色素上皮衍生因子修饰的人脐带间充质干细胞构建方法

Preparation of pigment epithelial-derived factor-modified human umbilical cord-derived mesenchymal stem cells

目的:构建携带色素上皮衍生因子(pigment epithelialderived factor,PEDF)基因的重组慢病毒,感染人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUCMSCs),探讨PEDF在hUCMSCs中的表达及重组慢病毒对hUCMSCs活性的影响。方法:利用pLenti-CMV-hChR2-EYFP载体系统,构建携带PEDF基因的重组慢病毒pLenti-CMV-PEDF-EYFP,进一步感染hUCMSCs,获得携带PEDF基因的hUCMSCs(PEDF-hUCMSCs),激光共聚焦显微镜和ELISA法检测PEDF-hUCMSCs的PEDF蛋白表达情况,采用CCK8法检测PEDF-hUCMSCs的活性。通过观察细胞形态和流式细胞术检测验证重组慢病毒pLenti-CMV-PEDFEYFP感染hUCMSCs后对细胞传代、细胞表型的影响。结果:成功构建携带PEDF基因的重组慢病毒pLentiCMV-PEDF-EYFP,高效感染hUCMSCs,获得的PEDFhUCMSCs能有效表达PEDF蛋白,检测结果显示重组病毒感染组细胞与空白对照组细胞活性无统计学差异(P>0.05)。重组慢病毒pLenti-CMV-PEDF-EYFP感染对hUCMSCs的增殖能力、细胞形态和细胞表型没有明显改变。结论:携带PEDF基因的重组慢病毒pLenti-CMV-PEDFEYFP修饰的PEDF-hUCMSCs能有效表达PEDF蛋白。

AIM： To construct the recombined pLenti-CMV-PEDF- EYFP vector to be infected into human umbilical cord- derived mesenchymal stern cells （hUCMSCs）, and then to identify the PEDF expression in hUCMSCs and the cell viability of hUCMSCs after infection with the recombined lentivirus. METHODS： The recombinant pLenti-CMV-PEDF-EYFP vector was constructed with and PEDF gene, and then obtain the PEDF- hUCMSCs. pLenti - CMV- hChR2 - EYFP infected into hUCMSCs to The expression of PEDF in hUCMSCs was identified by confocal microscopy and ELISA. The Cell Counting Kit （CCK8） was used to assess the cell viability of PEDF-hUCMSCs. The influences of pLenti-CMV-PEDF-EYFP infection on the passage and phenotypes of hUCMSCs were assessed by microscope and flow cytometry. RESULTS.. The recombinant pLenti- CMV- PEDF- EYFP was successfully constructed and efficiently infected into hUCMSCs. The expression of PEDF was positively detected in the PEDF- hUCMSCs, No significant difference was observed on cell viability between PEDF- modified hUCMSCs and hUCMSCs （P〉 0. 05）. The infection of pLenti- CMV-PEDF-EYFP had no obvious influences on the proliferation, morphology and phenotypes of hUCMSCs. CONCLUSION. PEDF was expressed effectively in the hUCMSCs modified with the recombinant lentivirus. The preliminary results from this study provide more evidences for further study of using PEDF-hUCMSCs to treat retinitis pigmentosa.