腺病毒介导的人骨形态发生蛋白-7基因转染大鼠骨髓基质干细胞及其表达

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,探讨基冈治疗的可能性.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-7 mRNA的表达,7 d后用Western blot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-7 mRNA的表达(470 bp),Western blot可检测到hBMP-7蛋白分泌(相对分子质量为15×103),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.

Transfection of rat bone marrow stromal cells with human bone morphogenetic protein-7 recombinant adenovirus and its expression

Objective To investigate the expression of human bone morphogenetic protein-7(hBMP-7)in rat bone malTOW stromal cells(BMSC)with a recombinant adenovirai vector carrying the hBMP-7 gene(Ad-hBMP-7) and study the effecta of Ad-hBMP-7 transfection on BMSC difierentiation.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 phosphatage (ALP)activity of the BMSC was observed.ResulIs In the Ad-hBMP-7 transduced treated group.hBMP-7 mRNA expression wag manifested detected by RT-PCR(470 bp),Westem blot analysis demonstrated that these cells indeed produced the hBMP-7 protein(Mr.15×103);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 therapv.