SCN10A基因SNP位点对一般人群疼痛敏感性的影响及其机制

发布时间：2018-04-13 07:04

  本文选题：SCN10A基因 + 单核苷酸多态性　； 参考：《华中科技大学》2016年博士论文

【摘要】：背景SCN10A基因编码的钠离子通道Nav1.8主要表达在背根神经节(Dorsal root ganglion, DRG)神经元,其生理和功能特性决定了Nav1.8通道能显著影响DRG神经元的兴奋性,进而影响人体疼痛敏感性。近年来,陆续有研究发现在特殊的疼痛相关疾病小纤维神经病变患者中存在罕见SCN10A基因突变,并且这些罕见突变可以影响DRG神经元的功能,从而引发患者剧烈疼痛的临床表现。然而,SCN10A基因对一般人群疼痛敏感性的影响作用尚不清楚,在一般人群至今尚无引起疼痛敏感性变化的SCN10A基因多态性位点发现,本研究旨在探索伴随错义突变的SCN10A基因单核苷酸多态性位点(Single nucleotide polymorphism, SNP)对一般人群疼痛敏感性的影响,并通过细胞电压钳和电流钳实验,验证在人群疼痛敏感性调节中起阳性作用的SCN10A基因SNP位点对DRG神经元电生理功能影响的具体机制。方法1.最初探索样本研究,本研究纳入508例健康在校大学生志愿者检测其实验性疼痛敏感性包括机械性疼痛刺激(钝压痛觉阈值,D-PPT和钝压耐痛阈值,D-PTO;锐压痛觉阈值,SPPT和锐压耐痛阈值,SPTO;刺痛觉阈值,QPT)和热痛刺激(热辐射疼痛反应阈值,WLT),同时我们采集受试者血样,并且基于已报道的可能引起功能改变的SNP位点和NCBI数据库中伴随氨基酸错义突变且等位基因频率大于0.05选择SCN10A基因SNP位点,提取受试者血液基因组DNA进行SNP分型检测,然后分析SCN10A基因SNP位点与该最初探索样本受试者疼痛敏感性的关联。2.复制验证样本研究,本研究纳入1025例女性妇科病人受试者作为复制验证样本,以此验证在最初探索样本中与受试者疼痛敏感性显著关联的SCN10A基因SNP位点,因为最初探索样本中仅机械性疼痛感觉阈值被发现与SCN10A基因SNP位点有显著关联,在复制验证样本中我们仅检测了受试者机械性疼痛感觉阈值(D-PPT, D-PTO, SPPT, SPTO, QPT),然后采集受试者血样同上步骤进行SNP分型检测和基因关联分析。3. SCN10A基因SNP位点功能验证研究,选取对上述人群疼痛敏感性影响有确定意义的SCN10A基因SNP位点rs6795970(GA,伴随1073号位置氨基酸Ala变化为Val)进行基因定点突变,然后分别将携带该野生型质粒Nav1.8-Ala 1073及突变型质粒Nav1.8-Val1073通过电转的方式导入SCN10A基因敲出的老鼠DRG神经元细胞中,再提取DRG神经元分别进行电压钳和电流钳实验进行电生理功能检测,比较其电生理功能参数差异,以此验证SCN10A基因SNP位点rs6795970对DRG神经元电生理功能影响的具体机制。结果1.在最初验证样本中,共有496例受试者和5个SCN10A基因SNP位点纳入最后分析,结果显示携带SCN10A基因多态性位点rs6795970突变型纯合子的个体机械性疼痛感觉阈值D-PPT (P0.05)和D-PTO (P0.05)显著大于杂合子和野生型纯合子,其他类型机械性疼痛感觉阈值差异尽管不显著但趋势跟D-PPT和D-PTO相似；另外rs12632942位点野生型纯合子个体机械性疼痛感觉阈值D-PPT (P0.05)和D-PTO (P0.05)显著大于杂合子和野生型纯合子；其余SCN10A基因SNP位点没有发现与实验性疼痛测量阈值存在显著关联(P0.05)。2.在复制验证样本中,共有1005例女性受试者和2个SCN10A基因SNP位点即rs6795970和rs12632942被纳入最后分析,基因关联分析结果显示,仅rs6795970位点对机械性疼痛感觉阈值的影响仍然具有显著统计学意义(P0.05),rs6795970突变型纯合子的个体5种机械性疼痛感觉阈值均显著大于杂合子和野生型纯合子,其中突变型rs6795970纯合子D-PPT和S-PPT与其他基因型个体统计学差异P值分别达到(1.7-2.4)×10-4和(7.6-7.8)×10-6。3.DRG神经元细胞电生理结果分析显示,电压钳结果中,转染突变型rs6795970位点A/A编码Nav1.8-Val 1073通道蛋白可引起通道失活时间显著小于野生型位点G/G编码的Nav1.8-Ala 1073通道蛋白(P0.05),即突变型Nav1.8-Val 1073通道蛋白更容易失活；另外,电流钳结果中,转染突变型rs6795970位点A/A的DRG神经元发生动作电位的频率显著低于野生型位点G/G(P0.05),即突变型Nav1.8-Val 1073通道蛋白可降低DRG神经元兴奋性。结论本研究发现并证实了SCN10A基因多杰性位点rs6795970对一般人群疼痛敏感性有显著影响,rs6795970突变位点可显著降低人群机械性疼痛敏感性,且该位点可显著影响DRG神经元电生理功能；这说明SCN10A基因位点不仅可以引起特殊疼痛疾病,还可影响一般人群疼痛敏感性,该结果可为人群疼痛敏感性预测提供参考,且该位点引起的Nav1.8通道结构变化区域可能作为疼痛干预的潜在靶点。
[Abstract]:The sodium channel Nav1.8 gene encoding the major background of SCN10A expression in the dorsal root ganglion (Dorsal root, ganglion, DRG) neurons, the physiological and functional characteristics of Nav1.8 is determined by the channel can significantly affect the excitability of DRG neurons, thereby affecting human sensitivity to pain. In recent years, many studies have found that rare mutations of SCN10A gene in patients with neuropathy small fiber special pain related diseases, and these rare mutations may affect the function of DRG neurons, causing severe pain in patients with clinical manifestations. However, the effect of SCN10A gene on the general population of pain sensitivity and the effects is not clear, found in the SCN10A gene polymorphism in the general population has no pain sensitivity in this study. In order to explore the site with single nucleotide polymorphism of SCN10A gene missense mutations (Single nucleotide, polymorphism, SNP) on The general population influence pain sensitivity, and the cell voltage clamp and current clamp experiments, verify the specific mechanism of SCN10A SNP gene positive effect on electrophysiological function of DRG neurons in the regulation of pain sensitivity in the population. Methods 1. samples of the initial exploration, the research into the detection of 508 cases of healthy college students volunteer experimental pain sensitivity including mechanical pain stimulation (blunt pressure pain threshold and pain tolerance threshold D-PPT blunt pressure, D-PTO; acute pressure pain threshold and pain tolerance threshold pressure SPPT sharp, SPTO; pain threshold, QPT) and heat pain stimuli (pain threshold, thermal radiation, and reaction WLT) we collected subjects the blood samples, and based on the reported amino acid may cause missense mutation and allele frequencies greater than 0.05 SCN10A SNP gene with altered function of the SNP locus and NCBI database, extract the subjects blood group For group DNA for SNP genotyping, and