绿色荧光蛋白报告基因转染示踪体内骨髓间充质干细胞的分化

Tracing the differentiation of bone marrow mesenchymal stem cells in vivo by GFP labeling

目的用绿色荧光蛋白(GFP)标记技术,观察组织工程化骨中种子细胞的变化与转归。方法用腺病毒载体Adeno-XTM将GFP基因转染新西兰大白兔骨髓间充质干细胞(BMSCs),成骨诱导培养3周后将其接种于磷酸钙/纤维蛋白胶复合支架材料,构建成组织工程化骨,回植于供体兔皮下。术后4周,激光共聚焦显微镜下示踪观察GFP,测定碱性磷酸酶(ALP)活性,免疫组化检测Ⅰ型胶原、骨钙素(OCN)和HE染色观察组织结构。结果4周后,大体可见组织工程化新骨形成;组织学显示新生骨围绕材料孔隙生成,磷酸钙/纤维蛋白胶复合材料部分降解;ALP活性明显增高;Ⅰ型胶原、OCN免疫组化染色阳性;新生组织内见GFP标记细胞。结论组织工程化骨组织结构与松质骨小梁类似;新生组织表达GFP,证实组织工程化骨组织的形成来源于接种细胞。

Objective To generate tissue engineered bone with cultured bone marrow mesenchymal stem cells （BMSCs） and trace it by green fluorescent protein （GFP） gene transfer. Methods Recombinant Adeno-XTM/GFP expression vector was used to infect BMSCs obtained from rabbits directly after packed by HEK293 cells. GPF-labeled BMSCs were then seeded onto the compound scaffold of calcium phosphate cement （CPC） and fibrin glue （FG） to form a new kind of tissue engineered bone after cultured in conventional osteogenic supplements for three weeks. The tissue engineered bone was then implanted into donor rabbit subcutaneously. GFP was observed by confocal microscope 4 weeks after operation. Newly formed bone was harvested at 4 weeks and evaluated by HE staining, ALP activity assay, immunohistochemistry staining of osteocalcin and CollagenⅠ . Results After 4 weeks, the newly formed tissue engineered bone could be observed. There were trabeculae formed around the pore of the compound scaffold. ALP activity assay and immunohistochemistry staining of osteocalcin and Collagen Ⅰ were positive. Confocal microscopy revealed that GFP-labeled cells existed in many newborn tissues andCPC was partialy degraded. Conclusion The results show that engineered bone is similar to spongy bone and it is originated from cultured BMSC.