摘要
目的利用人骨形态发生蛋白-7（hBMP-7）腺病毒表达载体（Ad—hBMP-7）转染大鼠骨髓基质干细胞（BMSC）的方法，观察Ad—hBMP-7转染后对BMSC分化的影响，探讨基因治疗的可能性。方法获取Wistar大鼠BMSC，并分为3组：Ad—hBMP-7转染组、Ad-绿色荧光蛋白（Ad—GFP）转染组、未转染组。将Ad—hBMP-7和Ad—GFP的病毒液分别转染体外培养的大鼠BMSC，用RT～PCR法检测hBMP-7mRNA的表达-7d后用Westernblot法检测hBMP-7的蛋白表达，同时进行碱性磷酸酶（ALP）染色。结果在Ad—hBMP-7转染组，RT—PCR可检测到hBMP-7mRNA的表达（470bp），Westernblot可检测到hBMP-7蛋白分泌（相对分子质量为15X10。），转染后第10天ALP染色多数细胞胞浆呈棕黑色阳性着色；而Ad—GFP转染组和未转染组ALP染色阳性细胞少见并且未检测到hBMP-7蛋白分泌。结论Ad—hBMP-7基因可高效转染BMSC，促进BMSC向成骨细胞转化，为利用hBMP-7开展局部基因治疗的研究奠定了基础。
Objective To investigate the expression of human bone morphogenetic protein-7（hBMP-7） in rat bone marrow stromal cells （BMSC） with a recombinant adenoviral vector carrying the hBMP-7 gene （Ad-hBMP-7） and study the effeets of Ad-hBMP-7 transfection on BMSC differentiation, in order to explore the possibility for hBMP-7 gene therpy. Methods The rat BMSC cultured in vitro. They were divided into 3 groups： untreated group, Ad-hBMP-7 and Ad-GFP transduced treated group. The rat BMSC were transfected by Ad-hBMP-7 and Ad-GFP. The expression of hBMP-7 was detected by RT-PCR and Western blot analysis, and the alkaline phosphatase （ALP） activity of the BMSC was observed. Results In the Ad-hBMP-7 transduced treated group, hBMP-7 mRNA expression was manifested detected by RT-PCR （470 bp）, Western blot analysis demonstrated that these cells indeed produced the hBMP-7 protein（Mr, 15 × 10^3）; 10 days after transduction treatment, most of the BMSC were had brown black particles stained positively by ALP activity. But in Ad-GFP transduced treated group and untreated group they did not. Conclusions Ad-hBMP-7 could efficiently transfect BMSC and promote the conversion to osteoblast. The expression of hBMP-7 in rat BMSC provides a basis for hBMP-7 gene therapy.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2009年第1期44-47,共4页
Chinese Jouranl of Endemiology
基金
基金项目：山东省优秀中青年科学家奖励基金（2006BS03062）
