本文主要研究内容
作者郭欢（2019）在《TRPV1与Nav1.6介导痛敏发育的表达调控机制》一文中研究指出：疼痛是一类复杂的感觉,是一种不愉快的感觉和情绪体验,往往伴随有实际的或潜在的组织伤害,是临床上最常见的病症之一,且具有显著年龄差异。慢性疼痛起源于周围神经系统的伤害感受器,目前关于热伤害感受的知识主要局限于温度传感器,包括背根神经节（DRG）伤害感受神经元中表达的瞬时受体电位阳离子通道TRPV1通道。伤害感受器神经元兴奋性升高或对刺激的反应提高是疼痛产生的重要机制,而神经元细胞膜上的电压门控钠离子通道被激活产生的动作电位也是疼痛产生的主要因素之一。热传感器损失只是部分损害了热伤害感受,伤害性信号的转导也需要钠通道（Nav）辅助,但其中存在的调控机制尚未阐明。因此,本论文主要聚焦在TRP通道与Na+通道的两种基因敲除小鼠,以基因敲除小鼠发育为背景,以基因敲除小鼠的周龄变化为基础,综合应用行为学、分子生物学（荧光定量PCR）、电生理学等技术,来探究在发育过程中TRP通道亚型与Na+通道亚型对机械痛和热痛敏的影响。更进一步揭示两者（机械痛和热痛）在发育过程中相互作用的细胞分子调控机制。实验方法:1.应用弗莱毛测痛法和热板法测定TRPV1基因型小鼠(即TRPV1+/+、TRPV1+/-、TRPV1-/-)和Nav1.6基因型小鼠(即Nav1.6+/+、Nav1.6+/-、Nav1.6-/-)的机械痛阈和热痛阈,由于Nav1.6-/-小鼠在大约21天自然死亡,因此无法进行后续实验。2.应用qRT-PCR实验方法,检测在正常生理状态下,1W-8W龄TRPV1敲除鼠和Nav1.6敲除鼠在DRG神经元上TRP通道各亚型和Na+通道各亚型的表达。3.采用全细胞膜片钳实验方法,检测Nav1.6通道和TRPV1通道DRG神经元早期发育电流变化。结果如下:1.43℃热刺激下对小鼠行为发育响应变化连续热刺激下,与野生型小鼠相比,TRPV1基因敲除后,小鼠体长发育不受影响,而小鼠体重减轻;与野生型小鼠相比,TRPV1+/-、TRPV1-/-小鼠在1W时,机械痛阈升高,且在1W时出现显著性差异（P<0.05）,3W-8W上述三种类型小鼠的机械痛阈无差异;而对于热刺激,TRPV1+/-、TRPV1-/-小鼠的左右缩足潜伏期均升高,在1W和2W时有显著性差异（P<0.05）,3W-8W上述三种类型小鼠的机械痛阈无差异。可见,连续43℃热刺激及TRPV1基因敲除后,小鼠体长发育无变化,而体重减轻,早期机械痛阈上调,热痛反应潜伏期增长,影响小鼠早期发育。小鼠出生1W-8W,连续43℃热刺激TRPV1野生型小鼠的体长和体重均高于不连续43℃热刺激TRPV1野生型小鼠,且存在显著性差异（P<0.05）。提示,不连续43℃热刺激影响小鼠的生长发育;在机械刺激实验中,不连续43℃热刺激机械痛更为敏感;而在检测热刺激引起的缩足潜伏期变化时,缩足潜伏期阈值逐渐增加;出生2W时,与连续43℃热刺激TRPV1野生型小鼠相比,不连续43℃热刺激TRPV1野生型小鼠的缩足潜伏期阈值显著增加（P<0.05）,可见不连续43℃热刺激影响小鼠的生长状况和对疼痛的感知。Nav1.6+/+和Nav1.6+/-两种基因型小鼠的机械阈值随周龄增长呈现递增趋势。与野生型小鼠比较,1W-7W龄Nav1.6+/-小鼠的机械痛阈降低,6W、7W均具显著性差异（P<0.01）,而到8W时,两种基因型小鼠痛阈无差异;连续43℃热刺激下,Nav1.6+/+,Nav1.6+/-小鼠体长、体重之间长幅一致,Nav1.6-/-小鼠体长、体重均明显小于Nav1.6+/+,约21天死亡;随周龄增长,两种基因型小鼠缩足潜伏期呈现降低趋势;连续43℃热刺激下,与野生型小鼠相比,Nav1.6基因缺失(Nav1.6+/-)小鼠热痛反应潜伏期显著降低（P<0.05）。在连续43℃热刺激下,Nav1.6基因缺失(Nav1.6+/-)或敲除(Nav1.6-/-)后,小鼠体长发育减缓;机械痛与热痛反应敏感度增加。2.痛敏相关基因在DRG上mRNA的表达发育变化通过qRT-PCR技术检测TRPV1+/+、TRPV1+/-、TRPV1-/-、Nav1.6+/+、Nav1.6+/-小鼠DRG神经元细胞中与痛敏相关基因mRNA表达情况。Na+通道各亚型与TRP通道各亚型在TRPV1三种转基因型小鼠DRG神经元细胞上均有表达。随周龄增长,TRP通道各亚型与Na+通道各亚型mRNA表达量总体呈现上调的趋势。TRPV1基因敲除后,与野生型小鼠相比,TRPA1基因与TRPV1基因表达下调,相反,Na+通道各亚型mRNA表达上调,以2周龄最为显著。提示,TRPV1基因敲除后差异性调节Na+通道和TRP通道的表达。Na+通道各亚型与TRP通道各亚型在Nav1.6敲除小鼠DRG神经元细胞上均有表达。随周龄增长,野生型小鼠mRNA表达量总体呈现下调趋势,而杂合型小鼠mRNA表达量总体呈现上调趋势,以出生后3周龄最为明显。Nav1.6基因敲除后,Na+通道亚型、TRPA1、TRPV1、TRPV2、TRPV4 mRNA表达量上调。提示,Nav1.6基因敲除后影响Na+通道和TRP通道的表达。因此,TRPV1与Nav1.6基因敲除后影响Na+通道和TRP通道的表达,推测两种通道亚型存在一种互作代偿机制。3.TRPV1与Na+通道电流的发育变化通过全细胞膜片钳技术检测TRPV1+/+、TRPV1+/-、TRPV1-/-小鼠DRG神经元细胞中的电流表达发育情况。