人骨形态发生蛋白-2基因转染人脂肪源性基质细胞的异位成骨作用  

Ectopic bone formation of hBMP-2 gene transfected human adipose tissue-derived stromal cells

作　　者：李慧武[1] 戴尅戎[1] 汤亭亭[1] 郁朝锋[1] 严孟宁[1] 李旭[1] 楼觉人[2] 

机构地区：[1]上海第二医科大学附属第九人民医院骨科 [2]美国华盛顿大学医学院骨科研究室

出　　处：《中华实验外科杂志》2005年第5期588-590,i003,共4页Chinese Journal of Experimental Surgery

基　　金：上海市教委科技资助项目(01JC14028);科技攻关项目(024119016)

摘　　要：目的探讨腺病毒介导的人骨形态发生蛋白-2(Adv-hBMP-2)基因转染人脂肪源性基质细胞(ADSCs)的可行性及其成骨作用。方法从人脂肪组织中提取间充质细胞,分别转染β-半乳糖苷酶(β-gal)基因和hBMP-2基因,通过X-gal染色明确转染效率,免疫沉淀+Westernblot法和ELISA法检测BMP-2表达,确定BMP-2表达量、表达时间及其相互关系,流式细胞仪观察基因转染对细胞凋亡的影响,并将转基因细胞注入裸鼠股部肌内行X线及组织学检查。结果X-gal染色显示转染效率达91%。免疫沉淀+Westernblot法和ELISA法显示转染hBMP-2的ADSC具有较高且稳定的BMP-2的表达,20d内表达量无明显减退。流式细胞表明腺病毒转染后细胞凋亡率上升,但上升幅度不大。裸鼠肌内注射转基因细胞后2周即显示有异位骨形成,4周明显增多,对照组无骨组织形成。结论ADSC是较好的基因治疗载体细胞,转染hBMP-2后可以诱导裸鼠体内骨形成。Objective To investigate the osteogenic potential of adipose tissue-derived stromal cells (ADSCs) exposed to adenovirus containing hBMP-2 cDNA (Adv-hBMP-2) and provide an optimal cell source for gene therapy.Methods ADSCs were obtained from patients who received orthopaedic surgery or liposuction.Human adipose tissue underwent proteolytic digestion to obtain ADSCs.After exposure to Adv-hBMP-2,immunoprecipitation and Western blotting and ELISA were performed for confirming BMP gene transduction.The apoptosis were observed by flow cytometry.ADSCs -Adv-hBMP-2 cells were injected into the hind limb of nude mice.Radiographic and histological evalution were performed on schdule to comfirm the bone formation.Results ADSCs were successfully isolated from human adipose tissue.The transfection efficiency was examined by X-gal staining.Western blotting and ELISA confirmed successful and persistent BMP-2 production by ADSCs -Adv-hBMP-2 cells.The flow cytometry showed there was some negative effect on apoptosis of ADSCs after exposed to adenovirus containing cDNA,but the rate of apoptosis is not so high that compromised the treatment.X-ray and HE sections from hind limb of nude mice subject to injection of Adv-hBMP-2 cells confirmed bone formation at 2nd week.Conclusion Adipose tissue contains abundant ADSCs that can be transfected with BMP-2 gene,with ectopic bone formation.ADSCs may be an ideal source of cells for gene therapy.

关 键 词：人骨形态发生蛋白-2 基因转染 成骨作用 基质细胞 源性 ELISA法检测 HBMP-2 BMP-2基因 β-半乳糖苷酶 X-gal BLOT法 转基因细胞 免疫沉淀 转染效率 ADSC 腺病毒介导 间充质细胞 流式细胞仪 组织学检查 细胞凋亡率 异位骨形成 

分 类 号：R346[医药卫生—基础医学]