wnt5a诱导心肌祖细胞向心肌细胞分化的作用研究  

Studies on wnt5a inducing differentiation of cardiac progenitor cells to myocardial cells

作　　者：吴良宇 向平[1] 刘玲娟[1] 何通川[2] 房松华 吕铁伟[1] 田杰[1] 李谧[1] WU Liang-yu;XIANG Ping;LIU Ling-juan;HE Tong-chuan;FANG Song-hua;LV Tie-wei;TIAN Jie;LI Mi(Heart Center, Children＇s Hospital of Chongqing Medical University, Key Laboratory of Developmental Disease in Childhood （Chongqing Medical University） of Ministry of Education, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Key Laboratory of Pediatrics of Chongqing Municipality, Chongqing 400014, China;Molecular Oncology Laboratory, University of Chicago, Chicago 60637, USA)

机构地区：[1]重庆医科大学附属儿童医院心内科、儿童发育疾病研究教育部重点实验室、重庆市重点实验室、儿童发育重大疾病国家国际科技合作基地,重庆400014 [2]芝加哥大学分子肿瘤实验室,美国芝加哥60637

出　　处：《解放军医学杂志》2018年第4期316-321,共6页Medical Journal of Chinese People's Liberation Army

摘　　要：目的探讨wnt5a诱导心肌祖细胞CP15-5a向心肌细胞分化的作用。方法利用HEK293细胞扩增重组腺病毒Ad-wnt5a和Ad-GFP;转染CP15-5a细胞后,设置wnt5a组、GFP组和空白对照组。采用流式细胞技术检测Ad-wnt5a和Ad-GFP的转染效率;1周后,通过实时定量PCR(q RT-PCR)检测经Ad-wnt5a诱导的CP15-5a细胞中心肌组织特异性转录因子GATA结合蛋白4(GATA4)、心肌增强因子2C(MEF2C)的表达;2周后,通过Western blotting及免疫荧光技术检测经Ad-wnt5a诱导的CP15-5a细胞中心肌连接因子43(CX43)、心肌肌钙蛋白T(cTnT)的表达;全细胞膜片钳技术检测钠电流(I_(Na))表达。结果 Ad-wnt5a和Ad-GFP转染效率分别为42.8%、44.3%。腺病毒转染CP15-5a细胞1周后,wnt5a组GATA4和MEF2C基因的表达量(分别为1.717±0.220、1.847±0.190)较GFP组和空白对照组(GATA4:1.003±0.087、0.961±0.063,P<0.05;MEF2C:0.456±0.042、0.500±0.095,P<0.05)明显升高,而GFP组与空白对照组比较差异无统计学意义;腺病毒转染2周后,wnt5a组CX43和c Tn T蛋白的表达量(分别为1.597±0.267、0.727±0.100)较GFP组和空白对照组(CX43:0.723±0.047、0.783±0.1333,P<0.05;cTnT:0.217±0.021和0.253±0.102,P<0.01)明显升高,而GFP组与空白对照组比较差异无统计学意义。与GFP组及空白对照组比较,wnt5a组可检测出I_(Na)。结论 wnt5a可诱导心肌祖细胞CP15-5a向心肌细胞分化。Objective To investigate the effect of Wnt5a on inducing differentiation of Cp15-5a cell to myocardial cell. Methods Recombinant adenovirus wnt5a（Ad-wnt5a） and Ad-GFP was amplified with human embryo kidney 293 cells（HEK293 cells）, and then transfected into CP15-5a cells and 3 experiment groups were set up： wnt5a group, GFP group and blank control group. Flow cytometry was used to detect the transfection efficiency of Ad-wnt5a and Ad-GFP. One week after transfection, the expressions of genes GATA binding protein 4（GATA4） and myocardial enhancement factor 2 C（MEF2C） were analyzed by realtime quantitative PCR（qRT-PCR）. Two weeks after transfection, the expressions of cardiac-specific connexin 43（Cx43） and cardiac troponin T（cTnT） in Ad-wnt5a-induced CP15-5a cells were detected by Western blotting and immunofluorescence techniques. The sodium current expression（I（Na）） was detected by whole cell patch clamp techniques. Results The transfection efficiency of Ad-wnt5a and Ad-GFP was 42.8% and 44.3%, respectively. One week after transduction, the expressions of GATA4 and MEF2C were significantly higher in wnt5a group（1.717±0.220 and 1.847±0.190） than in GFP group（1.003±0.087 and 0.456±0.042, P〈0.05） and blank control group（0.961±0.063 and 0.500±0.095, P〈0.05）, while no significant difference existed between GFP group and blank control group. Two weeks after transduction, the expressions of CX43 and cTnT were significantly higher in wnt5a group（1.597±0.267 and 0.727±0.100） than in GFP group（0.723±0.047 and 0.217±0.021, P〈0.05） and blank control group（0.783±0.1333 and 0.253±0.102, P〈0.01）, while no significant difference existed between GFP group and blank control group. I（Na） was detected in the wnt5a group compared with GFP group and blank control group. Conclusion wnt5a may induce differentiation of cp15-5a cell into myocardial cell.

关 键 词：WNT5A 心肌祖细胞 心肌细胞 

分 类 号：R541.6[医药卫生—心血管疾病]