Objective:To detect the CHRNA7 gene mutation and polymorphism in Southern Han Chinese patients with nocturnal frontal lobe epilepsy(NFLE).Methods:Blood samples were collected from 215 Southern Han Chinese patients wit...Objective:To detect the CHRNA7 gene mutation and polymorphism in Southern Han Chinese patients with nocturnal frontal lobe epilepsy(NFLE).Methods:Blood samples were collected from 215 Southern Han Chinese patients with NFLE and 200 healthy Southern Han Chinese control subjects.Genomic DNA was extracted,and CHRNA7 whole genome exons were amplified by the polymerase chain reaction and subjected to Sanger sequencing.Results:No CHRNA7 gene mutation was detected in all of the NFLE patients.However,five single nucleotide polymorphisms(SNPs)in sporadic cases were found,located in exons 5,6.and 7 of the CHRNA7 gene.Among them,c.690G>A and c.698A>G are known SNPs,while c.370G>A,c.654C>T,and c.497-498delTG were newly discovered SNPs.These SNPs were also found in some of the healthy controls.Conclusions:No CHRNA7 gene mutation was identified in Southern Han Chinese patients with NFLE.The CHRNA7 gene is probably not responsible for NFLE in this population.展开更多
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C...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。展开更多
基金supported by 2010 National Nature Science Foundation of China General Program(Program NO.8107I046)2012 Guangdong Provincial Science and Technology Program of China(Program NO.2012B032000009)2013 Guangdong Provincial Science and Technology Program of China(Program NO.2013B022000004)
文摘Objective:To detect the CHRNA7 gene mutation and polymorphism in Southern Han Chinese patients with nocturnal frontal lobe epilepsy(NFLE).Methods:Blood samples were collected from 215 Southern Han Chinese patients with NFLE and 200 healthy Southern Han Chinese control subjects.Genomic DNA was extracted,and CHRNA7 whole genome exons were amplified by the polymerase chain reaction and subjected to Sanger sequencing.Results:No CHRNA7 gene mutation was detected in all of the NFLE patients.However,five single nucleotide polymorphisms(SNPs)in sporadic cases were found,located in exons 5,6.and 7 of the CHRNA7 gene.Among them,c.690G>A and c.698A>G are known SNPs,while c.370G>A,c.654C>T,and c.497-498delTG were newly discovered SNPs.These SNPs were also found in some of the healthy controls.Conclusions:No CHRNA7 gene mutation was identified in Southern Han Chinese patients with NFLE.The CHRNA7 gene is probably not responsible for NFLE in this population.
文摘目的: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。
