体外培养时间对诱导多能干细胞源性心肌细胞成熟度的影响  

Effect of culture time in vitro on maturity of induced pluripotent stem cell-derived cardiomyocytes

作　　者：熊挺淋 张丽莎 王德伟 曹海平 杨燕 Xiong Tinglin;Zhang Lisha;Wang Dewei;Cao Haiping;Yang Yan(Department of Cardiology of Nanchong Central Hospital•Second Clinical Medical College of North Sichuan Medical College,Nanchong 637000,Sichuan Province,China;Sichuan Chidingshengtong Biotechnology Co.,Ltd.,Chengdu 610041,Sichuan Province,China)

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,四川省南充市637000 [2]四川驰鼎盛通生物科技有限公司,四川省成都市610041

出　　处：《中国组织工程研究》2025年第25期5304-5310,共7页Chinese Journal of Tissue Engineering Research

基　　金：四川省科学技术厅重点研发项目(2019YFS0314),项目负责人:杨燕。

摘　　要：背景:研究已证实诱导多能干细胞能够定向分化为心肌细胞,但目前很少有分化心肌细胞成熟度的研究报道。目的:探讨延长诱导分化时间对诱导多能干细胞源性心肌细胞的形态、肌节长度、双核细胞含量、心脏基因表达、心脏蛋白表达和线粒体功能的影响。方法:使用骨形态发生蛋白4、CHIR 99021和IWR1诱导多能干细胞向心肌细胞分化,分别在第20天和第40天收集分化心肌细胞;采用RT-PCR和免疫荧光检测分化心肌细胞中心脏基因和蛋白的表达水平;LAS X图像分析软件分析分化心肌细胞形态和肌节长度;MitoTracker Green FM线粒体染色检测线粒体总量,JC-1线粒体染色检测线粒体膜电位。结果与结论:与第20天的分化心肌细胞相比,第40天的分化心肌细胞的细胞周长和肌节长度更长、细胞面积更大(P<0.05);多核细胞比例从第20天的16%左右大幅上升至第40天的29%左右(P<0.05);第40天的分化心肌细胞具有与原代心肌细胞更相近的基因表达水平,SERCA2A、Cx-43和α-MHC基因表达水平明显高于第20天的分化心肌细胞(P<0.05);与第20天的分化心肌细胞相比,第40天的分化心肌细胞中TNNT2和α-MHC蛋白表达水平较高,线粒体分布密度更大,且功能性线粒体数量增加(P<0.05)。结果表明:延长诱导分化时间,可以通过增加肌节长度和功能性线粒体数量以及升高心脏基因和蛋白表达水平而提高分化心肌细胞的成熟度。BACKGROUND:It has been proved that induced pluripotent stem cells can differentiate into cardiomyocytes,but there are few reports on the maturity of differentiated cardiomyocytes.OBJECTIVE:To explore the effect of prolonging the induced differentiation time on the morphology,sarcomere length,binuclear cell content,cardiac gene expression,cardiac protein expression,and mitochondrial function of cardiomyocytes derived from induced pluripotent stem cells.METHODS:Bone morphogenetic protein 4,CHIR 99021,and IWR1 were used to induce pluripotent stem cells to differentiate into cardiomyocytes,and differentiated cardiomyocytes were collected on days 20 and 40 respectively.The expression levels of cardiac genes and proteins in differentiated cardiomyocytes were detected by RT-PCR and immunofluorescence,respectively.LAS X image analysis software was used to analyze the morphology and sarcomere length of differentiated cardiomyocytes.MitoTracker Green FM mitochondrial staining was used to detect total mitochondria.JC-1 mitochondrial staining was used to detect mitochondrial membrane potential.RESULTS AND CONCLUSION:Differentiated cardiomyocytes on day 40 had longer cell circumference and sarcomere length,and larger cell area than those on day 20(P<0.05).The proportion of multinucleated cells rose sharply from about 16%on day 20 to about 29%on day 40(P<0.05).Differentiated cardiomyocytes on day 40 had gene expression levels that were more similar to those of the primary cardiomyocytes,and the expression levels of SERCA2A,Cx-43,andα-MHC genes were significantly higher than on day 20(P<0.05).Compared with the differentiated cardiomyocytes on day 20,the expression density of TNNT2 andα-MHC protein was relatively higher,the distribution density of mitochondria was larger,and the number of functional mitochondria was greater on day 40(P<0.05).The results show that prolonging the induced differentiation time can increase the maturity of differentiated cardiomyocytes by increasing the length of sarcomere and the number of functio

关 键 词：诱导多能干细胞 分化心肌细胞 线粒体 心脏基因 心脏蛋白 成熟度 

分 类 号：R459.9[医药卫生—治疗学] R318.04[医药卫生—临床医学] R542.2