摘要
目的研究LIF/Stat3信号通路对小鼠胚胎干细胞(m ESCs)分化成心肌细胞的影响。方法利用m ESCs形成拟胚体(EBs)进行心肌分化。在野生型m ESCs形成EBs的过程中加入Janus激酶(JAK)抑制剂以抑制LIF/Stat3信号通路(JAKi组)。而在融合表达雌激素受体(ER)和Stat3的Stat3ER细胞株进行分化过程中添加4-羟基他莫西酚(4HT)以激活LIF/Stat3信号通路(4HT组)。用qRT-PCR检测心脏特异转录因子NKX2.5、α-肌球蛋白重链(α-MHC)和连接蛋白43(Cx43)以及m ESCs自我更新基因oct4、nanog和Stat3下游基因socs3的表达水平。用Western blot检测和免疫荧光染色法验证α-MHC和Cx43的相对表达。结果免疫荧光结果显示在自发搏动的EBs中出现α-MHC和Cx43的荧光,表明心肌细胞分化成功。随着分化时间的延长,NKX2.5、α-MHC和Cx43的mRNA表达量逐渐增加,至第15天时最为明显,且JAKi组高于对照组(P<0.01);对照组高于4HT组(P<0.01)。而nanog、oct4随着分化时间的延长逐渐降低,且JAKi组低于对照组(P<0.05),而4HT组高于其对照组(P<0.05)。Socs3的表达量在JAKi组一直较低且低于对照组,在4HT组表达较高且高于对照组(P<0.01)。Western blot检测结果显示第15天的Cx43和α-MHC蛋白表达量JAKi组明显高于对照组(P<0.05),4HT组低于其对照组(P<0.05)。结论抑制LIF/Stat3信号通路促进小鼠胚胎干细胞分化成心肌细胞;激活LIF/Stat3信号通路抑制小鼠胚胎干细胞的心肌细胞分化。
Objective To investigate the roles of LIF/Stat3 signaling pathway in differentiation of mouse embryonic stem cells（mESCs） into cardiomyocytes. Methods Inducing mESCs differentiate into cardiomyocytes through form- ing embryoid bodies（EBs）. In the process of wild type mESCs transforming to EBs administration with JAK inhibi- tor to inhibit LIF/Stat3 signaling pathways （JAKi group）. In the fusion expression of estrogen receptor （ER） and Stat3 （Stat3ER） cell lines 4-hydroxytamoxifen （4HT） to activate the LIF/Stat3 signaling pathways during the process of differentiation（4HT group）. The mRNA expression levels of NKX2.5, alpha myosin heavy chain （ α- MHC） and connection 43 （Cx43） as well as stem cell self-renewal marker genes nanog and oct4 and Star3 down- stream gene socs3 were analyzed through qRT -PCR. The relative expression of α-MHC and Cx43 were detected by Western blot and immunofluorescence staining. Results Spontaneously beating EBs were positively stained with α- MHC and Cx43, showed that myocardial cell differentiation. The mRNA expression levels of NKX2.5,α-MHC and Cx43 increase gradually, showing a peak of expression on the fifteenth day, during the differentiation. JAKi group produced higher gene expression compared with control group（P 〈 0.01 ）. The control group was higher than 4HT group（P 〈 0. 01 ）. Nanog and oct4 were reduced gradually, and JAKi group was lower than the control group（P 〈 0.05 ） , while 4HT group was higher than the control group （P 〈 0.05 ）. In JAKi group had low expression levels of socs3 and lower than that of control group（P 〈 0. 01 ） , whereas 4HT group had the opposite results. Western blot a- nalysis suggested that the expression of ~x-MHC and Cx43 in JAKi group were higher than those of control group（P 〈 0. 05） , and 4HT group was slightly lower than that of control group on the 15th day（ P 〈 0. 05 ）. Conclusion Inhibition of LIF/Stat3 signaling pathway promote mouse embryonic stem cells to diff
出处
《安徽医科大学学报》
CAS
北大核心
2017年第5期645-650,共6页
Acta Universitatis Medicinalis Anhui
基金
安徽省卫生厅医学科研重点项目(编号:2010B005)
