骨形态发生蛋白9基因修饰人羊膜间充质干细胞体外向韧带成纤维细胞分化研究

Effects of Bone Morphogentic Protein 9 Gene Modification on Differentiation of Human Amniotic Mesenchymal Stem Cells into Ligament Fibroblast Cells in vitro

目的:探讨骨形态发生蛋白9(BMP9)基因修饰对人羊膜间充质干细胞(h AMSCs)在体外向韧带成纤维细胞(LFs)分化是否有促进作用,并研究其分子机制。方法:体外分离培养h AMSCs及LFs,观察并比较两种细胞形态学差异。经Ad MAX系统体外构建BMP9基因腺病毒载体,经提纯及荧光法病毒滴度测定后转染P3代h AMSCs。实验分为三组,A组:携带绿色荧光蛋白(GFP)的空腺病毒载体转染h AMSCs(h AMSCs-GFP);B组:携带BMP9基因的腺病毒载体转染h AMSCs(h AMSCs-BMP9);C组:韧带成纤维细胞组(LFs)。于体外分别培养7天后使用荧光定量PCR检测并比较各组韧带相关基因Scleraxis(Scx)、Ⅰ型胶原(ColⅠ)、细胞连接素(TNC)、纤维连接蛋白(Fib)及腱调蛋白(Tnmd)m RNA的表达量。使用免疫荧光检测并比较三组细胞体外培养7天后ColⅠ的表达量。结果:在倒置相差显微镜下,h AMSCs原代呈多角形贴壁生长,传代后呈长梭状漩涡状生长。LFs原代呈长梭状贴壁生长,传代后呈漩涡状集落样生长。A、B组细胞转染8 h、24 h后,生长增殖缓慢,轮廓清晰,细胞形态未发生变化。荧光显微镜观察显示A组在转染8 h后可见GFP荧光表达,24 h后表达荧光的数量和强度均增多。实时荧光定量PCR显示,转染7天时B组SCX、Tnmd、ColⅠ、Fib及TNC的表达量均高于A组(P<0.05),而SCX、Fib、TNC及Tnmd的表达量低于C组(P<0.05),ColⅠ的表达量高于C组(P<0.05)。荧光免疫组化显示,转染7天后,B组ColⅠ的表达量均高于A组和C组。结论:BMP9基因可促进h AMSCs向LFs定向分化,并促进韧带特异性基因的表达和细胞外基质的产生,为h AMSCs作为韧带组织工程种子细胞提供了实验基础。

Objective To investigate the effect of bone morphogentic protein 9（BMP9） gene modifica-tion on the differentiation of human amniotic mesenchymal stem cells（h AMSCs） into ligament fibroblastcells（LFs） in vitro. Methods HAMSCs and LFs were isolated and cultured in vitro, and observed fortheir morphological differences using the inverted phase contrast microscopy（IPCM）. The BMP9 geneadenovirus was constructed using Ad MAX system and purified in vitro. After the virus titer was mea-sured under the fluorescence microscope, BMP9 gene was introduced into h AMSCs at passage 3 viap Double Ex-EGFP-BMP9 plasmid. Then h AMSCs transfected by green fluorescent protein-carrying adenovirus vector（h AMSCs-GFP） were selected into group A, those transfected by BMP9-carrying adenovirus（h AMSCs-BMP9） were chosen into group B, while LFs were assigned into group C. The m RNAs ofthe ligament-related genes including Scleraxis（Scx）, Collagen type I（Col I）, Tenascin-C（TNC）, Fibro-nectin（Fib） and Tenomodulin（Tnmd） were measured for each group after 7-day culture in vitro usingthe real-time fluorescence quantitative PCR. The expression of Col I was also measured using the im-munofluorescence. Results IPCM showed the round or polygon-shaped h AMSCs at primary or early cul-ture, and spindle-shaped spiral growth after passage. However, primary LFs were spindle-shaped, andshowed spiral colony-like growth after passage. No morphological change was observed in the BMP9-transfected h AMSCs at 8 hours and 24 hours after the transfection. Positive GFP expression was foundin group A 8 hours after transfection by fluorescence microscopy, with increased quantity and intensityat 24 hours after transfection. On Day 7 after transfection, the real-time fluorescent quantitative PCRshowed that the m RNA expressions of SCX, Tnmd, Fib and TNC of group B were significantly higherthan group A（P〈0.05） but lower than group C（P〈0.05）, while the m RNA expression of ColⅠwas sig-nificantly higher than group A and C（P〈0.05 for both）. Fluorescence immunohistochemistry showed sig-nificantly higher Col I expression in group B than the other groups 7 days after the transfection（P〈0.05）. Conclusion BMP9 can facilitate the differentiation of h AMSCs into ligament cells and enhancethe expression of ligament-related genes and extracellular matrix, suggesting the potential application ofh AMSCs in the ligament tissue engineering.