高效靶向激活肝细胞内源基因直接重编程为胰岛样细胞  

Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes

作　　者：王晗月 李富荣[2] 杨晓菲 胡巢凤[1] Wang Hanyue;Li Furong;Yang Xiaofei;Hu Chaofeng(Department of Pathology and Pathophysiology,School of Medicine,Jinan University,Guangzhou 510632,Guangdong Province,China;Translational Medicine Collaborative Innovation Center,Second Clinical Medical College(Shenzhen People’s Hospital)of Jinan University,Shenzhen 518020,Guangdong Province,China)

机构地区：[1]暨南大学基础医学院病理生理学系,广东省广州市510632 [2]暨南大学第二临床医学院(深圳市人民医院)转化医学协同创新中心,广东省深圳市518020

出　　处：《中国组织工程研究》2021年第7期1056-1063,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学青年基金(81800685),项目负责人:杨晓菲;国家自然科学基金(81670702),项目负责人:李富荣;广东省自然科学基金项目(2018A030310039),项目负责人:杨晓菲;深圳市科创委基础研究自由探索项目(JCYJ20170307100154602),项目负责人:杨晓菲。

摘　　要：背景:胰岛细胞移植是治疗糖尿病最有效的方法之一,然而移植细胞来源短缺限制了其临床应用。在体外进行肝细胞向胰岛β细胞的直接重编程是解决该问题的一个新思路,但肝细胞向胰岛β细胞分化是一个复杂的过程。目的:利用构建的CRISPR/dCas9-Pumilio系统(Casilio系统),通过改造向导RNA序列,与转录激活子PUFa-P65-HSF1结合,实现高效靶向激活肝细胞内源基因直接重编程为胰岛样细胞。方法:采用脂质体转染法将Casilio体系(PNM)转染至HEK293T细胞系,靶向激活肝细胞内源PNM(Pdx1,Ngn3及MafA)基因,利用实时荧光定量PCR及免疫荧光检测内源PNM的表达。利用携带Ins-promoter-EGFP的慢病毒构建增强型绿色荧光蛋白稳定表达的Ins-EGFP HepG2细胞系,PiggyBac(PB)转座系统在此细胞系中构建核酸内切酶失活的Cas9(dCas9)和PUFa-P65-HSF1稳定表达的稳转细胞系。用脂质体向稳转细胞系分别转染针对PNM的向导RNA,然后实时荧光定量PCR和免疫荧光检测内源基因PNM的表达水平,并观察重编程效率。结果与结论:①实验在293T细胞系中验证了Casilio体系对内源基因的激活作用;②RT-PCR、Western Blot及免疫荧光检测验证了稳转细胞系中EGFP、dCas9和PUFa-P65-HSF1的表达;③实时荧光定量PCR证实靶向激活PNM的新型Casilio体系可实现内源PNM基因表达明显上调,直接重编程效率为10%-15%;④上述数据说明基于CRISPR/dCas9的新型Casilio体系,可成功激活肝脏内源PNM基因,可初步实现肝细胞系直接重编程为胰岛样细胞。BACKGROUND:Islet cell transplantation is one of the most effective methods to treat diabetes.However,the shortage of transplanted cells has limited its clinical application.Direct reprogramming of hepatocytes to isletβcells in vitro is a new idea to solve this problem,but differentiation of hepatocytes to isletβcells is a complicated process.OBJECTIVE:Direct reprogramming hepatocytes into islet-like cells by efficient targeting and activating the endogenous genes of hepatocytes with Casilio system(constructed CRISPR/Cas9-Pumilio system)through modifying guide RNA sequence combined with the transcriptional activator PUFa-P65-HSF1.METHODS:The endogenous PNM(Pdx1,Ngn3,MafA)genes of hepatocytes were targeted and activated by using the Casilio system,which was transfected to the HEK293T cell line by liposome transfection.The expression of endogenous PNM was detected by real-time fluorescence quantitative PCR and immunofluorescence.The Ins-EGFP cell line with stable expression of enhanced green fluorescent protein was constructed by the lentivirus carrying Inspromoter-EGFP.The Ins-EGFP-HepG2-Cas9-PUFa-p65-HSF1 cell line(referred to as stable translocated cell lines)with stable expression of dCas9 and PUFa-P65-HSF1 in Ins-EGFP-HepG2 cell line was constructed by the PiggyBac(PB)transposon system.By using liposome,the gRNAs were transfected to stable translocated cell line,and the expression level of endogenous gene PNM was detected by real-time fluorescence quantitative PCR and immunofluorescence.Simultaneously,reprogramming efficiency was observed.RESULTS AND CONCLUSION:(1)The activation of endogenous genes by Casilio system was verified in 293T cell line.(2)The expression of EGFP,dCas9 and PUFa-P65-HSF1 in stable cell line was detected by RT-PCR,western blot assay and immunofluorescence.(3)The real-time fluorescence quantitative PCR results confirmed that this new Casilio system could target and activate the PNM which led to the up-regulation of the endogenous PNM gene expression.The efficiency of direct reprogrammin

关 键 词：体细胞 胰岛样细胞 肝 细胞基因转染 糖尿病 CRISPR/dCas9 因子 蛋白 通路 

分 类 号：R459.9[医药卫生—治疗学] R363[医药卫生—临床医学]