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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,为进一步探索应用PEDF-hUCMSCs治疗视网膜色素变性疾病奠定了实验基础和理论依据
OBJECTIVE: To construct recombinant lentivirus carrying pigment epithelialderived factor (PEDF) gene and infect human umbilical cord-derived mesenchymal stem cells (hUCMSCs) to investigate the expression and recombination of PEDF in hUCMSCs Effect of lentivirus on hUCMSCs activity. Methods: Recombinant lentiviral vector pLenti-CMV-PEDF-EYFP carrying PEDF gene was constructed by using pLenti-CMV-hChR2-EYFP vector system, and hUCMSCs were further infected with PEDF gene to obtain PEDF-hUCMSCs. Laser confocal microscopy The PEDF protein expression in PEDF-hUCMSCs was detected by ELISA and the activity of PEDF-hUCMSCs was detected by CCK8. Cell morphology and flow cytometry were used to test the effect of hUCMSCs infected by recombinant lentivirus pLenti-CMV-PEDFEYFP on cell passage and cell phenotype. Results: Recombinant lentiviral vector pLentiCMV-PEDF-EYFP carrying PEDF gene was successfully constructed and PEDFhUCMSCs were efficiently infected with hUCMSCs. The results showed that there was no significant difference in the cell viability between PEDF-infected cells and blank control group (P > 0.05). Recombinant lentivirus pLenti-CMV-PEDF-EYFP infection hUCMSCs proliferation, cell morphology and cell phenotype did not change significantly. CONCLUSION: PEDF-hUCMSCs modified with recombinant lentivirus pLenti-CMV-PEDFEYFP carrying PEDF gene can effectively express PEDF protein, which lays the foundation for the further exploration of the application of PEDF-hUCMSCs in the treatment of retinitis pigmentosa