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目的 研究增强型绿色荧光蛋白(EGFP)转染人骨髓基质细胞(MSC),对其体外诱导后表达成骨表型的影响,探讨EGFP作为骨组织工程种子细胞示踪剂的可行性。方法 构建携带增强型绿色荧光蛋白(EGFP)重组逆转录病毒载体,并转染骨髓基质细胞,G418进行筛选。将转染后稳定表达绿色荧光蛋白的骨髓基质细胞(MSC-EGFP)进行成骨诱导扩增,以未转染的MSC为对照组,分别检测成骨诱导后碱性磷酸酶(AKP)活性,骨钙蛋白(OCN)含量和成骨细胞转录因子(Cbfal)的表达。结果 构建携带增强型绿色荧光蛋白(EGFP)重组逆转录病毒载体,转染后获得稳定表达绿色荧光蛋白的MSC-GFP,其体外经成骨诱导后,同样能表达成骨特征性表型,AKP,OCN,Cbfal表达和未转染组无明显差别。结论 MSC转染EGFP后,不影响其体外成骨诱导后成骨表型的表达,EGFP可作为骨组织工程种子细胞(MSC)良好的示踪剂。
Objective To investigate the effect of enhanced green fluorescent protein (EGFP) transfection on human bone marrow stromal cells (MSC) and its effect on the osteogenic phenotype after induction in vitro, and to explore the feasibility of EGFP as a tracer for bone tissue engineering seed cells. Methods Recombinant retroviral vector carrying enhanced green fluorescent protein (EGFP) was constructed and transfected into bone marrow stromal cells, G418 for screening. Bone marrow stromal cells (MSC-EGFP) stably expressing green fluorescent protein (EGFP) after transfection were used for osteoinduction amplification. The untransfected MSCs were used as the control group to detect the activity of alkaline phosphatase (AKP) after osteogenic induction, Osteocalcin (OCN) content and osteoblast transcription factor (Cbfal) expression. Results Recombinant retroviral vector carrying enhanced green fluorescent protein (EGFP) was constructed and transfected to obtain stable expression of green fluorescent protein (MSC-GFP). After osteogenic induction, MSC-GFP could express osteogenic phenotype, AKP , OCN, Cbfal expression and non-transfected group no significant difference. Conclusion Transfection of EGFP with MSC did not affect the osteogenic phenotype after osteogenic induction, and EGFP could be used as a good tracer for bone tissue engineering seed cells (MSC).