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目的:探讨整联蛋白β1(Integrinβ1)在维生素C(Vc)诱导小鼠诱导性多能干细胞(induced pluripotent stemcells,iPSCs)向心肌样细胞(cardiomyocytes,CMs)分化中的作用。方法:在立式显微镜下,分别摘取胚胎(12.5 d、14.5 d及16.5 d)BALB/c胎鼠、新生(出生1 d,P1)和成年BALB/c小鼠的心脏,提取总RNA,半定量及实时定量PCR分析心脏发育不同时期Integrinβ1的表达。利用悬滴培养形成拟胚体(embryoid body,EB)的方法体外诱导iPSCs向心肌样细胞分化,诱导过程中分别添加Vc和integrinβ1抑制剂(HMβ1-1)处理,共分为3组:对照组、Vc处理组和Vc+HMβ1-1处理组。诱导分化的第3、5、7天,半定量及实时定量PCR检测Oct4、integrinβ1和心肌特异性因子(α-MHC、MLC2a)。用免疫荧光染色法检测心肌特异性结构蛋白心肌肌钙蛋白I(cTnI)的表达。结果:Inte-grinβ1在心脏胚胎发育时期(E12.5、E14.5、E16.5、P1)的表达递增(P<0.01),成年期表达有所回落(P<0.01)。半定量及实时定量PCR的结果提示,Vc组α-MHC、MLC2a的表达显著高于对照组(P<0.01),加integrinβ1抑制剂HMβ1-1后表达量降低(P<0.01)。跳动EBs计数的结果提示,诱导分化第10、14、18、22、26天,Vc组跳动EBs的百分率显著高于对照组(P<0.01);而Vc+HMβ1-1组跳动EBs的百分率相对于Vc组显著降低(P<0.01)。免疫荧光染色显示,Vc组有较强的cTnI表达,添加integrinβ1抑制后,Vc+HMβ1-1组cTnI的表达明显减弱。结论:Vc可促进iPSCs向心肌细胞分化,其机制可能是通过integrinβ1介导。
Objective: To investigate the role of Integrinβ1 in differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) induced by vitamin C (Vc). Methods: The hearts of neonatal (born 1 d, P1) and adult BALB / c mice were harvested from the BALB / c embryos (12.5 d, 14.5 d and 16.5 d) under the microscope, Semi - quantitative and real - time quantitative PCR analysis of expression of Integrinβ1 in different stages of cardiac development. Using suspension culture to form embryoid body (EB), iPSCs were induced to differentiate into cardiomyocyte-like cells in vitro. Vc and integrinβ1 inhibitor (HMβ1-1) were added during induction. The cells were divided into 3 groups: control group , Vc treatment group and Vc + HMβ1-1 treatment group. Induction of differentiation on day 3, 5, 7, semi-quantitative and real-time quantitative PCR detection of Oct4, integrinβ1 and myocardial specificity factor (α-MHC, MLC2a). The expression of cardiac troponin I (cTnI), a specific structural protein of myocardium, was detected by immunofluorescence staining. Results: The expression of Inte-grinβ1 increased during cardiac embryonic development (E12.5, E14.5, E16.5, P1) (P <0.01), and decreased in adulthood (P <0.01). The results of semi-quantitative and real-time quantitative PCR showed that the expression of α-MHC and MLC2a in Vc group was significantly higher than that in control group (P <0.01), and decreased after HMβ1-1 (P <0.01). The results of beating EBs count suggested that the percentage of beating EBs in Vc group was significantly higher than that in control group on the 10th, 14th, 18th, 22th and 26th day (P <0.01), while the percentage of beating EBs in Vc + HMβ1-1 group was In Vc group was significantly lower (P <0.01). Immunofluorescence staining showed that the expression of cTnI in Vc group was stronger than that in Vc + HMβ1-1 group after addition of integrinβ1. Conclusion: Vc can promote iPSCs to differentiate into cardiomyocytes, which may be mediated by integrin β1.