活血通络药物对膝骨关节炎小鼠凋亡软骨细胞体外干预的影响  

An observation of the effect of extraorgan medicine intervention on chondrocyte apoptosis of mice with knee osteoarthritis

作　　者：韦佳佳 廖建青 张英杰 王琦 WEI Jiajia;LIAO Jianqing;ZHANG Yingjie;WANG Qi(The First Affiliated Hospital of Yunnan University of Chinese Medicine,Kunming 650021,China;Yunnan University of Chinese Medicine,Kunming 65050,China)

机构地区：[1]云南中医药大学第一附属医院,昆明650021 [2]云南中医药大学,昆明650500

出　　处：《吉林中医药》2021年第12期1636-1640,共5页Jilin Journal of Chinese Medicine

基　　金：国家自然科学基金-地区基金项目(81560781)

摘　　要：目的体外培养膝骨关节炎(knee osteoarthritis,KOA)小鼠凋亡软骨细胞模型,对凋亡软骨细胞模型进行形态学观察和鉴定,传代培养第一代凋亡软骨细胞模型。培养成功后,分别以不同浓度的骨痹合剂以及氨糖美辛.生理盐水干预后,进行形态学观察和绘制细胞生长曲线,确定凋亡软骨细胞变化最明显的时间点以及促进凋亡软骨细胞增殖的最佳给药浓度,观察活血通络药物对凋亡软骨细胞的影响。方法取SPF级小鼠80只,20只用于软骨细胞取材,60只用于含药血清制备。首先取正常小鼠、KOA造模小鼠各10只,处死后取软骨细胞,对原代细胞进行传代培养,培养成功后对小鼠正常软骨细胞和小鼠KOA退变软骨细胞进行形态特征染色观察以及细胞比较鉴定。取SPF小鼠60只,随机分为骨痹合剂组、氨糖美辛组、生理盐水组,每组20只,分别以3种药物灌胃3d后取含药血清保存备用。以第一代软骨细胞,加入5%、10%、20%不同浓度骨痹合剂、氨糖美辛、生理盐水含药血清进行细胞干预,2、4、6、8 d后,同时观察3组血清不同药含药浓度分别对凋亡软骨细胞模型增殖的影响。结果由甲苯胺蓝染色后发现,正常软骨细胞以及KOA软骨细胞均成功培养及传代。检测后发现,KOA模型分离出的软骨细胞凋亡程度较高。经含药血清干预后发现,骨痹合剂和氨糖美辛均抑制细胞凋亡,促进细胞增殖,对细胞起保护作用。经MTT检测后发现骨痹合剂组中,10%含药血清相对于5%和20%含药血清浓度更加促进细胞增殖,说明10%含药血清浓度相对为最佳浓度,2d的时间点各组增殖变化最为明显。结论10%的含骨痹合剂血清相较其余组别可更好地抑制小鼠KOA软骨细胞凋亡,促进软骨细胞增殖。Objective To observe the effect of extraorgan medicine intervention on chondrocyte apoptosis of mice with knee osteoarthritis by using drugs that act to promote the blood circulation for removing obstructions in collaterals.Methods A total of 20 SPF mice were used for chondrocyte extraction,and sixty for medicated serum preparation.Firstly,10 normal mice and 10 KOA mice were sacrificed and their chondrocytes were taken for subculture of primary cells.After it,the morphological characteristics of the mice normal chondrocytes and KOA degenerative chondrocytes were observed by staining and cell comparison and identification.A total of 60 SPF mice were randomly divided into Gubi mixture group,aminomethacin group and normal saline group,with 20 mice in each group,which were respectively gavaged with the three drugs for 3 days and their medicated serum was taken and stored for later use.The first generation chondrocytes were added with different concentrations of Gubi mixture,aminomethacin and normal saline medicated serum for cell intervention(5%,10%,20%).After 2.4,6 and 8 days,the effects of different concentrations in the medicated serum of the three groups on the proliferation of the apoptotic chondrocyte model were observed.Results As revealed by toluidine blue staining,normal chondrocytes as well as KOA chondrocytes were successfully cultured and passaged.After the detection,it was found that the chondrocytes isolated from the KOA model had a higher degree of apoptosis.After the intervention with medicated serum,it was found that both Gubi mixture and aminomethacin inhibited cell apoptosis,promoted cell proliferation and protected cells.After testing by MTT assay,it was found that the 10%medicated serum promoted cell proliferation more significantly than the 5%or 20%medicated serum,indicating that 10%medicated serum concentration was the best,and the proliferation change in each group was the most obvious at the time point of 2 days.Conclusion The 10%Gubi mixture serum can better inhibit mice KOA chondrocyte apop

关 键 词：软骨细胞 连续传代 骨痹合剂 中药 膝骨关节炎 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]