旋转微重力细胞培养系统下Indianhedgehogd转染兔BMSCs促进成软骨分化并抑制老化的实验研究  

EFFECT OF Indianhe dgehog GENE TRANSFECTION INTO RABBIT BONE MARROW MESENCHYMAL STEM CELLS IN PROMOTING CHONDROGENIC DIFFERENTIATION AND INHIBITING CARTILAGE AGING IN ROTARY CELL CULTURE SYSTEM

作　　者：刘鹏程[1] 刘宽[1] 刘俊峰[1] 夏阔 陈礼阳 吴兴[1] 

机构地区：[1]同济大学附属上海市第十人民医院骨科,上海200072

出　　处：《中国修复重建外科杂志》2016年第7期892-902,共11页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金面上项目(81371950)~~

摘　　要：目的探讨模拟微重力环境下,Indianhedgehog(IHH)基因过表达对兔BMSCs成软骨分化的影响。方法取第2代兔BMSCs,分为旋转微重力细胞培养系统(rotary cell culture system,RCCS)组和传统组2个大组,每个大组进一步分为3个亚组,即IHH基因腺病毒载体转染组(RCCS 1组和传统1组)、绿色荧光蛋白腺病毒载体转染组(RCCS 2组和传统2组)及空白对照组(RCCS 3组和传统3组)。RCCS组细胞均在模拟微重力环境下进行成软骨细胞诱导分化;常规组在6孔板中进行常规细胞培养及成软骨细胞诱导分化。诱导分化过程中,检测IHH蛋白表达(ELISA法)及ALP活性;实时荧光定量PCR检测软骨及软骨肥大相关基因表达;Wertern blot法检测Ⅱ型胶原、聚集蛋白聚糖(aggrecan,ANCN)蛋白表达;并取细胞爬片行甲苯胺蓝染色及膜联蛋白V(Annexin V)-cy3免疫荧光染色观察。结果转染后荧光显微镜下RCCS 1、2组可见明显绿色荧光,转染效率约95%。ELISA检测示,RCCS及传统1组IHH蛋白均明显高于2、3组(P<0.05);传统1组各时间点ALP活性均高于传统2、3组(P<0.05),RCCS 1组ALP活性仅于诱导分化3、7 d稍高于RCCS 2、3组(P<0.05)。实时荧光定量PCR检测示,传统1组在诱导分化早期高表达Ⅱ型胶原、ANCN,但在后期高表达Ⅹ型胶原、ALP及AnnexinⅤ(P<0.05);RCCS1组在诱导分化各时期均高表达软骨相关基因Ⅱ型胶原、ANCN、SOX9,且低表达软骨肥大相关基因Ⅹ型胶原、ALP及AnnexinⅤ(P<0.05)。Wertern blot法检测示,诱导21 d时传统1组Ⅱ型胶原蛋白表达量明显低于传统2、3组(P<0.05);RCCS 1组各时间点Ⅱ型胶原、ANCN蛋白表达量均明显高于RCCS 2、3组(P<0.05)。甲苯胺蓝染色示,诱导21 d时传统1组染色浅于传统2、3组,RCCS 1组各时间点染色均明显深于RCCS 2、3组;Annexin V-cy3免疫荧光染色示,各时间点传统1组红色荧光均强于传统2、3组,RCCS各亚组红色荧光表达均较弱且组间无明显差异。结论在模拟微重Objective To investigate the effect of overexpressing the Indianhedgehog（IHH） gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells（BMSCs） in a simulated microgravity environment. Methods The 2nd generation BMSCs from rabbit were divided into 2 groups： the rotary cell culture system（RCCS） group and conventional group. Each group was further divided into the IHH gene transfection group（RCCS 1 group and conventional 1 group）, green fluorescent protein transfection group（RCCS 2 group and conventional 2 group）, and blank control group（RCCS 3 group and conventional 3 group）. RCCS group cells were induced todifferentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase（ALP） activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen type II, agreecan（ANCN） protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. Results After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups（P〈0.05）; at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups（P〈0.05）; ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days（P〈0.05）. Conventional 1 group expressed high levels of cartilage-related genes, such as collagen type II and ANCN at the early stage of differentiation induction, and expressed high level

关 键 词：BMSCS Hedgehog信号 Indianhedgehog 旋转微重力细胞培养系统 软骨组织工程 基因转染 

分 类 号：R68[医药卫生—骨科学]