增强型绿色荧光蛋白(EGFP)转染对人骨髓基质细胞体外成骨诱导的影响

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。

An experimental study on effects of EGFP transfection on osteoblastic phenotype in vitro induced by mesenchymal stem cells

Objective To determine whether the Iransfection of EGFP could effect inducement to os-leublasl in vitro by ihe MSC,and whether it is feasible to use EGFP as a monitor of stem cells for bone tissue engineering.Methods A recombinant retovirat vector EGFP-PLNCX2 was constructed by standard molecular cloning techniques.hMSC-EGFP (the human mesenchymal stem cells which were transfected with EGFP) was induced to osteublast wilh ossifie medium in vitro.The AKP activity,osleocalcin synthesis and cbfal expression were compared with those of non-transfceted MSC.Results The construction of a recomhinant retoviral vector EGFP-PI.NCX2 could stably transfer! hMSC and express enhanced green fluorescent protein.There was no significant difference between the hMSC-EGFP and hMSC in ihe AKP activity,osteucalcin synthesis and cbfal expression.Conclusion The transfection of EGFP has no effect on the MSC inducement to osteoblastic phenotype in vitro.EGFP could be used as a monitor of stem cells for bone tissue engineering.