LIN Xinzhen;XU Ningda(Shenzhen Hospital of Guangzhou University of Chinese Medicine(Futian),Shenzhen 518034,Guangdong,China;Shenzhen Hospital of Traditional Chinese Medicine,Shenzhen 518033,Guangdong,China)

机构地区：[1]广州中医药大学深圳医院(福田),广东深圳518034 [2]深圳市中医院,广东深圳518033

出　　处：《湖南中医杂志》2021年第7期141-144,共4页Hunan Journal of Traditional Chinese Medicine

基　　金：广东省中医药管理局项目(20191295)。

摘　　要：目的:观察牛膝总皂苷(TSA)体外干预人膝骨关节炎软骨细胞增殖及Ⅱ型胶原(CollagenⅡ)表达水平及最佳浓度。方法:将人膝骨关节炎软骨细胞进行分离、培养、鉴定,并将其分为6组,分别为0、0.01、0.05、0.1、0.5、1 mg/ml组。制备TSA存储液及6组不同TSA浓度DMEM/F12(D/F12)培养基,分别用6组培养基对P3代软骨细胞培养并进行细胞形态学观察;采用CCK-8法检测软骨细胞P1、P2、P3传代增殖情况,观察其生长曲线;采用Western-blot法检测P3代软骨细胞Collagen II蛋白表达、RT-PCR法检测CollagenⅡmRNA表达。结果:软骨细胞体外分离并培养成功,经甲苯胺蓝染色鉴定细胞为软骨细胞来源;CCK-8法绘制的生长曲线符合Logistic生长曲线规律,P3代软骨细胞增殖速度高于P1、P2代;Western-Blot法检测结果显示,Collagen II蛋白表达量浓度为0.5 mg/ml时达最高,RT-PCR法检测结果显示,各组浓度AST能促进软骨组织Ⅱ型胶原mRNA表达上调,以0.5 mg/ml组最明显(P<0.05)。结论:TSA能体外有效促进软骨细胞增殖和Ⅱ型胶原mRNA表达上调,以0.5 mg/ml浓度最明显,具有促进软骨修复的作用,具体作用机制有待进一步研究。Objective: To investigate the in vitro intervention and optimal concentration of total saponins from Achyranthis Bidentatae Radix(TSA) on the expression of type II collagen(Collagen II) in chondrocytes of human knee osteoarthritis.Methods: Chondrocytes of human knee osteoarthritis were isolated, cultured, identified, and then divided into 0,0.01,0.05,0.1,0.5,and 1 mg/ml groups.TSA stock solution and DMEM/F12 medium with the above six concentrations of TSA were prepared for the culture of P3 chondrocytes, and cell morphology was observed;CCK-8 assay was used to observe the passage and proliferation of P1,P2,and P3 chondrocytes and their growth curves;Western blot was used to measure the protein expression of collagen II in P3 chondrocytes, and RT-PCR was used to measure the mRNA expression of collagen II.Results: Chondrocytes were successfully isolated and cultured in vitro, and the origin of cells was identified as chondrocytes by toluidine blue staining.The growth curve plotted based on CCK-8 assay was consistent with the logistic growth curve, and P3 chondrocytes had a higher proliferation rate than P1 and P2 chondrocytes.Western blot showed the highest protein expression of collagen II at the concentration of 0.5 mg/ml, and RT-PCR showed that TSA at each concentration promoted the upregulation of the mRNA expression of collagen II,with the most significant effect at the concentration of 0.5 mg/ml(P<0.05).Conclusion: TSA in vitro can effectively promote the proliferation of chondrocytes and the upregulation of the mRNA expression of collagen II,with the most significant effect at the concentration of 0.5 mg/ml, and it can also promote cartilage repair.Further studies are needed to clarify its mechanism of action.

关 键 词：膝骨关节炎 软骨细胞 牛膝总皂苷 体外干预 实验研究 

分 类 号：R285.5[医药卫生—中药学]