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目的通过对5-aza诱导人脐带间充质干细胞分化为心肌样细胞后行免疫组化检测cTnI表达,以确定心肌样细胞的形态结构。方法取传2代hUC-MSCs,分别加入终浓度为2.5、5、10、20、40、80μmo1/L的5-aza,培养4周之后观察诱导后的细胞形态学的变化以及生长情况。在诱导后的第二周进行免疫组织化学染色检测cTnI的表达,并计算阳性细胞转化效率。之后采用RT-PCR技术,在诱导后的第1、2、3、4周,对hUC-MSCs的ANP、BNP、a-skeletalactin的基因表达进行检测。结果 5-氮胞苷诱导7d之后,细胞形态出现变化。细胞多呈现紧密生长、平行排列,细胞的体积逐渐增大,梭形细胞的比例逐渐下降。40μmol/L,80μmol/L组细胞形态变化明显。4周时,40,80μmo1/L组诱导的细胞折光性降低、细胞活性减弱,并且出现死亡的细胞。诱导后2周,5、10,20,40,80μmo1/L组hUC-MSCs可见表达阳性的cTnI。cTnI阳性染色细胞数率比较,10μmol/L,20μmol/L,40μmol/L,80μmol/L组之间无明显差异,均明显高于5μmol/L组(P﹤0.05)。RT-PCR检测到ANP、BNP、a-skeletalactin基因表达增加;10μmol/L组基因的表达量在不同时间点与其他各组的基因量相比较在数值上均高于其他各组,且在大部分的时间点上差异有统计学意义(P﹤0.05),仅少部分差异无统计学意义(P﹥0.05)。结论经5-aza诱导的人脐带间充质干细胞有cTnI表达,具有心肌细胞的特异的免疫组化表现。
Objective To detect the expression of cTnI by 5-aza-induced human umbilical cord mesenchymal stem cells into cardiomyocyte-like cells and immunohistochemistry to determine the morphology and structure of cardiomyocyte-like cells. Methods The passage 2 hUC-MSCs were harvested, and 5-aza was added to the final concentration of 2.5, 5, 10, 20, 40 and 80μmol / L, respectively. After 4 weeks, the morphological changes and growth of the cells were observed. In the second week after induction, the expression of cTnI was detected by immunohistochemical staining and the positive cell transformation efficiency was calculated. Afterwards, the gene expression of ANP, BNP, a-skeletalactin in hUC-MSCs was detected by RT-PCR at the first, second, third and fourth weeks after induction. Results After 7 days of 5-azacytidine treatment, cell morphology changed. The cells showed close growth, parallel arrangement, the volume of cells gradually increased, the proportion of spindle cells gradually decreased. The morphological changes of 40μmol / L and 80μmol / L groups were obvious. At 4 weeks, the cells induced by 40 and 80μmol / L decreased the refraction of cells, decreased the cell viability and died of cells. Two weeks after the induction, expression of cTnI was observed in hUC-MSCs of 5, 10, 20, 40 and 80μmol / L groups. The number of cTnI positive stained cells showed no significant difference between 10μmol / L, 20μmol / L, 40μmol / L and 80μmol / L groups, which were significantly higher than that of 5μmol / L group (P <0.05). The mRNA expression of ANP, BNP and a-skeletalactin was increased by RT-PCR. The gene expression of 10μmol / L group was higher than that of other groups at different time points, The difference was statistically significant (P <0.05) at some of the time points, with only a few of them showing no significant difference (P> 0.05). Conclusion 5-aza induced human umbilical cord mesenchymal stem cells have cTnI expression, with specific immunohistochemical expression of myocardial cells.