Wnt3a对小鼠胚胎干细胞心肌细胞分化的影响

Effects of Wnt3a on differentiation of mouse embryonic stem cells into cardiomyocytes

目的：观察Wnt3a信号对小鼠胚胎干细胞（embryonicstemcell，ESC）心肌细胞分化的影响。方法：用悬滴培养法促进ESCs形成拟胚体（embryoidbodies，EBs）。用免疫荧光染色法检测心肌特异性蛋白cTnT的表达。在不同分化时间加入Wnt3a及Wnt信号通路抑制剂Dkk一1观察对搏动EBs百分率的影响。用RT—PCR检测Nk同源异型盒5（Nkx2．5）、锌指转录因子-4（GATA．4）、B．肌球蛋白重链（[~-MHC）及心房钠尿肽（ANP）基因表达水平的变化。用Westernblot检测cTnT表达水平的变化。将不加入Wnt3a，分化第0—5天及5～lO天加入Wnt3a的组分别命名为对照组，D0-5组及D5．10组。加入Dkk．1的组命名为Dkk．1组。结果：通过悬滴培养法形成的EBs能够出现自发性搏动并且有TnT表达。EBs形成过程中即分化第0—5天加入Wnt3a与对照组相比具有更高的搏动EBs百分率，Nkx2．5、GATA4、B．MHC和ANP基因表达的水平，以及cTnT蛋白表达水平，而EBs形成后即分化第5—10天加入Wnt3a的结果相反。分化第5—10天加入Dkk．1与对照组相比具有更高的搏动EBs百分率及cTnT蛋白表达水平，并且在分化第0—5天分别加入Wnt3a及Dkk．1与单独加入Wnt3a及Dkk-1相比，搏动EBs百分率及cTnT蛋白表达水平更高。结论：Wnt3a对ESCs向心肌细胞分化的调控呈时间依耐性，EBs形成过程中激活Wnt3a信号及EBs形成后阻断Wnt3a信号能够获得更多的心肌细胞。

AIM： To investigate the roles of Wnt3a in differentiation of mouse embryonic stem cells （ESC） into cardiomyocytes. METHODS： Hanging drop culture was used to induce the differentiation of ESCs into cardiomyocytes through detected by immunocytochemistry points. The percentage of formation of embryoid bodies （EBs）. Cardiac-specific protein TnT was and Wnt3a and its inhibitor Dkk-1 were administered at different time beating EBs was calculated at different time points, mRNA expression of Nkx2.5, GATA4 and 13-MHC were detected by RT-PCR, and the protein expression of TnT was detected by Western blot. The untreated group, the Wnt3a-treated group during days 0 to 5 and the Wnt3a-treated group during days 5 to 10 were referred to as control group, 190-5 group and 1）5-10 group, respectively. The group treated with Dkk-lwas referred to as Dkk-1 group. RESULTS： Spontaneously beating EBs were positively stained with TnT. Wnt3a treatment during EBs formation （days 0 to 5 ） produced higher percentage of beating EBs, higher gene expression of Nkx2.5, GATA4, （3-MHC, ANP, and higher pro- tein expression of TnT compared with those in control group, whereas Wnt3a treatment after EB formation （ days 5 to 10） had the opposite results. Dkk-1 treatment during days 5 to 10 produced a higher percenttime-dependent manner. The activation of Wnt3a during EB formation and the inhibition of Wnt3a after EB formation produce a higher degree of myocardial differentiation.