目的:应用增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)和CM-Dil标记技术,观察组织工程骨在体内形成过程中种子细胞的变化和转归.方法:分别用EGFP慢病毒表达和CM-Dil染料的方法标记比格犬骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs),MTT法检测标记细胞的体外增殖能力.BMSCs接种珊瑚支架体外成骨诱导7天后,将未标记组、EGFP组和CM-Dil组分别植入裸鼠背部皮下,空白支架作为阴性对照.术后4、8、12周取材,HE染色观察成骨情况,EGFP组采用GFP免疫组化、CM-Dil组冰冻切片荧光显微镜下示踪BMSCs在体内的变化.结果:两种标记技术能高效标记BMSCs,标记前后细胞的体外增殖无显著性差异(P>0.05).细胞-支架复合物植入体内12周后有新生骨形成,标记细胞数量随时间延长而逐渐减少,12周后仍显示有部分标记细胞存活.结论:EGFP和CM-Dil可用于示踪组织工程种子细胞,通过示踪说明BMSCs在体内组织工程骨成骨过程中发挥了重要作用.%Objective To observe the effect of BMSCs in fabricating tissue engineering bone in vivo using EGFP andCM-Dil labeling technology. Methods BMSCs isolated from Beagle Dogs were labeled using EGFP and CM -Dilseparately and the proliferation abilities were analyzed by MTT assay. BMSCs were seeded onto the coral scaffolds andcultured in the osteogenic medium for 7 days.Then the BMSCs/Coral constructs were implanted into the nude micessubcutaneously.The constructs were divided into three groups:Unlabeled group,EGFP group,CM -Dil group.Thespecimens were collected respectively at 4,8 and 12 weeks after implantation and tissue engineered bone wasevaluated by HE staining.The seeded BMSCs were traced by immunohistochemistry staining of GFP in the EGFP groupand directly observed in the frozen section under the fluorescence microscope in the CM-Dil. Results BMSCs werelabeled efficiently by both GFP and CM-Dil labeling technology and there was no statistical significance in BMSCsproliferation after labeling (P>0.05).The histological observation of three groups showed that there were engineered boneformed around the pores of the scaffolds after 12 weeks implantation. The labeled BMSCs were decreased graduallyand could be detected up to 12 weeks in vivo. Conclusion EGFP and CM-Dil can be used to label and trace theseeded BMSCs in tissue engineered bone formation in vivo.
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