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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(AnnexinV)-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、7d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Annexin V(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Annexin V(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21d时传统1组染色浅于传统2、3组,RCCS1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 在模拟微重力环境下,IHH基因转染BMSCs可有效促进软骨生成,并抑制软骨老化或向成骨发展,适合软骨组织工程的需要.“,”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 to differentiate 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 levels of cartilage hypertrophy-related genes,such as collagen type X,ALP,and Annexin V at the late stage (P＜0.05).RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages.The expression of collagen type Ⅱ protein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P＜0.05);RCCS 1 group expressed high levels of collagen type Ⅱ and ANCN proteins at all stages (P＜0.05).Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction;while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups.Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point.The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups.Conclusion Under the simulated microgravity environment,transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis.Therefore,this technique is suitable for cartilage tissue engineering.