5-氮胞苷诱导脐带间充质干细胞分化为心肌细胞表达cTnI的研究  

Study on cTnI expression of cardiomyocytes induced with 5-azacytidine from human umbilical cord mesenchymal stem cells

作　　者：郑学峰[1] 

机构地区：[1]辽宁医学院附属第一医院消化内科,辽宁锦州121001

出　　处：《现代预防医学》2012年第23期6264-6265,6268,共3页Modern Preventive Medicine

摘　　要：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 confirm the morphology of cardiomyocytes from hUC-MSCs induced with 5-azacytidine, expression of cTnI was identified by immuno-histochemistry method. METHODS The medium of hUC-MSCs of the second passage was changed to complete medium containing 5-azacytidine （ 2.5, 5, 10, 20, 40, 80 μmol / L, respectively）. At second week, the expression of cTnI was identified by immuno-histochemistry method. Thereafter, the cells were cultivated for four weeks. The cellular morphology was observed. At 1st, 2nd, 3rd, 4th week, mRNA of ANP, BNP and a-skeletal actin in hUCMSCs were tested by RT PCR. RESULTS After hUC-MSCs were treated with 5-azacytidine for 7 days, the cellular morphology began to change. The cells were close, parallel and became bigger. The cells with fusiform became less. The cellular morphology of 40μmol / L and 80μmol / L group changed obviously. After 4 weeks, the speed of cellular proliferation declined, some of the cells died. cTnI was positive in 5, 10, 20, 40, 80 μmol / L groups 2 weeks after induced. On the number of cTnI positive cells, there were no significant differences among 10, 20, 40, 80 μmol / L groups, but higer than 5μmol / L group significantly （P﹤0.05）. The mRNA expression of ANP, BNP, a-skeletal actin increased. The levels of l0μmol/ L group were higher than other groups in different points. There were significant differences in most of points （P﹤0.05）. CONCLUSION hUC-MSCs induced with 5-azacytindine show positive cTnI expression, represent special immunohistochemical phenotype of cardiac muscle cells.

关 键 词：脐带间充质干细胞 心肌细胞 基因表达 

分 类 号：R392.1[医药卫生—免疫学]