尼古丁受体CHRNA5亚基突变对其功能影响的初步研究  

A Preliminary Research about the Mutation of CHRNA5 on Effects of nAchR Function

作　　者：彭芳[1] 王懿春 廖广园[1] 洪玮[2] 杨嘉琳[1] 高元妹[1] PENG Fang;WANG Yi-chun;LIAO Guang-yuan;HONG Wei;YANG Jia-lin;GAO Yuan-mei(Intensive Care Unit,The Third Affiliated Hospital of Guangzhou Medical University,Guangdong,510150,China;The State Key Laboratory of Respiratory Diseases,The First Affiliated Hospital of Guangzhou Medical University,Guangdong,510120,China)

机构地区：[1]广州医科大学附属第三医院重症医学科,广东广州510150 [2]广州医科大学附属第一医院呼吸疾病国家重点实验室,广东广州510120

出　　处：《现代生物医学进展》2021年第21期4033-4037,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(82000045)。

摘　　要：目的:研究CHRNA5亚基突变对尼古丁受体功能的影响。方法:通过RT-PCR克隆CHRNA5基因片段,并通过重叠延伸PCR方法对CHRNA5亚基基因定点突变,从而构建CHRNA5突变型及野生型表达载体,然后转染至HEK293T细胞中,检测其基因表达。并将其分别与CHRNA3和CHRNB4共转至HEK293T细胞中,检测三质粒共转后的基因表达;通过采用尼古丁持续灌流细胞10 min,然后检测细胞内钙离子内流峰值的变化情况,接下来检测突变对转染后细胞活力的影响。结果:构建的CHRNA5表达载体在转染HEK293T细胞48 h后,能够检测到绿色荧光蛋白的表达,这证明重组载体已成功转染进HEK293T细胞;通过RT-PCR检测出转染细胞CHRNA5 m RNA表达,这证明CHRNA5突变型和野生型均在HEK293T细胞中成功进行了表达。通过尼古丁灌流细胞实验显示突变组和野生型组F340/F380峰值变化均值分别为0.865±0.048和0.447±0.127,突变体组峰值显著高于野生型组(P<0.05)。细胞活力检测实验发现0.01 m M和0.1 m M尼古丁刺激下突变组的细胞活力峰值分别为139%和137%,显著高于野生组的124%和126%,有显著差异(P<0.05)。结论:本研究成功构建了CHRNA5突变及野生型真核表达载体,发现CHRNA5的突变会导致其受体功能性改变,并影响细胞活力。Objective:This study investigated the effect of CHRNA5 mutant on nicotinic acetylcholine receptors.Methods:To cloned the fragments of CHRNA5 by RT-PCR.Overlap extension PCR was utilized for site-directed mutagenesis,to construct eukaryotic expression vector of CHRNA5 mutant.The vectors were transfected into the HEK293 T cell,to detect the gene expression.Co-transfected either all three wild-type humanα3,β4 andα5 DNAs or theα5 SNP and wild typeα3,β4 into HEK293 T cells.Nicotine was used to continuously perfuse the cells for 10 minutes,to detect the changes in the peak value of intracellular calcium influx and the effect of mutation on cell viability.Results:CHRNA5 mutation and wild gene fragments were successfully expressed in HEK293 T cells,the expression of green fluorescent protein was successfully detected after 48 hours.The CHRNA5 m RNA expression in the transfected cells was detected by RT-PCR,indicating that the mutant and wild-type of CHRNA5 gene were successfully expressed in HEK293 T cells.The nicotine perfusion cell experiment showed that the mean values of F340/F380 peak changes in the mutant group and the wild-type group were 0.865±0.048 and 0.447±0.127,respectively.The peak values of the mutant group were significantly higher than those of the wild-type group(P<0.05).Cell viability testing experiments found that the peak values of cell viability after the stimulation of 0.01 m M and 0.1 m M nicotine in the mutant group were 139%and 137%,which were significantly higher than those of 124%and 126%in the wild-type group(P<0.05).Conclusions:This study successfully constructed the eukaryotic expression vectors of mutant and unmutated CHRNA5 gene.It confirmed that the mutation of CHRNA5 may cause functional changes related to nicotine receptors and affected the cell viability.

关 键 词：尼古丁受体 CHRNA5 细胞活力 

分 类 号：R-33[医药卫生] Q593[生物学—生物化学]