一种新的分化心肌细胞筛选策略:稳定表达αMHC-Bla^r-2A-EGFP-Rex-mCherry-2A-neo融合基因的人胚胎干细胞系的建立及其向心肌细胞定向分化的鉴定  

Stable Expression of α-MHC-Bla^r-2A-EGFP-Rex-mCherry-2A-neo Fusion Gene in Human Embryonic Stem Cells for Screening the Induced Differentiation of Cardiomyocytes

作　　者：魏晋[1] 牟晓宁[2,3] 马跃[2] 蒋曹德[4] 

机构地区：[1]西南大学生命科学学院,重庆400715 [2]中国科学院生物物理研究所,北京100101 [3]中国科学院研究生院,北京100080 [4]西南大学动物科技学院,重庆400715

出　　处：《中国生物化学与分子生物学报》2012年第3期276-282,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划(973计划;No.2010CB945024)~~

摘　　要：心肌梗死是威胁人类健康的重要疾病之一,胚胎干细胞来源的心肌细胞移植是目前治疗心肌梗死的研究热点.但是由于受到分化的心肌细胞纯度的影响,限制了心肌分化的机理研究以及临床应用.本实验构建含有心肌特异性启动子α-MHC启动的灭瘟素(Blasticidin,简称Blar)抗性基因及增强绿色荧光蛋白(EGFP)的慢病毒表达载体αMHC-Blar-2A-EGFP-Rex-mCherry-2A-neo.应用慢病毒转染技术将慢病毒转染到人胚胎干细胞(hESCs),胚胎干细胞特异性启动子Rex启动mCherry和neo抗性蛋白的表达.经过G418药物筛选,建立G418和mCherry阳性的细胞系.通过PCR及流式细胞术,进一步鉴定稳定转染的hESCs细胞系;核型分析表明,该细胞系在建立过程中仍保持细胞核型的稳定.在诱导稳定转染的hESCs向心肌细胞分化的实验中,分化的心肌细胞表达心肌细胞特异的肌钙蛋白(cTnT),同时具有EGFP和Blasticidin药物筛选的双标记.本实验建立的这一细胞系可用于心肌细胞的纯化,为深入研究心肌发生发育的关键调控机制及临床应用奠定基础.Human embryonic stem cells（hESCs）-derived cardiomyocytes provide a promising research material for cardiac differentiation and transplantation therapy.However,the studies hindered by the impurity of differentiated cardiomyocytes.We constructed a blasticidin resistant,and hESC-specific Rex promoted EGFP expressing lentiviral vector,which also contained a cardiac-specific alpha-myosin heavy chain（α-MHC）-promoter driven Neomycin/mCherry cassettes.We infected hESCs with the produced lentivirus and selected hESCs by G418.After selection of mCherry expressing of stable cells and confirmed of normal karyotypes by PCR and cytometry,the obtained hESC lines were subjected for differentiation of cardiomyocytes.Blasticidin was used for the selection of cardiomyocytes,and were also screened for EGFP expression.Immunostaining analysis showed the expression of typical cardiac marker cTnT could be found in the formed cardiomyocytes spheroids.Our study indicated the feasibility of a practical approach to select hESCs differentiated cardiomyocytes isolation,and therefore could be useful to study the regulatory mechanism in cardiogenesis.

关 键 词：心肌特异性 α肌球蛋白重链启动子 慢病毒 人胚胎干细胞 心肌细胞 

分 类 号：Q78[生物学—分子生物学] Q28