基因修饰人多能干细胞的高效单克隆建系方法  

An Efficient Monoclonal Establishment Method of Genetically Modified Human Pluripotent Stem Cells

作　　者：钱昱 丁晓雨 刘志强[2] 袁增强 QIAN Yu;DING Xiao-yu;LIU Zhi-qiang;YUAN Zeng-qiang(College of Graduate Studies,University of South China,Hengyang 421001,China;Institute of Military Cognition and Brain Sciences,Beijing 100850,China)

机构地区：[1]南华大学研究生院,衡阳421001 [2]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《中国生物工程杂志》2021年第8期33-41,共9页China Biotechnology

摘　　要：目标:提供一种能够显著提高慢病毒稳定转染人多能干细胞的方法,并建立一种简便无损的转染细胞筛选方法。方法:在慢病毒转染人多能干细胞过程,分别比较添加与不添加Y-27632情况下细胞形态的动态变化规律,以及细胞不同形态下对慢病毒颗粒的摄入能力差异,优化建立高效的慢病毒转染方法。随后,设计并研制可视化的简便显微操作装置,探索在荧光显微镜辅助下挑取转染的阳性单克隆细胞建系的技术,建立较为简便的转染细胞纯化新方法。结果:正常培养的人多能干细胞(hESC、hiPSC),添加Y-27632后6 h集落形态发生明显变化,细胞呈现出明显长梭形,集落松弛,细胞表面积显著增加;去除后6 h集落恢复正常;常规培养的多能干细胞克隆,慢病毒主要倾向于进入集落外围或局部细胞;经Y-27632提前处理6 h,细胞集落松驰、表面积显著增加的多能干细胞,慢病毒能够较为均匀地感染集落外围与内部细胞,显著提高慢病毒转染效率。利用毛细玻璃管,自主设计制作了一款显微镜下可视化的细胞单集落挑选器件,在显微镜辅助下能够简便地进行阳性克隆细胞的挑选建系,从而在常规实验室即可完成,取代具有一定细胞损伤效应的嘌呤霉素筛选及需要专业设备的流式分选方法。结论:在慢病毒转染过程中,常规培养的hESC/i PSC集落较为致密,对慢病毒感染具有一定抵抗性;小分子化合物Y-27632使得hESC/i PSC克隆集落结构相对松散,表面积增加,显著提高了对慢病毒的易感性,提高了感染效率;成功设计了一种简便且对细胞无毒性的显微操作器件,在常规实验室条件下,可有效取代流式分选及药物筛选,实现细胞单克隆的挑选建系。Objective:To provide a method that can significantly improve the stable transfection of lentivirus into human pluripotent stem cells,and establish a simple and a non-invasive screening method for transfected cells.Methods:In the process of lentivirus transfection of human pluripotent stem cells,we compared the dynamic changes of cell morphology with and without Y-27632,and the differences of lentivirus particles uptake ability under different cell morphologies,so as to optimize and establish an efficient lentivirus transfection method.After that,a visualized and simple micromanipulation device was designed and developed to explore the technology of picking up the transfected positive monoclone cells to establish a line with the aid of a fluorescence microscope,and establish a relatively simple new method for purification of transfected cells.Results:The morphology of normal cultured human pluripotent stem cells colony changed significantly 6 hours after Y-27632 was added.The cells were in loose colony showing a long spindle shape,and an increased cell surface area;The colonies returned to normal 6 hours after removal;In conventionally cultured pluripotent stem cells,lentivirus tended to enter the colony periphery or partial cells;after Y-27632 was treated for 6 hours in advance,the pluripotent stem cells showed a loose colony and a significant increased surface,making the lentivirus infect more evenly into the periphery and internal of the colony.It significantly improved the efficiency of lentiviral transfection.Using the capillary glass tube,we designed and manufactured independently a single colony selection device that was visualized under a microscope.With the aid of a microscope,the selection and establishment of successfully transfected colony can be easily performed in the laboratory.It can replace puromycin screening with certain cell damage and the flow cytometry which requires professional equipment.Conclusions:In the process of lentiviral transfection,the hESC/IPSC colonies cultured in conventional co

关 键 词：慢病毒转染 胚胎干细胞 诱导性多能干细胞 细胞筛选 

分 类 号：Q819[生物学—生物工程]