Rad对心肌营养素1诱导心肌细胞肥大作用的实验研究

摘要

目的：探讨小G蛋白Rad对心肌营养素1（CT-1）诱导心肌细胞肥大作用的影响。方法2011年6月—2013年3月，选取新生健康雄性SD乳鼠60只。构建载有Rad基因的腺病毒载体（ Ad-Rad）、含有编码绿色荧光蛋白基因的空白腺病毒载体（ Ad-GFP）。取乳鼠心脏并剪碎，进行心肌细胞原代培养。采用随机数字表法将提取的原代心肌细胞平均分为6组，即空白对照组〔转染磷酸盐缓冲液（ PBS），刺激物为PBS〕、 Ad-GFP组（转染Ad-GFP，刺激物为PBS）、 Ad-Rad组（转染Ad-Rad，刺激物为PBS）、 CT-1对照组（转染PBS，刺激物为CT-1）、 Ad-GFP＋CT-1组（转染Ad-GFP，刺激物为CT-1）、 Ad-Rad＋CT-1组（转染Ad-Rad，刺激物为CT-1）。转染腺病毒载体48 h后，采用流式细胞仪检测腺病毒载体转染率，并采用反转录－聚合酶链式反应（ RT-PCR）法检测Rad、心房钠因子（ ANF）、β肌球蛋白重链（β-MHC） mRNA表达水平， Western blotting法检测Rad表达水平。结果腺病毒载体转染率为95.51％。Ad-Rad组Rad mRNA 表达水平高于空白对照组、 Ad-GFP组、 CT-1对照组、 Ad-GFP ＋CT-1组（ P ＜0.05）； Ad-Rad＋CT-1组Rad mRNA表达水平高于空白对照组、 Ad-GFP组、 CT-1对照组、 Ad-GFP＋CT-1组（ P＜0.05）。 CT-1对照组、Ad-GFP＋CT-1组ANF mRNA 表达水平高于空白对照组、 Ad-GFP组、 Ad-Rad组（ P ＜0.05）； Ad-Rad ＋CT-1组 ANF mRNA表达水平低于空白对照组、 Ad-GFP组、 Ad-Rad组、 CT-1对照组、 Ad-GFP＋CT-1组（ P＜0.05）。 CT-1对照组、Ad-GFP＋CT-1组β-MHC mRNA表达水平高于空白对照组、 Ad-GFP组、 Ad-Rad组（P＜0.05）； Ad-Rad＋CT-1组β-MHC mRNA表达水平低于CT-1对照组、 Ad-GFP＋CT-1组（ P＜0.05）。 Ad-Rad组Rad表达水平高于空白对照组、 Ad-GFP组、CT-1对照组、 Ad-GFP＋CT-1组（ P＜0.05）； Ad-Rad＋CT-1组Rad表达水平高于空白对照组、 Ad-GFP组、 CT-1对照组、Ad-GFP＋CT-1组（ P＜0.05）。结论 Rad具有负性调节CT-1诱导心肌细胞肥大的作用。%Objective To explore the effects of small G -protein Rad on cardiotrophin -1 ( CT-1 ) induced myocardial cells hypertrophy.Methods From June 2011 to March 2013, we selected 60 newborn healthy male SD rats.Ad-Rad with Rad genes and Ad-GFP containing gene encoding green fluorescent protein were made.Hearts were taken out from rats and were cut into pieces to conduct myocardial cell primary culture.Using random number table method, we divided the primary myocardial cells into 6 groups: blank control group 〔transfection by phosphatebuffer solution ( PBS ) with PBS as irritant〕, Ad-GFP group (transfection by Ad-GFP with PBS as irritant), Ad-Rad group (transfection by Ad-Rad with PBS as irritant), CT-1 control group (transfection by PBS with CT-1 as irritant), Ad-GFP+CT-1 group (transfection by Ad-GFP with CT-1 as irritant) and Ad-Rad+CT-1 group ( transfection by Ad-Rad with CT-1 as irritant ) .After adenovirus vector transfection for 48 hours, we detected transfection rate of adenovirus vector by flow cytometry, detected the mRNA expression levels of Rad, ANF andβ-MHC by RT-PCR method and measured Rad expression by Western blotting method.Results The transfection rate of adenovirus vector was 95.51%.The expression level of Rad mRNA in Ad-Rad group was higher than that in control group, Ad-GFP group, CT-1 control group and Ad-GFP +CT-1 group ( P <0.05 ); the expression level of Rad mRNA in Ad-Rad+CT-1 group was higher than that in control group, Ad-GFP group, CT-1 control group and Ad-GFP+CT-1 group (P<0.05). The expression level of ANF mRNA in CT-1 control group and Ad-GFP +CT-1 group was higher than that in control group, Ad-GFP group and Ad-Rad group (P<0.05); the expression level of ANF mRNA in Ad-Rad+CT-1 group was lower than that in control group, Ad-GFP group, Ad-Rad group, CT-1 control group and Ad-GFP+CT-1 group (P<0.05).The expression level ofβ-MHC mRNA in CT-1 control group and Ad-GFP+CT-1 group was higher than that in control group, Ad-GFP group and Ad-Rad group ( P<0.05); the expression level ofβ-MHC mRNA in Ad-Rad+CT-1 group was lower than that in CT-1 control group and Ad-GFP+CT-1 group ( P<0.05 ) .The expression level of Rad in Ad-Rad group was higher than that in control group, Ad-GFP group, CT-1 control group and Ad-GFP +CT-1 group ( P <0.05 ); the expression level of Rad in Ad-Rad+CT-1 group was higher than that in control group, Ad-GFP group, CT-1 control group and Ad-GFP+CT-1 group (P<0.05). Conclusion Rad down-regulates the hypertrophic response of myocardial cells induced by CT-1.