慢病毒介导的绿色荧光蛋白报告基因对人多能干细胞的生物风险评估研究

摘要 目的：探讨慢病毒介导的绿色荧光蛋白（GFP）标记人诱导多能干细胞（hiPSC）是否影响其细胞生物学特性，为多角度评价生物学风险提供实验基础。方法：慢病毒感染hiPSC后24小时，通过抗性基因表达筛选成功标记GFP的hiPSC。利用流式细胞分析法（FACS）检测GFP阳性的细胞比例。通过碱性磷酸酶染色检验干细胞的多能性，并通过免疫荧光染色检测多能性标记基因OCT4，NANOG，SOX2，SSEA4的表达情况。体外拟胚体分化实验检测GFP标记的hiPSC分化为不同胚层细胞的能力。结果：慢病毒感染不仅可以成功hiPSC标记上GFP，而且抗性基因表达筛选使GFP阳性细胞比例从37.5%提高到97.4%。AP染色和多能性标记基因的免疫染色证明标记后的细胞能维持多能性。体外分化实验显示感染后hiPSC可以形成拟胚体并实现三个胚层细胞共存。结论：慢病毒能够高效的标记hiPSC，并且不影响其多能性和拟胚体形成能力，可以用于后续的分化和细胞示踪研究。

Green Fluorescence Protein Reporter Gene Labeled Human Induced Pluripotent Stem Cells Using Lentivirus

ABSTRACT Objective: To explore the effect of green fluorescence protein (EGFP) labeling mediated by lentivirus on the biophysical properties of human induced pluripotent stem cells (hiPSC), and provides experimental basis for multi angle evaluation of biological risk. Methods: HiPSC labeled with GFP was successfully screened by resistance gene expression 24 h after lentivirus infection. After GFP expression was observed under fluorescence microscope, Positive rate of GFP were measured with fluorescent-Activated cell scanning (FACS) analysis. The pluripotency of the cells was tested by alkaline phosphatase staining, and the expressions of pluripotent marker genes OCT4, NANOG, SOX2 and SSEA4 with immunostaining. Finally, in vitro embryoid body experiments were performed to detect differentiate ability of GFP labeled hiPSC into different cell layer. Results: Lentivirus infection can not only successfully label hiPSC with GFP, but through expression screening of resistant genes, the proportion of GFP positive cells were improved from 37.5% to 97.4%. Both AP staining and immunostaining of the pluripotent marker genes demonstrated that the pluripotent feature were maintained in the labeled cells. In vitro differentiation experiments showed that GFP labeled hiPSC could form the embryoid body and further differentiate into three embryo layers. Conclusion: Lentiviruses can efficiently label hiPSC and has no significant effect on the pluripotency and differentiation of embryoid body, and can be used for subsequent differentiation and cell tracer studies.