绿色荧光蛋白标记骨髓间质干细胞示踪技术在体外构建组织工程骨中的应用研究

Application of Tracing Technique of Labeling Bone Marrow Mesenchymal Stem Cells with Green Fluorescent Protein in Tissue-engineered Bone Construction in Vitro

探讨绿色荧光蛋白(hrGFP)标记技术在组织工程骨体外构建中对大鼠骨髓间质干细胞(rBMSCs)的示踪作用。用293FT细胞包装含hrGFP基因的慢病毒载体,收集病毒液转染rBMSCs,转染后细胞持续表达绿色荧光,流式细胞仪检测转染率81.3%。将hrGFP标记的rBMSCs与生物玻璃-胶原蛋白-透明质酸-磷酸丝氨酸支架复合培养体外构建组织工程骨。荧光显微镜下观察到绿色荧光的细胞在支架表面很好地黏附、增殖。成骨诱导后,表达绿色荧光的细胞内部及支架表面均有COLⅠ的阳性表达。说明HrGFP标记技术能直观有效地观察组织工程骨体外构建过程中细胞的黏附、生长及成骨分化等行为。

This study was designed to label rat bone marrow mesenchymal stem cells（rBMSCs） with humanized renillar green fluorescent protein（hrGFP） for exploring the detectable effects of hrGFP on the construction of tissue-engineered bone in vitro.The hrGFP expression plasmid was packed into lentivirus by 293FT cells and transduced into rat BMSCs.After transduction,81.3% rBMSCs successfully expressed green florescence.The hrGFP-rBMSCs were statically loaded on Bioglass-Collagen-Hyaluronic Acid-Phosphatidylserine（BG-COL-HYA-PS） scaffold in complete L-DMEM medium or osteogenic medium for 14 days.At 6h,a number of cells expressing green fluorescence can be observed by fluorescent microscopy.At day 7 and 14 after co-culture,the number of cells on the scaffold gradually increased.After 14 days for osteogenic induction,the hrGFP-rBMSCs and the interior of scaffold can be detected the expression of type Ⅰ collagen.The results demonstrate that hrGFP labelling technique can detect visualizedly and effectively cell adhesion,proliferation and osteogenic differentiation in the construction of tissue-engineered bone in vitro.