T-cadherin在脂联素抑制缺氧/复氧导致的乳鼠心肌细胞凋亡中的作用*

 目的： 通过腺病毒介导的RNA干扰技术沉默一种新的脂联素（APN）受体T-cadherin的表达，并建立心肌细胞缺氧/复氧(H/R)模型, 观察T-cadherin对H/R所致心肌细胞凋亡的影响。方法： 选用原代培养72 h的心肌细胞进行实验，随机分为5组:（1）空白对照组:心肌细胞正常培养;（2）H/R组;（3）APN+H/R组;（4）Ad-T-cadherin-siRNA+APN+H/R组;（5）Ad-HK（腺病毒阴性对照)+APN+H/R组。首先,确定最适合的腺病毒感染滴度，RT-PCR和Western blotting检测腺病毒介导的干扰RNA对T-cadherin表达的影响效果；其次,通过流式细胞术和TUNEL检测心肌细胞的凋亡。结果： 原代培养获得高纯度的乳鼠心肌细胞。腺病毒感染的最佳MOI值为100，48 h后腺病毒的感染效率和红色荧光蛋白表达最强,其效率高于90%。RT-PCR和Western blotting证实腺病毒介导的siRNA转染的心肌细胞T-cadherin mRNA和蛋白水平均明显下降(P<0.05)。Ad-T-cadherin-siRNA+APN+H/R组心肌细胞凋亡率与APN+H/R组相比显著升高(P<0.05)，与H/R组相比差异无统计学意义(P>0.05)。结论： 重组腺病毒Ad-T-cadherin-siRNA能够在体外高效感染原代心肌细胞,并成功降低T-cadherin在心肌细胞的表达；低表达的T-cadherin阻断了脂联素对缺氧/复氧诱导的乳鼠心肌细胞凋亡的抑制作用。

Effects of APN/T-cadherin against hypoxia/reoxygenation (H/R) induced myocardial apoptosis

ABSTRACT] The aim of the prenent study was to silence a new adiponectin receptor T - cadherin through the adenovirus mediated interference RNA technology. Then establish myocardial cell hypoxia/reoxygenation (H/R) model, observe the influence of myocardial cell injury. METHODS::We isolated primary cardiomyocytes from neonatal rats and established an model of hypoxia/reoxygenation(H/R) in vitro. The cardiomyocytes were randomly divided into 5 groups:(1)the control group: (2) H/R group; (3) H/R +APN group, (4) H/R +APN group + Ad-T-cadherin- siRNA group; (5) H/R group + APN + Ad-HK group (Adenovirus negative control).First of all, the transfection ability and efficiency was examined at various MOI by invert fluorescence microscope. Then the expression of T –cadherin mRNA and protein was detected by RT - PCR and Western blot.Finally, the rate of apoptosis was analyzed through the flow cytometry and TUNEL . RESULTS: High purity neonatal rat cardiomyocytes were obtained by primary culture. After 48h,over 90 % of myocardiocytes were infected at MOI 100. RT - PCR and Western blotting analysis showed that the transfected myocardiocytes lower the expression of T-cadherin under normal physiological condition. Compared with H/R+APN group: cell apoptosis rate significantly increased in silencing T - cadherin gene group,the difference was statistically significant(P<0.05),compared with H/R group, the difference was not statistically significant(P>0.05). CONCLUSION::the Ad-T-cadherin-siRNA could efficientively infection myocardial cells in vitro and successfully reduce the expression of T-cadherin in myocardial cell. The protective effect of adiponectin to hypoxia/reoxygenation (H/R) induced myocardial apoptosis were attenuated by low expression of T-cadherin receptor.