摘要
目的观察钙调神经磷酸酶Aβ亚基(Cn Aβ)基因沉默的乳鼠肥大心室肌细胞内向整流钾电流(Ik1)和动作电位的变化。方法 1天龄SD乳鼠,分离心室肌细胞,培养48 h后换为无血清培养基,将细胞分为A、B、C、D组。A组加入MOI=50的腺病毒空载体,感染6 h时更换成2倍体积新鲜无血清DMEM,培养48 h。B组加入MOI=50的腺病毒空载体,感染6 h换为2倍体积新鲜无血清DMEM,感染24 h时加入终浓度为100μmol/L的PE,培养48 h。C组加入MOI=50的A1,感染6 h时换成2倍体积新鲜无血清DMEM,感染24 h时加入终浓度为100μmol/L的PE,培养48 h。D组加入MOI=50的重组腺病毒sh RNA干扰载体介导沉默编码Cn Aβ的基因(AdCn Aβsh RNA1,A1),感染6 h时换成2倍体积新鲜无血清DMEM,培养48 h。采用Western blotting法检测各组心室肌细胞Cn Aβ蛋白,采用全细胞膜片钳实验检测Ik1,观察并记录动作电位复极20%、50%和90%(APD20、APD50、APD90)时的动作电位。结果 A、B、C、D组心室肌细胞Cn Aβ蛋白相对表达量分别为1. 0±0. 01、2. 59±0. 30、1. 00±0. 16、0. 82±0. 16; B组与A组比较,P <0. 05; C组与B组比较,P <0. 05。与A组比较,B组-140 m V～-90m V时心室肌细胞Ik1电流密度降低(P均<0. 05);与B组比较,C组心室肌细胞Ik1电流密度升高(P均<0. 05)。与A组比较,B组心室肌细胞APD20、APD50、APD90均延长(P均<0. 05);与B组比较,C组B组心室肌细胞APD20、APD50、APD90均缩短(P均<0. 05)。结论 Cn Aβ基因沉默的乳鼠肥大心室肌细胞Ik1电流密度升高,心室肌细胞APD20、APD50、APD90均降低。Cn Aβ可能通过调节Ik1电流密度减小、APD延长参与心室肥大的发生发展。
Objective To observe the change of inward rectifier potassium current(Ik1)and action potential in the calcineurin Aβsubunit(CnAβ)gene silencing hypertrophic ventricular myocytes from neonatal rats.Methods The ventricular myocytes of 1-day-old Sprague-Dawley rats were isolated and cultured for 48 h.The cells were then transferred to serum-free medium and divided into groups A,B,C,and D.The cells in the group A were infected with empty adenovirus vector at an MOI of 50 for 6 h,then the cells were cultured in two volumes of fresh serum-free DMEM for 48 h.The cells in the group B were infected with adenovirus empty vector at an MOI of 50 for 6 h,then the cells were cultured in two volumes of fresh serum-free DMEM for 48h,a total of 100μM of phenylephrine(PE)was added into the DMEM when cultured for 24 h.The cells in the group C were infected with A1 at an MOI of 50 for 6 h,then the cells were cultured in two volumes of fresh serum-free DMEM for 48 h,a total of 100μM of PE was added into the DMEM when cultured for 24 h.The cells in the group D were infected with the recombinant adenovirus shRNA interference vector for silencing the A subunitβsubtype of calcineurin(Ad-CnAβshRNA1,A1)at an MOI of 50 for 6 h,then the cells were cultured in two volumes of fresh serum-free DMEM for 48 h.The protein expression of CnAβwas assayed by Western blotting.Whole cell patch clamp technique was used to record Ik1 and action potential.Results The relative expression levels of CnAβprotein in the groups A,B,C and D were 1.0±0.01,2.59±0.30,1.00±0.16,and 0.82±0.16,respectively.The CnAβprotein expression in cells of group B increased as compared with that of group A(P<0.05),and the CnAβprotein expression in cells of the group C decreased as compared with that of the group B(P<0.05).At a stimulation voltage from-140mV to-90 mV,Ik1 current density in the group B was significantly lower than that in the group A(P<0.05,respectively),and the Ik1 current density in the group C was significantly higher than that in the group B(P<0.0
作者
扶泽南
杨龙
夏桂玲
何炯红
黄勇淇
田野
FU Zenan;YANG Long;XIA Guiling;HE Jionghong;HUANG Yongqi;TIAN Ye(Guizhou Provincial People's Hospital,Guiyang 550002,China)
出处
《山东医药》
CAS
2018年第43期31-34,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81260040)
国家临床重点专科建设项目(2013-544)
贵州省科学技术厅临床研究中心项目[(2017)5405]
关键词
钙调神经磷酸酶
心肌动作电位
内向整流钾电流
心室肥大
calcineurin
myocardial potential
inward rectifier potassium current
ventricular hypertrophy
