Apelin-13对5-Aza诱导脐带间充质干细胞向心肌细胞分化的影响

Influence of apelin-13 on 5-azacytidine inducing differentiation of umbilical cord mesenchymal stem cells to cardiomyocytes

目的探讨apelin-13蛋白在体外对5-氮胞苷(5-Aza)诱导人脐带间充质干细胞(hUC-MSCs)向心肌细胞分化的影响。方法采用组织块贴壁法培养hUC-MSCs,胰酶消化传代;取P2代细胞置于不同分化条件的培养液中分化培养14d,实验分为A、B、C、D组(apelin-13浓度分别为0、1×10-6、2×10-6和10×10-6mol/L),各组培养基中5-Aza浓度均为10×10-6mol/L。采用流式细胞仪检测hUC-MSCs表面免疫标记,MTT法检测细胞在570nm处的吸光度(A)值,RTPCR检测肌钙蛋白T(cTnT)、GATA-4 mRNA表达水平,免疫组织化学染色法检测心肌细胞表面标志物cTnT的表达。结果流式细胞仪鉴定结果表明,hUC-MSCs表达CD44、CD90、CD105、CD73、经典人类白细胞抗原Ⅰ类抗原(HLAABC),不表达CD34、CD45、经典人类白细胞抗原Ⅱ类抗原(HLA-DR)。MTT检测显示,0、10×10-6mol/L apelin-13处理的hUC-MSCs在570nm处的A值分别为0.841±0.290、0.875±0.325,差异无统计学意义(P>0.05)。B、C、D组cTnT mRNA表达水平分别是A组的2.09±1.35、5.24±1.30、1.17±0.63倍,B、C、D组GATA-4 mRNA表达水平分别是A组的2.68±1.18、4.82±0.14、2.14±0.27倍;C组与B、D组比较,cTnT、GATA-4 mRNA表达差异具有统计学意义(P<0.05)。免疫组化染色显示,诱导14d后C组细胞cTnT蛋白表达呈阳性。结论 10×10-6mol/L apelin-13对hUC-MSCs无毒性作用,一定浓度的apelin-13蛋白可增强5-Aza诱导hUC-MSCs向心肌细胞分化的效率,浓度为2×10-6mol/L时增强作用最明显。

Objective To explore the influence of apelin-13 on 5-azacytidine （5-Aza） inducing differentiation of human umbilical cord mesenchymal stem cells （hUC-MSCs） to cardiomyocytes. Methods hUC-MSCs were cultivated by tissue adherent method, and subcultured after digested with trypsin. The second passage cells （P2） were cultured for 14 days with different concentrations of apelin-13 （0, 1×10–6mol/L, 2×10–6 and 10×10–6mol/L apelin-13, called as group A, B, C and D respectively）, and the concentration of 5-Aza was 10×10–6mol/L in each group. Immune markers on the surface of hUC-MSCs were detected by flow cytometry, the absorbance （Avalue） of cultured cells at 570nm was detected by MTT method, the mRNA expression levels of troponin T （cTnT） and GATA-4 were determined by RT-PCR, and the expression of cTnT （myocardial cell surface marker） was observed by immunohistochemistry. Results HUC-MSCs expressed CD44, CD90, CD105, CD73 and HLA-ABC, but did not express CD34, CD45 or HLA-DR. The Avalue at 570nm of hUC-MSCs treated with 10×10–6mol/L apelin-13 was 0.875±0.325 and similar to that of hUC-MSCs treated with 0 mol/L apelin-13 （0.841±0.290, P〉0.05）. The expression levels of cTnT mRNA and GATA-4 mRNA in groups B, C and D were 2.09±1.35, 5.24±1.30 and 1.17±0.63 and 2.68±1.18, 4.82±0.14 and 2.14±0.27 times those in group A. The expression levels of cTnT and GATA-4 mRNA in group C were significantly higher than those in groups B and D （P〈0.05）. Immunohistochemical staining showed cTnT protein was positively expressed in group C after induction for 14 days. Conclusions There is no toxic effects of apelin-13 at concentration of 10×10–6mol/L on hUC-MSCs. Certain concentrations of apelin-13 could promote the 5-Aze inducing differentiation of hUC-MSCs to cardiomyocytes, especially at 2×10–6mol/L.