摘要
目的观察透明质酸(HA)复合腺病毒介导的人骨形态发生蛋白(AdvhBMP)2基因转染的兔骨髓基质干细胞(BMSCs)的体内外诱导成骨活性。方法(1)体外成骨活性研究方法分成4组,AdvhBMP2转染细胞+HA组、AdvhBMP2转染细胞组、未转染细胞+HA组和未转染细胞组,采用免疫沉淀法(IP)、Western印迹法检测hBMP2表达、逆转录聚合酶链反应(RTPCR)法检测骨涎蛋白mRNA表达、ALP和vonKossa染色及ALP定量检测;(2)裸鼠肌内诱导成骨实验裸鼠12只(共24侧)分4组注射AdvhBMP2转染细胞+HA组(n=8)、注射AdvhBMP2转染细胞组(n=8)、注射未转染细胞组(n=4)和单纯注射HA组(n=4),观察方法采用X线、成骨计量对照、组织学观察法。结果(1)AdvhBMP2转染细胞+HA组和AdvhBMP2转染细胞组BMSCs表达hBMP2和骨涎蛋白mRNA,两组的ALP活性均高于未转染细胞+HA组和未转染细胞组(P<0.01),并有明显的钙结节形成;(2)AdvhBMP2转染细胞+HA组和AdvhBMP2转染细胞组均有成骨,两组差异有显著性(P<0.01),未转染细胞和单纯注射HA组均未见成骨,局部以纤维和脂肪组织为主。结论HA复合AdvhBMP2基因转染的BMSCs在体外有较好的诱导成骨活性,在裸鼠肌内有良好的诱导成骨和成软骨的作用,是可注射性的骨缺损修复方法。
Objectives To evaluate in vitro and vivo osteogenetic effectiveness of hyaluronic acid mixed with adenovirus mediated human bone morphogenetic protein 2 gene (Adv hBMP 2) transferred bone marrow derived mesenchymal stem cells (BMSCs) of rabbit.Methods In the in vitro test,the BMSCs were collected from bone marrow of rabbits and cultured in vitro.There were 4 groups in the test:Ⅰ.Adv hBMP 2 transfected BMSCs+HA;Ⅱ.Adv hBMP 2 transfected BMSCs;Ⅲ.Untransfected BMSCs+HA:Ⅳ.Untransfected BMSCs.These BMSCs were evaluated by immunoprecipitation (IP)/Western blotting,RT PCR,ALP activity assay,and von Kossa staining.In in vivo test,12 node mice (24 sides) subject to the injection of the cultured cells into the muscle were divided into four groups:Ⅰ.Adv hBMP 2 transfected BMSCs+HA ( n =8);Ⅱ.Adv hBMP 2 transfected BMSCs ( n =8);Ⅲ.Untransfected BMSCs ( n =4);Ⅳ.HA ( n =4).The osteoinductivity in muscle of nude mice was investigated by X ray examination,osteogenesis quantification and histological examination.Results Results of in vitro test:hBMP 2 and bone sialoprotein mRNA were expressed,and the high ALP activities and formation of calcified nodes were detected in the groups of Adv hBMP 2 transfected BMSCs+HA and Adv hBMP 2 transfected BMSCs,both of which were more significant than those in the groups of untransfected BMSCs+HA and untransfected BMSCs;Results of in vivo test:Under X ray examination the ectopic bones in the muscles of nude mice were observed in the groups of Adv hBMP 2 transfected BMSCs+HA and Adv hBMP 2 transfected BMSCs,and the amount of ectopic bones in the groups of Adv hBMP 2 transfected BMSCs+HA Adv hBMP 2 transfected BMSCs was (155.0±11.5) mg and (83.4±13.5) mg respectively,with the difference being statistically significant ( P <0.01).Histological examination revealed that there were chondrogenesis and osteogenesis in the groups of Adv hBMP 2 transfected BMSCs+HA Adv hBMP 2 transfected BMSCs and it seemed that chondrogenesis in the former was more than that in the latter.There was no ec
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2004年第7期845-847,i001,i002,共5页
Chinese Journal of Experimental Surgery
基金
上海市教委科技资助项目(03979029)
国际合作项目(014307021)
