诱导多能性干细胞与心肌细胞的融合细胞遗传表型特征研究  

Genetic phenotype of fusion cells of induced pluripotent stem cells and cardiacmyocytes

作　　者：熊挺淋 张丽莎 张晓刚[2] 刘涛 

机构地区：[1]南充市中心医院心内科川北医学院第二临床医学院,四川南充637000 [2]重庆医科大学附属第一院心内科,重庆400016

出　　处：《北京生物医学工程》2017年第5期451-458,477,共9页Beijing Biomedical Engineering

基　　金：重庆市卫生局医学科学技术研究重点项目(20090150)资助

摘　　要：目的体外构建诱导多能性干细胞(induced pluripotent stem cells,i PSCs)与心肌细胞(cardiomyocytes,CMs)的融合细胞,探讨其细胞遗传学以及定向分化等生物学特点。方法 i PSCs与原代心肌细胞通过聚乙二醇(PEG-4000)诱导融合,检测融合细胞染色体数分布情况,融合细胞碱性磷酸酶染色,鉴定亲本细胞特异性基因和蛋白在融合细胞中的表达情况,以及当融合细胞失去亲本心肌细胞表型时,其向心肌细胞分化的能力。结果融合细胞表现为i PSCs样的特点;融合细胞主要表现为四倍体或者近似四倍体的核型(超过75%的融合细胞染色体数目在76~80条之间);碱性磷酸酶的阳性率低于同时间点的i PSCs;干细胞特异性基因Oct-4和Nanog的表达量在融合细胞与i PSCs中没有差异,而心肌细胞特异性基因α-MHC和β-MHC在融合细胞中的表达量明显低于心肌细胞;Oct-4蛋白在不同代数的融合细胞中均有表达,而随着时间的延长,c Tn T蛋白逐渐呈阴性表达;失去亲本心肌细胞表型的融合细胞能够向心肌细胞分化,且分化率高于同时间点的i PSCs。结论 i PSCs与原代心肌细胞的融合细胞,初期表现出双向重建,P5代后完全表现出干细胞显型,并且能向心肌细胞分化。Objective To construct fusion cells with induced pluriopotent stem cells（i PSCs） and cardiomyocytes in vitro,and to investigate biological characteristics of cytogenetic and directed differentiation of fusion cells. Methods Polyethylene glycol（PEG-4000） was used to mediate the cell fusion of i PSCs and primary cardiomyocytes,the distribution of fusion cells chromosome number and alkaline phosphatase staining were detected. Specific genes and proteins of parental cells in fusion cells were identified,and ability to differentiation into cardiomyocytes when fusion cells lose parental cardiomyocytes phenotype. Results The fusion cells showed i PSCs-like morphology,mainly showed tetraploid or near tetraploid karyotype（more than 75% of the fusion cells had chromosomes between 76 and 80）.The positive rate of alkaline phosphatase in fusion cells was lower than i PSCs at the same time point. There was nodifference expression of stem cell-specific gene Oct-4 and Nanog in the fusion cells and i PSCs,but the expression of cardiomyocytes-specific gene α-MHC and β-MHC in fusion cells was significantly lower than cardiomyocytes. Oct-4 protein was expressed in different generations of fusion cells,whereas with time,c Tn T protein negative expression gradually. The fusion cells that lost the parental cardiomyocytes phenotype were able to differentiate into cardiomyocytes,and the differentiation rate was higher than i PSCs at the same time point.Conclusions Fusion cells from i PSCs and primary cardiomyocytes,initially showed bidirectional reprogramming,fully exhibit stem cell phenotype after fifth generation and can differentiate into cardiomyocytes.

关 键 词：诱导多能性干细胞 心肌细胞 细胞融合 融合细胞 重编程 

分 类 号：R318.04[医药卫生—生物医学工程]