骨形态发生蛋白2基因转染诱导异位成骨的机制研究

目的观察骨形态发生蛋白2基因直接及间接转染诱导异位成骨的效果和机制。方法1.将携带骨形态发生蛋白-2基因的腺病毒载体(Ad-BMP-2)直接注射到裸鼠肌内诱导成骨;2.Ad-BMP-2体外转染人骨髓间质干细胞(hBMSC)后种植异种骨支架(BCB)植入裸鼠皮下。实验分为5组:①BMP-2基因转染细胞 BCB;②对照基因转染细胞 BCB;③未转染细胞 重组BMP-2 BCB;④未转染细胞 BCB;⑤BCB。术后行X线、免疫组化及原位杂交染色观察BMP-2基因诱导下的成骨方式及成骨机制。结果1.裸鼠肌内注射后有异位成骨,其主要为软骨化骨。2.Y染色体探针荧光原位杂交法(FISH)示踪植入细胞命运,见部分转基因细胞直接参与成骨;骨钙素免疫组化检测见基因表达产生的BMP-2诱导宿主间质细胞向成骨细胞转化。结论BMP-2基因转染的hBMSC不仅作为运载生长因子的工具,而且直接参与成骨,但BMP-2的诱导趋化作用占主导地位。

Mechanism of heterotopic osteogenesis induced by adenovirus mediated BMP-2 gene transfection

Objective To evaluate the mechanism of heterotopic osteogenesis induced by adenovirus mediated BMP-2 gene (Ad-BMP-2) transfection. Methods (1) Ad-BMP-2 was directly injected into the muscle of nude mice to induce bone formation; (2) Human mesenchymal stem cells (hBMSC), after being transfected by Ad-BMP-2, were implanted subcutaneously into the back of the nude mice in five groups respectively, ie. Ad-BMP-2 infected hBMSC plus antigen-free bovine cancellous bone (BCB)(Group A), hBMSC-BCB plus reconstructed hBMP-2 (Group B), Ad-LacZ infected hBMSC-BCB (Group C), hBMSC- BCB (Group D) and only BCB scaffolds (Group E). X-ray and histological examination were conducted after operation. Results (1) Heterotopic osteogenesis was found after intramuscular injection in nude mice, most of which were endochondral ossification. (2) Implanted cell fates were traced by Y chromosome fluorescence in situ hybridization and some transduced gene cells, direct involvement in bone formation was observed; host mesenchymal cells, conversion into osteoblasts induced by BMP-2 from gene expression was displayed by osteocalcin immunohistochemical staining analysis. Conclusions hBMSC infected by BMP-2 gene not only is used as the tool of growth factor delivery, but also directly involve in bone formation. However, the induction of BMP-2 is in the leading place.