骨关节炎患者滑膜细胞对于成骨细胞骨向分化的影响及机制研究  

Effects and significance of miR-21 derived from osteoarthritis synovial cells on osteoblast in vitro.

作　　者：陈铿[1] 方浩[1] 徐苏洋[1] 徐天阳 CHEN Keng;FANG Hao;XU Su-yang(Department of Orthopedics,Shanghai Eighth People's Hospital,Shanghai 200233,China.)

机构地区：[1]上海市第八人民医院骨科,上海200233

出　　处：《临床和实验医学杂志》2020年第12期1303-1307,共5页Journal of Clinical and Experimental Medicine

基　　金：上海市公共卫生项目(编号:SHDC2015034)。

摘　　要：目的探索骨关节炎患者滑膜细胞对成骨细胞骨向分化的影响及其相关机制。方法取骨关节炎患者滑膜组织及正常滑膜组织,采用酶消化法分离培养滑膜细胞,并传代,取第2代滑膜细胞和成骨细胞进行实验。首先将滑膜细胞与成骨细胞在体外进行共培养,实验分为两组,取P2代骨关节炎患者滑膜细胞与成骨细胞transwell共培养作为实验组(A组),正常滑膜细胞与成骨细胞transwell共培养作为对照组(B组),共培养36 h后,利用CCK-8法检测成骨细胞存活率,实时荧光定量PCR检测成骨细胞miR-21表达。然后,体外培养成骨细胞,构建过表达miR-21的重组慢病毒表达载体并转染入成骨细胞,实验分为两组,转染对照组(C组),miR-21转染组(D组),体外培养7 d后,实时荧光定量PCR检测成骨细胞miR-21表达以及成骨相关基因骨钙素(OCN)、RUNX2及I型胶原(Collagen I)mRNA表达,茜素红S法检测钙离子吸光度,ELISA法检测碱性磷酸酶(ALP)活性,western blot法检测OCN、RUNX2蛋白表达。结果共培养36 h后,A组与B组成骨细胞存活率无显著差异[(98.21±1.07)%vs.(96.14±0.93)%,P>0.05],而A组miR-21表达量明显高于B组(4.61±0.47 vs.0.32±0.11,P<0.05);体外培养7 d后,D组miR-21表达量明显高于C组(11.02±0.97 vs.0.41±0.08,P<0.05),而D组OCN、RUNX2及Collagen I基因表达显著低于C组(0.33±0.06 vs.1.04±0.27,0.27±0.03 vs.0.99±0.12,0.73±0.08 vs.1.05±0.08,P<0.05),D组钙离子吸光度和ALP活性显著低于C组(P<0.05),此外,D组OCN、RUNX2蛋白相对表达量均明显低于C组(1.02±0.16 vs.15.66±1.37,1.03±0.11 vs.8.47±0.94,P<0.05)。结论骨关节炎成骨细胞内miR-21水平呈高表达,而成骨细胞存活率却未受明显影响。骨关节炎患者滑膜细胞miR-21可抑制成骨细胞的矿化作用。Objective To explore the effect of microRNA-21(miR-21)derived from osteoarthritis synovial cells on osteoblast,and to study the relationship between miR-21 and osteoblast differentiation in vitro.Methods Synovial cells were isolated by enzyme digestion method from osteoarthritis synovial tissue and normal synovial tissue.P2 of synovial cells and osteoblast were used for transwell co-culture experiment.Synovial cells from osteoarthritis synovial tissue were co-cultured with osteoblast as Group A,and normal synovial cells were co-cultured with osteoblast as Group B.After co-cultured for 36 hours,cell activity was valued by CCK-8 method,besides,miR-21 expression was detected via real-time PCR.Then,osteoblast was cultured in vitro and transferred with lenti-microRNA-21 by DNA transfection as the test group(group D),osteoblast cultured with DMEM medium was regarded as control group(group C).After cultred for 7 days,miR-21,as well as osteocalcin(OCN),RUNX2,Type I collagen(Collagen I)expression were detected by real-time PCR.Besides,the content of calcium by Alizarin red S method and the activity of alkaline phosphatase(ALP)were detected via elisa.Meanwhile,the protein expression of OCN and RUNX2 were valued by western blot.Results After co-cultured for 36 hours,there is no significant difference of cell activity between group A and group B(P>0.05),while,miR-21 expression in group A was markedly higher than that in group B(P<0.05).After cultred for 7 days,miR-21 expression in group D was markedly higher than that in group C(P<0.05),while,the mRNA expression of OCN,RUNX2 and collagen I in group D were significantly lower than that in group C(P<0.05).Meanwhile,the content of calcium and the activity of ALP in group D were significantly lower than that in group C(P<0.05).What was more,the protein expression of OCN and RUNX2 in group D were markedly lower than that in group C(P<0.05).Conclusion Synovium-derived MicroRNA-21 showed inhibitory effect on osteoblast mineralization.

关 键 词：骨性关节炎 MICRORNA-21 滑膜细胞 成骨细胞 矿化作用 

分 类 号：R684.3[医药卫生—骨科学]