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目的探讨三碘甲腺原氨酸(thyroxine,T3)在诱导小鼠诱导多能干细胞(mi PSC)向心肌细胞分化过程中非基因的机制作用。方法培养Oct4-GFP+mi PS细胞,利用悬滴培养形成拟胚体(embryoid body,EB)的方法诱导mi PSC向心肌细胞分化,在诱导过程中分别添加甲状腺素T3和甲状腺素类似物三碘甲腺乙酸(triiodothyroacetic acid,Triac)。实验共分对照组、T3处理组、T3+Triac处理组。分别在诱导分化第4、6和12天,在倒置显微镜下观察细胞的分化情况,采用免疫荧光染色法检测分化第12天心肌细胞特异性结构蛋白α-actinin及肌钙蛋白(cardiac troponin,c Tn)T的表达,Western blot法检测c Tn T和p-JNK/JNK的表达情况。结果诱导分化第4、6和12天,T3处理组跳动EB数量明显多于对照组和T3+Triac组(P<0.05);免疫荧光染色显示T3处理组的α-actinin和c Tn T表达量较对照组和T3+Triac组明显增强(P<0.05);Western blot结果表明T3显著提高c Tn T的表达量(P<0.05);细胞内信号分子JNK蛋白的磷酸化水平明显大于对照组和T3+Triac组(P<0.05)。结论甲状腺素通过的非基因机制促进mi PS细胞向心肌细胞分化,这种作用机制可能跟JNK信号通路有关。
Objective To investigate the non-gene mechanism of thyroxine (T3) in inducing the differentiation of mouse induced pluripotent stem cells (mi PSCs) into cardiomyocytes. Methods Oct4-GFP + mi PS cells were cultured and induced by suspension culture of embryoid body (EB). Mi PSCs were induced to differentiate into cardiomyocytes. During the induction, thyroid hormone T3 and triiodothyronine analogues Triiodothyroacetic acid (Triac). The experiment was divided into control group, T3 treatment group, T3 + Triac treatment group. Differentiation of cells was observed under an inverted microscope on the 4th, 6th and 12th day after differentiation respectively. The expression of α-actinin and cardiac troponin (cTnI) on the 12th day after differentiation was detected by immunofluorescence staining Tn) T expression was detected by Western blot cTn T and p-JNK / JNK expression. Results On the 4th, 6th and 12th day after differentiation, the number of EBs in T3 group was significantly higher than that in control group and T3 + Triac group (P <0.05). Immunofluorescence staining showed that the expression of α-actinin and cTnT (P <0.05). Compared with control group and T3 + Triac group, the level of cTn T was significantly increased by Western blot (P <0.05). The phosphorylation level of JNK protein was significantly higher than that of control group and T3 + Triac group T3 + Triac group (P <0.05). Conclusions Thyroxine can promote the differentiation of mi PS cells into cardiomyocytes through the non-gene mechanism, which may be related to the JNK signaling pathway.