逆转录酶基因过表达或沉默对HL-1细胞钠电流的作用

Effects of TERT gene overexpression or silencing on sodium current in HL-1 cells

目的研究逆转录酶(TERT)基因过表达或沉默对HL-1细胞钠电流(INa)的作用。方法构建TERT基因过表达或沉默慢病毒载体,感染HL-1细胞,分为过表达慢病毒载体(TERT)组、对照载体(Vector)组、TERT RNA干扰慢病毒载体(shTERT)组、对照载体(shNC)组。应用定量荧光原位杂交(Q-Fish)方法测定端粒长度,应用CCK-8法绘制细胞生长曲线,应用全细胞膜片钳技术记录INa。结果TERT组HL-1细胞端粒长度明显延长,从3.33±1.01延长至4.03±0.94(n=5,P<0.01),且增殖能力明显增强;-35 mV去极化时,与Vector组比较,TERT组HL-1细胞INa电流密度从(-149.5±12.6)pA/pF增至(-213.3±17.2)pA/pF(n=10,P<0.01)。而与shNC组比较,shTERT组INa电流密度从(-141.3±13.5)pA/pF降至(-87.3±9.8)pA/pF(n=10,P<0.01)。TERT组钠通道稳态失活曲线右移,通道关闭态失活时间延长,失活后恢复加速,shTERT组的效应恰好相反。而TERT组、shTERT组钠通道稳态激活和中间态失活变化不大。结论TERT基因对HL-1细胞INa有明显的调控作用,这可能是其改变心房肌细胞电生理的机制之一。

Objective To study the effect of telomerase reverse transcriptase(TERT)gene overexpression or silencing on sodium current(INa)in HL-1cells.Methods HL-1cells were infected with lentivirus vector containing TERT gene overexpression or silencing,which were divided into 4group:TERT group,Vector group,shTERT group and shNC group.Telomere lengths were measured by quantitative fluorescence in situ hybridization(Q-Fish),cell growth curves were plotted by cell counting KIT-8(CCK-8)and INawas recorded by whole-cell patch-clamp technology.Results The telomere length of HL-1cells in the TERT group was significantly prolonged from 3.33±1.01to 4.03±0.94 (n=5,P<0.01),and the proliferation ability was significantly enhanced.At-35 mV depolarization,the INapeak current density in the TERT group increased from(-149.5±12.6)pA/pF to (-213.3±17.2)pA/pF (vs.Vector group;n=10,P<0.01).And the INacurrent density in the shTERT group decreased from (-141.3±13.5)pA/pF to(-87.3±9.8)pA/pF (vs.shNC group;n=10,P<0.01).Furthermore,TERT group could shift the steady-state INaactivation curve of the channel to the right,prolong the INaactivation time of channel closed state and accelerate the recovery after INaactivation.And shTERT group had the opposite effect.However,the over-expression or silencing of TERT had no effect on channel steady-state activation and intermediate-state INaactivation.Conclusion TERT gene has a significant regulatory effect on INain HL-1cells,which may be one of the mechanisms changing the electrophysiology of atrial myocytes.