腺病毒介导的骨形态发生蛋白-2基因转染脂肪间充质干细胞的实验研究

Experimental Studies of Human Adipose Tissue-derived Stromal Cells Transfected with Ad-hBMP-2 Gene

目的探讨腺病毒介导的人骨形态发生蛋白-(2Ad-hBMP-2)基因转染大鼠脂肪间充质干细胞(ADSCs)的可行性及其成骨特性。方法取4周龄SD大鼠腹股沟的脂肪组织,经体外分离培养的方法获得ADSCs,分别转染以腺病毒为载体的绿色荧光蛋白(Ad-GFP)基因和Ad-hBMP-2基因。用荧光显微镜每隔12h观察转染Ad-GFP的细胞,并确定转染率,观察其形态变化,绘制生长曲线;利用免疫细胞化学法对转染Ad-hBMP-2的细胞作碱性磷酸酶(ALP)染色,用免疫细胞化学法作骨钙素(OC)检测确定成骨活性,Westernblot法检测hBMP-2的表达。结果12h后荧光显微镜观察转染Ad-GFP的细胞,有52%的阳性表达细胞,48h后转染率达95%;转染Ad-hBMP-2后倍增时间延长,ALP、OC检测为阳性表达,hBMP-2蛋白转染后48h为阳性表达,并持续至第5代。结论ADSCs可以作为腺病毒转染的载体,转染Ad-hBMP-2基因后有明显的成骨作用,可以作为骨组织工程的种子细胞。

Objective To investigate the possibility of adipose-derived stromal cells （ADSCs） transfeced by adenovirus containing human bone morphogenetic protein-2 （Ad-hBMP-2） gene and their osteogenic potential. Methods ADSCs were obtained from inguinal fat tissue of 4 weeks old SD rats. After exposure to adenovirus containing green fluorescent protein（Ad-GFP）, fluorescent microscope was used to observe gene transfeetion effect once 12 hours. After transfeeted with Ad-hBMP-2, eytoehemistry, immueytochemistry and Western blot were used to examine the expression of alkaline pbosphatase （ALP）, osteocalcin （OC） and hBMP-2. Results After exposed to Ad-GFP 12 hours, 52% ADSCs were observed being transfected and 48 hours later reached 95%. The double number time belonged after transfeeting with Ad-hBMP-2, and eytochemistry, immueytoehemistry and Western blot examines indicated positive results of ALP, OC, hBMP-2 after 48 hours. Conclusion Adipose tissue contains abundant ADSCs which could be transfected as gene vectors by adenovirus, ADSCs transfeeted with Ad-hBMP-2 can convert to ostoeblasts, and can act as a kind of seed cells for osteo-tissue engineering.