结果显示,2W龄TRPV1+/-小鼠DRG神经元细胞的峰值电流约为2300 pA,反转电位在30 mV,激活电位在50 mV。TRPV1+/+小鼠4W的电流峰值电流在约4000 pA,反转电位在18 mV,激活电位在60 mV。TRPV1+/+小鼠5W的电流峰值电流在约2400 pA,反转电位在25 mV,激活电位在60 mV。TRPV1+/+小鼠4W和5W相比,5W小鼠的峰值电流变小,失活减慢。TRPV1三种基因型小鼠2W电流比较,TRPV1基因敲除后,峰值电流减小,Na+通道抑制激活,延缓失活。结论:1.揭示了连续43℃热刺激及TRPV1基因敲除后降低小鼠的基础代谢,且对有害刺激的感知能力降低。其中1W-2W为其关键时期。2.揭示了不连续43℃热刺激影响小鼠体长和体重的生长,对有害刺激的感知能力降低。其中1W-2W为其关键时期。3.揭示了连续43℃热刺激及Nav1.6基因敲除后,小鼠体长不受影响,而体重减轻,对疼痛的感知更为敏感。4.揭示了TRPV1与Nav1.6基因敲除后影响Na+通道和TRP通道的表达,推测两种通道存在一种互作代偿机制,Na+通道不仅可以介导动作电位的传播,也可以响应热。
Abstract
teng tong shi yi lei fu za de gan jiao ,shi yi chong bu yu kuai de gan jiao he qing xu ti yan ,wang wang ban sui you shi ji de huo qian zai de zu zhi shang hai ,shi lin chuang shang zui chang jian de bing zheng zhi yi ,ju ju you xian zhe nian ling cha yi 。man xing teng tong qi yuan yu zhou wei shen jing ji tong de shang hai gan shou qi ,mu qian guan yu re shang hai gan shou de zhi shi zhu yao ju xian yu wen du chuan gan qi ,bao gua bei gen shen jing jie （DRG）shang hai gan shou shen jing yuan zhong biao da de shun shi shou ti dian wei yang li zi tong dao TRPV1tong dao 。shang hai gan shou qi shen jing yuan xing fen xing sheng gao huo dui ci ji de fan ying di gao shi teng tong chan sheng de chong yao ji zhi ,er shen jing yuan xi bao mo shang de dian ya men kong na li zi tong dao bei ji huo chan sheng de dong zuo dian wei ye shi teng tong chan sheng de zhu yao yin su zhi yi 。re chuan gan qi sun shi zhi shi bu fen sun hai le re shang hai gan shou ,shang hai xing xin hao de zhuai dao ye xu yao na tong dao （Nav）fu zhu ,dan ji zhong cun zai de diao kong ji zhi shang wei chan ming 。yin ci ,ben lun wen zhu yao ju jiao zai TRPtong dao yu Na+tong dao de liang chong ji yin qiao chu xiao shu ,yi ji yin qiao chu xiao shu fa yo wei bei jing ,yi ji yin qiao chu xiao shu de zhou ling bian hua wei ji chu ,zeng ge ying yong hang wei xue 、fen zi sheng wu xue （ying guang ding liang PCR）、dian sheng li xue deng ji shu ,lai tan jiu zai fa yo guo cheng zhong TRPtong dao ya xing yu Na+tong dao ya xing dui ji xie tong he re tong min de ying xiang 。geng jin yi bu jie shi liang zhe （ji xie tong he re tong ）zai fa yo guo cheng zhong xiang hu zuo yong de xi bao fen zi diao kong ji zhi 。