利用Tet-On系统构建稳定表达Pdx1的小鼠ESC细胞株  

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

作　　者：钟娃[1] 夏忠胜[1] 于涛[1] 倪楚燕 黎洁瑶[1] 陈其奎[1] ZHONG Wa;XIA Zhong-sheng;YU Tao;NI Chu-yan;LI Jie-yao;CHEN Qi-kui(Department of Gastroenterology,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510120,Chinam)

机构地区：[1]中山大学孙逸仙纪念医院消化内科,广东广州510120

出　　处：《中国病理生理杂志》2021年第1期187-192,共6页Chinese Journal of Pathophysiology

基　　金：广东省科技社会发展项目(No.2012B031800030);广东省自然科学基金资助项目(No.2017A030313600)。

摘　　要：目的:利用Tet-On系统构建稳定表达胰十二指肠同源异形框1(Pdx1)的小鼠胚胎干细胞(ESC)株,为进一步研究Pdx1+定型内胚层细胞向胰腺细胞分化奠定了基础。方法:采用Tet-On系统构建具有绿色荧光蛋白标记及嘌呤霉素抗性的Pdx1过表达慢病毒载体并感染胚胎干细胞。实验分为空白对照组(ESC组)、空载慢病毒对照组(PDX1−ESC组)和Pdx1慢病毒转染组(PDX1+ESC组)。流式细胞术检测多西环素(DOX)筛选后转染细胞的阳性率;检测Tet-On系统功能及Pdx1的mRNA和蛋白表达。流式细胞分选仪分选转染细胞,构建稳定表达Pdx1基因的ESC株及阴性对照ESC株。结果:(1)DOX筛选后PDX1−ESC组的转染细胞阳性率为90.72%,PDX1+ESC组的转染细胞阳性率为94.01%。用流式细胞分选仪分选后PDX1−ESC组的转染细胞阳性率为97.84%,PDX1+ESC组为98.13%。(2)加入DOX后,PDX1−ESC组和PDX1+ESC组可见绿色荧光。PDX1+ESC组Pdx1的mRNA和蛋白表达明显增高(P<0.05)。不加DOX,则3组细胞均未见绿色荧光,且Pdx1 mRNA和蛋白表达的差异无统计学显著性(P>0.05)。(3)细胞株冻存3个月后复苏培养仍然存活,并受DOX调控。结论:利用Tet-On系统成功构建可诱导表达Pdx1的小鼠ESC株,为研究Pdx1+定型内胚层细胞向胰腺细胞分化提供了有效的细胞模型。AIM:To construct the mouse embryonic stem cell(ESC)line with stable pancreatic and duodenal homeobox 1(Pdx1)expression by Tet-On system,which may lay a foundation for further research on the differentiation of Pdx1+definitive endoderm cells into pancreatic cells.METHODS:The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC.The cells were divided into 3 groups:blank control group(ESC group),empty lentivirus control group(PDX1−ESC group)and Pdx1 lentivirus transfection group(PDX1+ESC group).Flow cytometry was used to detect the transfected cells after screening by doxycycline(DOX).The function of Tet-On system and the expression of Pdx1 gene were detected.The transfected cells in PDX1−ESC group and PDX1+ESC group were sorted by flow cytometry,and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified.RESULTS:(1)The positive rates of transfected cells in PDX1−ESC group and PDX1+ESC group were 90.72%and 94.01%after screening by DOX,respectively.The positive rates of transfected cells in PDX1−ESC group and PDX1+ESC group was 97.84%and 98.13%after sorting by flow cytometry,respectively.(2)With DOX,green fluorescence was observed in PDX1−ESC group and PDX1+ESC group.The mRNA and protein expression of Pdx1 was significantly increased in PDX1+ESC group(P<0.05).Without DOX,no green fluorescence was observed in the cells of the 3 groups,and no significant difference in the mRNA and protein expression of Pdx1 was observed(P>0.05).(3)After 3 months of cryopreservation,the cell lines still survived in resuscitation culture and were regulated by DOX.CONCLUSION:Using Tet-On system,the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1+definitive endoderm cells into pancreatic cells.

关 键 词：Tet-On系统 胰十二指肠同源异形框1 胚胎干细胞 胰腺细胞 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R33-33[医药卫生—基础医学]