TRPC对被动致敏的人气道平滑肌细胞的增殖及下游基因表达的作用  

Effects of TRPC on the Proliferation and Expression of Downstream Genes in Passively Sensitized Human Airway Smooth Muscle Cells

作　　者：闫文丽 张晓宇 唐学义 YAN Wen-li;ZHANG Xiao-yu;TANG Xue-yi(Department of Respiratory Crisis,Anyang People's Hospital No.6,He'nan Anyang 455000,China;Department of Respiratory and Critical Care Medicine,He'nan People's Hospital and Zhengzhou University People's Hospital,Zhengzhou 450003,China)

机构地区：[1]河南省安阳市第六人民医院呼吸危重症科,河南安阳455000 [2]河南省人民医院(郑州大学人民医院)呼吸与危重症医学科,郑州450003

出　　处：《循证医学》2021年第5期283-288,308,共7页The Journal of Evidence-Based Medicine

基　　金：国家自然科学基金资助项目(U1304801)。

摘　　要：目的探究经典瞬时感受器电位通道(transient receptor potential channels,TRPC)对被动致敏的人气道平滑肌细胞(human airway smooth muscle cells,HASMC)的增殖及其表达和作用。方法本实验设置分组情况如下:实验组TRPC6阻断剂SKF96365处理的HASMC细胞;TRPC6 siRNA转染的HASMC细胞;对照组TRPC6野生型的HASMC细胞。对不同处理的细胞利用实时荧光定量聚合酶链反应、western blot、细胞计数以及氚胸腺嘧啶核苷掺入试验检测其增殖及其表达和作用。结果利用western blot检测TRPC6蛋白发现TRPC6阻断剂SKF96365和TRPC6 siRNA转染均能够显著减少HASMC中TRPC6的表达(P<0.05)。氚胸腺嘧啶核苷(tritium thymine nucleoside,3H-TdR)掺入实验对TRPC6对HASMC细胞增殖的检测发现10μmol/L的SKF96365处理72 h或TRPC6 siRNA转染细胞48h后,3H-TdR标记的细胞数量显著减少(P<0.05)。细胞计数实验发现,TRPC6阻断剂SKF96365使TRPC6基因表达减少后,细胞数目随着SKF96365浓度的升高而减少(P<0.01),在单一浓度10μmol/L的作用下,细胞数目随着SKF96365处理时间的延长增长速度缓慢(P<0.01),同时通过TRPC6siRNA转染使TRPC6敲除后细胞增殖明显减少。TRPC6阻断剂SKF96365和TRPC6 siRNA转染抑制了TRPC6的表达后,均能够显著降低HASMC中PI3K、AKT、p-AKT、mTOR的表达(P<0.05)。结论TRPC6通过PI3K/Akt/mTOR通路对被动致敏的HASMC的增殖及其表达具有显著的抑制作用,因此本研究为哮喘病的治疗提供新的参考依据。Objective To explore the proliferation,expression and effect of TRPC on passively sensitized human airway smooth muscle cells(HASMC).Methods HASMC cells treated with TRPC6 blocker SKF96365 in the experimental group were grouped as follows:TRPC6 siRNA transfected HASMC cells;Control group TRPC6 wild type HASMC cells.The proliferation,expression and function of tritium thymine nucleoside were detected by real-time fluorescence quantitative polymerase chain reaction,western blot,cell count and tritium thymine nucleoside(3 H-TdR)addition test.Results Western blot analysis of TRPC6 protein showed that TRPC6 blocker SKF96365 and TRPC6 siRNA transfection could significantly reduced the expression of TRPC6 in HASMC(P<0.05).When 3 H-TdR was added to HASMC,it was found that the number of 3 H-TdR labeled cells decreased significantly after 10 mol/L SKF96365 treatment for 72 h or TRPC6 siRNA transfection for 48 h(P<0.05).Cell count experiment found that TRPC6 blocker SKF96365 reduced TRPC6 gene expression,the number of cells with the rising of SKF96365 concentration reduced(P<0.01),in a single concentrations under the action of 10μmol/L,cell number as SKF96365 processing time slowed growth rate(P<0.01),and at the same time through TRPC6 siRNA transfection cell proliferation significantly reduced after the TRPC6 knocked out.After TRPC6 blocking agents SKF96365 and TRPC6 siRNA were transfected,the expression of PI3 K,Akt,p-Akt and mTOR in HASMC were significantly reduced.Conclusions TRPC6 had a significant inhibitory effect on the proliferation and expression of downstream genes in passively sensitized HASMC,through PI3 K/Akt/mTOR pathway.Therefore,this study provided a new reference for the treatment of asthma.

关 键 词：哮喘 TRPC6 HASMC 细胞增殖 

分 类 号：R56[医药卫生—呼吸系统] R329[医药卫生—内科学]