Ghrelin以非经典受体依赖性方式促进人胚胎干细胞定向分化为心肌细胞的研究

目的 探讨Ghrelin对人胚胎干（hES）细胞定向分化为心肌细胞的影响. 方法 以不同浓度Ghrelin诱导hES细胞定向分化为心肌细胞,显微镜下计数搏动细胞团的比例,应用实时PCR检测心肌特异性标志物的表达.应用RT-PCR、Western blot及免疫荧光染色检测分化细胞中生长激素促分泌物受体1α（GHSR1α）的表达,添加GHS-R1α拮抗剂[D-lys3]-GHRP-6,观察其对Ghrelin促分化作用的影响. 结果 与对照组比较,10-10、10-9及10-8 mol/L Ghrelin均可增加搏动细胞团的比例[搏动比例分别为（12.9±1.4）％％,（19.5±2.2）％及（13.5±3.1）％vs（10.3±2.2）％,P＜0.05].RT-PCR分析显示,Ghrelin可上调α-肌球蛋白重链（α-MHC）和心肌肌钙蛋白I（cTnI）的表达[α-MHC：100％ vs （40.1±13.2）％,P＜0.001;cTnI：100％ vs （52.6±9.8）％,P＜0.001].RT-PCR、Western blot及免疫荧光染色均显示GHS-R1α在不同阶段的分化细胞中表达,但酰基化和非酰基化Ghrelin的促分化作用相似,且添加GHS-R1α拮抗剂不能阻断Ghrelin的促分化作用. 结论 Ghrelin可促进hES细胞定向分化为心肌细胞,该作用可能由GHS-R1α以外的信号途径所介导.

Ghrelin induces cardiomyocyte differentiation of human embryonic stem cells in a non-classical receptor-independent manner

Objective To investigate the effects of ghrelin on cardiomyocyte differentiation of human embryonic stem （hES） cells.Methods The hES cells were differentiated into cardiomyocytes with or without different concentrations of ghrelin.The appearance of spontaneously contracting clusters was counted microscopically and the expression of cardiomyocyte-specific markers was determined through real-time PCR（RT-PCR）.The expression of growth hormone secretagogue receptor （GHS-R） 1α was detected by RT-PCR,Western blot and immunostaining.Furthermore,GHS-R1α antagonist [D-lys3]-GHRP-6 was used to determine whether GHS-R1α was involved in the ghrelin-induced cardiomyocyte differentiation.Results Compared with control group,the proportion of spontaneously contracting clusters was increased by 10-10,10-9 and 10-8 mol/L of ghrelin [percentages of beating EBs were （12.9± 1.4）％,（19.5±2.2）％ and （13.5±3.1）％ vs （10.3±2.2）％ respectively,P＜0.05].RT-PCR analysis showed that the mRNA levels of α-myosin heavy chain （α-MHC） and cardiac troponin Ⅰ （cTnI） were upregulated in ghrelin-treated group [α-MHC：100％ vs （40.1 ± 13.2）％,P＜0.001 ;cTnI：100％ vs （52.6±9.8）％,P＜0.001].RT-PCR,Western blot and immunostaining analyses revealed that GHS-R1α was detected in the differentiated cells at different stages.However,the proportion of spontaneously contracting clusters and the mRNA level of α-MHC showed similarity between the des-acyl ghrelin-and ghrelin-treated groups.Moreover,the effect of ghrelin on the cardiomyocyte differentiation was unchanged after the addition of the GHS-R1α antagonist.Conclusion Ghrelin enhances cardiomyocyte differentiation of hES cells.The promoting effect of ghrelin on the cardiomyocyte differentiation of hES cells might be mediated via GHS-R1α-independent pathway.