本文選題：SCN10A基因 + 單核苷酸多態(tài)性��； 參考：《華中科技大學》2016年博士論文

【摘要】：背景SCN10A基因編碼的鈉離子通道Nav1.8主要表達在背根神經節(jié)(Dorsal root ganglion, DRG)神經元,其生理和功能特性決定了Nav1.8通道能顯著影響DRG神經元的興奮性,進而影響人體疼痛敏感性。近年來,陸續(xù)有研究發(fā)現(xiàn)在特殊的疼痛相關疾病小纖維神經病變患者中存在罕見SCN10A基因突變,并且這些罕見突變可以影響DRG神經元的功能,從而引發(fā)患者劇烈疼痛的臨床表現(xiàn)。然而,SCN10A基因對一般人群疼痛敏感性的影響作用尚不清楚,在一般人群至今尚無引起疼痛敏感性變化的SCN10A基因多態(tài)性位點發(fā)現(xiàn),本研究旨在探索伴隨錯義突變的SCN10A基因單核苷酸多態(tài)性位點(Single nucleotide polymorphism, SNP)對一般人群疼痛敏感性的影響,并通過細胞電壓鉗和電流鉗實驗,驗證在人群疼痛敏感性調節(jié)中起陽性作用的SCN10A基因SNP位點對DRG神經元電生理功能影響的具體機制。方法1.最初探索樣本研究,本研究納入508例健康在校大學生志愿者檢測其實驗性疼痛敏感性包括機械性疼痛刺激(鈍壓痛覺閾值,D-PPT和鈍壓耐痛閾值,D-PTO;銳壓痛覺閾值,SPPT和銳壓耐痛閾值,SPTO;刺痛覺閾值,QPT)和熱痛刺激(熱輻射疼痛反應閾值,WLT),同時我們采集受試者血樣,并且基于已報道的可能引起功能改變的SNP位點和NCBI數(shù)據(jù)庫中伴隨氨基酸錯義突變且等位基因頻率大于0.05選擇SCN10A基因SNP位點,提取受試者血液基因組DNA進行SNP分型檢測,然后分析SCN10A基因SNP位點與該最初探索樣本受試者疼痛敏感性的關聯(lián)。2.復制驗證樣本研究,本研究納入1025例女性婦科病人受試者作為復制驗證樣本,以此驗證在最初探索樣本中與受試者疼痛敏感性顯著關聯(lián)的SCN10A基因SNP位點,因為最初探索樣本中僅機械性疼痛感覺閾值被發(fā)現(xiàn)與SCN10A基因SNP位點有顯著關聯(lián),在復制驗證樣本中我們僅檢測了受試者機械性疼痛感覺閾值(D-PPT, D-PTO, SPPT, SPTO, QPT),然后采集受試者血樣同上步驟進行SNP分型檢測和基因關聯(lián)分析。3. SCN10A基因SNP位點功能驗證研究,選取對上述人群疼痛敏感性影響有確定意義的SCN10A基因SNP位點rs6795970(GA,伴隨1073號位置氨基酸Ala變化為Val)進行基因定點突變,然后分別將攜帶該野生型質粒Nav1.8-Ala 1073及突變型質粒Nav1.8-Val1073通過電轉的方式導入SCN10A基因敲出的老鼠DRG神經元細胞中,再提取DRG神經元分別進行電壓鉗和電流鉗實驗進行電生理功能檢測,比較其電生理功能參數(shù)差異,以此驗證SCN10A基因SNP位點rs6795970對DRG神經元電生理功能影響的具體機制。結果1.在最初驗證樣本中,共有496例受試者和5個SCN10A基因SNP位點納入最后分析,結果顯示攜帶SCN10A基因多態(tài)性位點rs6795970突變型純合子的個體機械性疼痛感覺閾值D-PPT (P0.05)和D-PTO (P0.05)顯著大于雜合子和野生型純合子,其他類型機械性疼痛感覺閾值差異盡管不顯著但趨勢跟D-PPT和D-PTO相似；另外rs12632942位點野生型純合子個體機械性疼痛感覺閾值D-PPT (P0.05)和D-PTO (P0.05)顯著大于雜合子和野生型純合子；其余SCN10A基因SNP位點沒有發(fā)現(xiàn)與實驗性疼痛測量閾值存在顯著關聯(lián)(P0.05)。2.在復制驗證樣本中,共有1005例女性受試者和2個SCN10A基因SNP位點即rs6795970和rs12632942被納入最后分析,基因關聯(lián)分析結果顯示,僅rs6795970位點對機械性疼痛感覺閾值的影響仍然具有顯著統(tǒng)計學意義(P0.05),rs6795970突變型純合子的個體5種機械性疼痛感覺閾值均顯著大于雜合子和野生型純合子,其中突變型rs6795970純合子D-PPT和S-PPT與其他基因型個體統(tǒng)計學差異P值分別達到(1.7-2.4)×10-4和(7.6-7.8)×10-6。3.DRG神經元細胞電生理結果分析顯示,電壓鉗結果中,轉染突變型rs6795970位點A/A編碼Nav1.8-Val 1073通道蛋白可引起通道失活時間顯著小于野生型位點G/G編碼的Nav1.8-Ala 1073通道蛋白(P0.05),即突變型Nav1.8-Val 1073通道蛋白更容易失活；另外,電流鉗結果中,轉染突變型rs6795970位點A/A的DRG神經元發(fā)生動作電位的頻率顯著低于野生型位點G/G(P0.05),即突變型Nav1.8-Val 1073通道蛋白可降低DRG神經元興奮性。結論本研究發(fā)現(xiàn)并證實了SCN10A基因多杰性位點rs6795970對一般人群疼痛敏感性有顯著影響,rs6795970突變位點可顯著降低人群機械性疼痛敏感性,且該位點可顯著影響DRG神經元電生理功能；這說明SCN10A基因位點不僅可以引起特殊疼痛疾病,還可影響一般人群疼痛敏感性,該結果可為人群疼痛敏感性預測提供參考,且該位點引起的Nav1.8通道結構變化區(qū)域可能作為疼痛干預的潛在靶點。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R614
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本文編號：1743424