【摘要】 目的探讨nesprin-1在胚胎干细胞分化为心肌细胞过程中的作用。方法采用悬滴-悬浮-贴壁三步法,5-氮杂胞苷(5-aza)与丹参共同诱导胚胎干细胞分化为心肌细胞,用Western blot与免疫荧光技术检测nesprin-1蛋白表达水平和在分化成熟过程中的改变。同时利用RNA干扰(RNAi)技术抑制nesprin-1蛋白的表达:Ⅰ组(RNA干扰目的序列AAAGCCAAGCACGCAACTA),Ⅱ组(RNA干扰目的序列GGGAACCAACAGTGAGATT),Ⅲ组(RNA干扰目的序列ACCAGGACATTGCGTACTA),对照组(使用无意义的ShRNA干扰序列),观察对胚胎干细胞分化的影响。结果 Nesprin-1各亚型在胚胎干细胞分化为心肌细胞过程中的表达水平有所变化。RNAi后,Ⅰ、Ⅱ、Ⅲ组和对照组心肌细胞分化率分别为(17.78±1.92)%、(36.67±3.34)%、(44.42±5.08)%和(77.78±1.92)%,各干扰组与对照组相比,差异具有统计学意义(P<0.05),且各干扰组分化后心肌组织分化成熟的蛋白标志肌球蛋白(myosin)的表达量减少。结论 Nesprin-1在胚胎干细胞分化为心肌细胞过程中发挥重要作用,不同分子大小的nesprin-1亚型在此分化过程中可能有着不同的功能。更多还原

【Abstract】 Objective To investigate the role of nesprin-1 in mouse embryonic stem cells differentiation into cardiomyocyte.Methods Hanging drop-suspension-adherence method was applied for the differentiation of mouse embryonic stem cells into cardiomyocytes under the inducing of salvia miltorrhiza and 5-azacytidine.Changes in nesprin-1 gene expression were detected by using Western blotting and immunofluorescent assay.RNA interference was used to reduce nesprin-1 protein levels to further investigate the importance of nesprin-1 in mouse embryonic stem cells differentiation,group Ⅰ(target sequence AAAGCCAAGCACGCAACTA),group Ⅱ(target sequence GGGAACCAACAGTGAGATT),group Ⅲ(target sequence ACCAGGACATTGCGTACTA),and group Ⅳ(control group).Results The nesprin1 isoform profile was altered in mouse embryonic stem cells differentiation.The rates of differentiation of the four groups were(17.78±1.92)%,(36.67±3.34)%,(44.42±5.08)%,(77.78±1.92)%;The rate of differentiation of group Ⅳ was higher than RNAi groups and the difference was significant(P<0.05).In addition,compared with the control group,myosin in RNAi groups were dramatically reduced.Conclusion Nesprin-1 played important roles in mouse embryonic stem cells differentiation into cardiomyocyte.Nesprin-1 isoforms might perform different functions in the process of mouse embryonic stem cells differentiation.更多还原