shi yan fang fa :1.ying yong fu lai mao ce tong fa he re ban fa ce ding TRPV1ji yin xing xiao shu (ji TRPV1+/+、TRPV1+/-、TRPV1-/-)he Nav1.6ji yin xing xiao shu (ji Nav1.6+/+、Nav1.6+/-、Nav1.6-/-)de ji xie tong yu he re tong yu ,you yu Nav1.6-/-xiao shu zai da yao 21tian zi ran si wang ,yin ci mo fa jin hang hou xu shi yan 。2.ying yong qRT-PCRshi yan fang fa ,jian ce zai zheng chang sheng li zhuang tai xia ,1W-8Wling TRPV1qiao chu shu he Nav1.6qiao chu shu zai DRGshen jing yuan shang TRPtong dao ge ya xing he Na+tong dao ge ya xing de biao da 。3.cai yong quan xi bao mo pian qian shi yan fang fa ,jian ce Nav1.6tong dao he TRPV1tong dao DRGshen jing yuan zao ji fa yo dian liu bian hua 。jie guo ru xia :1.43℃re ci ji xia dui xiao shu hang wei fa yo xiang ying bian hua lian xu re ci ji xia ,yu ye sheng xing xiao shu xiang bi ,TRPV1ji yin qiao chu hou ,xiao shu ti chang fa yo bu shou ying xiang ,er xiao shu ti chong jian qing ;yu ye sheng xing xiao shu xiang bi ,TRPV1+/-、TRPV1-/-xiao shu zai 1Wshi ,ji xie tong yu sheng gao ,ju zai 1Wshi chu xian xian zhe xing cha yi （P<0.05）,3W-8Wshang shu san chong lei xing xiao shu de ji xie tong yu mo cha yi ;er dui yu re ci ji ,TRPV1+/-、TRPV1-/-xiao shu de zuo you su zu qian fu ji jun sheng gao ,zai 1Whe 2Wshi you xian zhe xing cha yi （P<0.05）,3W-8Wshang shu san chong lei xing xiao shu de ji xie tong yu mo cha yi 。ke jian ,lian xu 43℃re ci ji ji TRPV1ji yin qiao chu hou ,xiao shu ti chang fa yo mo bian hua ,er ti chong jian qing ,zao ji ji xie tong yu shang diao ,re tong fan ying qian fu ji zeng chang ,ying xiang xiao shu zao ji fa yo 。xiao shu chu sheng 1W-8W,lian xu 43℃re ci ji TRPV1ye sheng xing xiao shu de ti chang he ti chong jun gao yu bu lian xu 43℃re ci ji TRPV1ye sheng xing xiao shu ,ju cun zai xian zhe xing cha yi （P<0.05）。di shi ,bu lian xu 43℃re ci ji ying xiang xiao shu de sheng chang fa yo ;zai ji xie ci ji shi yan zhong ,bu lian xu 43℃re ci ji ji xie tong geng wei min gan ;er zai jian ce re ci ji yin qi de su zu qian fu ji bian hua shi ,su zu qian fu ji yu zhi zhu jian zeng jia ;chu sheng 2Wshi ,yu lian xu 43℃re ci ji TRPV1ye sheng xing xiao shu xiang bi ,bu lian xu 43℃re ci ji TRPV1ye sheng xing xiao shu de su zu qian fu ji yu zhi xian zhe zeng jia （P<0.05）,ke jian bu lian xu 43℃re ci ji ying xiang xiao shu de sheng chang zhuang kuang he dui teng tong de gan zhi 。Nav1.6+/+he Nav1.6+/-liang chong ji yin xing xiao shu de ji xie yu zhi sui zhou ling zeng chang cheng xian di zeng qu shi 。