应用GFP标记技术示踪裸鼠体内组织工程化骨的形成  

Use of GFP labelling technique to monitor tissue engineered bone formation in nude mice

作　　者：袁捷[1] 刘德莉[1] 周广东[1] 苗春雷[1] 崔磊[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学第九人民医院整复外科上海市组织工程研究重点实验室,上海200011

出　　处：《上海第二医科大学学报》2004年第4期235-237,266,共4页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家重点基础研究发展规划资助项目("973"项目)(G1999054308);国家高技术研究发展计划资助项目("863"计划)(2002AA205011);上海市"重中之重"重点学科基金资助.

摘　　要：目的 应用绿色荧光蛋白(GFP)标记技术,观察组织工程化骨体内形成过程中种子细胞的变化与转归。方法 用GFP重组逆转录病毒载体(GFP-RV)转染犬骨髓基质干细胞(BMSCs),将其接种于β-磷酸三钙(β-TCP),形成细胞一材料复合物,移植于裸鼠皮下。术后8周,HE染色观察组织结构,碱性磷酸酶(AKP)染色和骨钙素(OCN)免疫组化检测功能蛋白,激光共聚焦显微镜下对GFP进行示踪观察。结果 8周后,大体可见组织工程化新骨形成,组织学示新生骨小梁围绕材料孔隙生成,AKP染色和OCN免疫组化结果阳性,并可见新生组织内有呈绿色的GFP标记细胞,β-TCP部分降解。结论组织工程化骨组织学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供体细胞。Objective To reconstruct tissue engineered bone with cultured bone marrow stromal cells (BMSCs) and trace it by green fluorescent protein(GFP) gene transfer. Methods Recombinant RV-GFP expression vector was used to infect canine BMSCs directly. GPF-labeled BMSCs were seeded onto beta-tricalciumphosphate(β-TCP) to form a cell-scaffold construct, then implanted into nude mice subcutaneously. Formed tissues were harvested at 8 weeks and evaluated by HE staining, AKP staining, immunohistochemistry of osteocalcin and confocal microscope. Results The tissue engineered bone had been formed over a 8-week period. There were newly formed trabeculae a-round the pores of β-TCP, AKP staining and mmunohistochemistry of osteocalcin being positive. Confocal microscope revealed that GFP-labeled cells existed in many newborn tissues,β-TCP was partly degraded. Conclusion The results show that engineered bone are similar to spongy bone and the composed cells originated from cultured BMSC.

关 键 词：绿色荧光蛋白标记技术 组织工程 骨组织 重组逆转录病毒载体 Β-磷酸三钙 供体细胞 

分 类 号：R318.1[医药卫生—生物医学工程]