association analysis of.2. replication samples SCN10A gene SNP and the initial exploration of the sample subjects of pain sensitivity, this study included 1025 cases of female patients undergoing gynecological subjects as replication samples to verify in the initial exploration and the subjects in the sample of SCN10A gene SNP site of pain sensitivity was significantly associated, because the initial exploration in the sample only mechanical pain threshold was found to be significantly associated with the SCN10A gene SNP locus in replication samples we only tested mechanical pain threshold (D-PPT, D-PTO, SPPT, SPTO, QPT), and then collected the subjects blood samples were the same step for SNP genotyping and genetic association analysis to verify the.3. SCN10A SNP gene function, selection of the population have pain sensitivity determined rs67 SCN10A gene SNP locus significance 95970 (GA, with the number 1073 position amino acid Ala changes to Val) by site directed mutagenesis, and then carrying the wild type and mutant plasmid Nav1.8-Ala 1073 plasmid Nav1.8-Val1073 by electroporation method into SCN10A gene knockout mouse DRG neurons, DRG neurons were extracted from the voltage clamp and current clamp test electrophysiological function, the electrophysiological function parameter differences, in order to verify the specific mechanism of SCN10A gene SNP locus rs6795970 effect on electrophysiological function of DRG neurons. The results in the first 1. samples, a total of 496 subjects and 5 SCN10A gene SNP sites included in the final analysis, the results show the individual mechanical pain perception threshold D-PPT carrying SCN10A gene polymorphism in rs6795970 mutation homozygote (P0.05) and D-PTO (P0.05) was significantly greater than the heterozygous and homozygous wild type, other types of The mechanical pain threshold while no significant difference feeling but the trend with D-PPT and D-PTO and rs12632942 loci similar; wild homozygous individual mechanical pain perception threshold (P0.05) and D-PPT D-PTO (P0.05) was significantly higher than heterozygotes and wild-type homozygotes; the rest of the SCN10A gene SNP locus showed no significant association with experimental pain threshold measurement there is (P0.05).2. in the replication sample, a total of 1005 cases of female subjects and 2 SCN10A SNP gene rs6795970 and rs12632942 were included in the final analysis, gene association analysis showed that only rs6795970 loci on mechanical pain threshold is statistically significant (P0.05), rs6795970 mutation individuals homozygous 5 mechanical pain threshold were significantly greater than heterozygotes and wild-type homozygotes, the mutant homozygous D-PPT and S-PPT and rs6795970 Individual difference other genotypes P value respectively (1.7-2.4) * 10-4 (7.6-7.8 * 10-6.3.DRG) and neuronal cell electrophysiological results show that the voltage clamp results, Nav1.8-Ala 1073 channel protein transfection of mutated rs6795970 loci A/A encoding Nav1.8-Val 1073 channel protein can cause the inactivation time was significantly less than that of wild type G/G loci encoding (P0.05), the mutant Nav1.8-Val 1073 channel protein more susceptible to inactivation; in addition, the current clamp results, transfection of mutant rs6795970 sites of A/A DRG neurons action potential frequency was significantly lower than that of wild type G/G (P0.05), the site of mutant Nav1.8-Val 1073 channel protein can reduce the excitability of DRG neurons in this study. Conclusion found and confirmed the SCN10A gene loci rs6795970 Dorje has a significant impact on the general population sensitivity to pain and rs6795970 mutations can significantly reduce the population of machinery Pain sensitivity, and the site can significantly affect DRG neural electrophysiological function; this shows that SCN10A gene can not only cause special pain disease, can also affect the general population sensitivity to pain, the results can provide references for people with pain sensitivity prediction, a potential target for the changes of the Nav1.8 channel structure and the regional sites may be caused by pain intervention.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R614
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本文编号：1743424