yu ye sheng xing xiao shu bi jiao ,1W-7Wling Nav1.6+/-xiao shu de ji xie tong yu jiang di ,6W、7Wjun ju xian zhe xing cha yi （P<0.01）,er dao 8Wshi ,liang chong ji yin xing xiao shu tong yu mo cha yi ;lian xu 43℃re ci ji xia ,Nav1.6+/+,Nav1.6+/-xiao shu ti chang 、ti chong zhi jian chang fu yi zhi ,Nav1.6-/-xiao shu ti chang 、ti chong jun ming xian xiao yu Nav1.6+/+,yao 21tian si wang ;sui zhou ling zeng chang ,liang chong ji yin xing xiao shu su zu qian fu ji cheng xian jiang di qu shi ;lian xu 43℃re ci ji xia ,yu ye sheng xing xiao shu xiang bi ,Nav1.6ji yin que shi (Nav1.6+/-)xiao shu re tong fan ying qian fu ji xian zhe jiang di （P<0.05）。zai lian xu 43℃re ci ji xia ,Nav1.6ji yin que shi (Nav1.6+/-)huo qiao chu (Nav1.6-/-)hou ,xiao shu ti chang fa yo jian huan ;ji xie tong yu re tong fan ying min gan du zeng jia 。2.tong min xiang guan ji yin zai DRGshang mRNAde biao da fa yo bian hua tong guo qRT-PCRji shu jian ce TRPV1+/+、TRPV1+/-、TRPV1-/-、Nav1.6+/+、Nav1.6+/-xiao shu DRGshen jing yuan xi bao zhong yu tong min xiang guan ji yin mRNAbiao da qing kuang 。Na+tong dao ge ya xing yu TRPtong dao ge ya xing zai TRPV1san chong zhuai ji yin xing xiao shu DRGshen jing yuan xi bao shang jun you biao da 。sui zhou ling zeng chang ,TRPtong dao ge ya xing yu Na+tong dao ge ya xing mRNAbiao da liang zong ti cheng xian shang diao de qu shi 。TRPV1ji yin qiao chu hou ,yu ye sheng xing xiao shu xiang bi ,TRPA1ji yin yu TRPV1ji yin biao da xia diao ,xiang fan ,Na+tong dao ge ya xing mRNAbiao da shang diao ,yi 2zhou ling zui wei xian zhe 。di shi ,TRPV1ji yin qiao chu hou cha yi xing diao jie Na+tong dao he TRPtong dao de biao da 。Na+tong dao ge ya xing yu TRPtong dao ge ya xing zai Nav1.6qiao chu xiao shu DRGshen jing yuan xi bao shang jun you biao da 。sui zhou ling zeng chang ,ye sheng xing xiao shu mRNAbiao da liang zong ti cheng xian xia diao qu shi ,er za ge xing xiao shu mRNAbiao da liang zong ti cheng xian shang diao qu shi ,yi chu sheng hou 3zhou ling zui wei ming xian 。Nav1.6ji yin qiao chu hou ,Na+tong dao ya xing 、TRPA1、TRPV1、TRPV2、TRPV4 mRNAbiao da liang shang diao 。di shi ,Nav1.6ji yin qiao chu hou ying xiang Na+tong dao he TRPtong dao de biao da 。yin ci ,TRPV1yu Nav1.6ji yin qiao chu hou ying xiang Na+tong dao he TRPtong dao de biao da ,tui ce liang chong tong dao ya xing cun zai yi chong hu zuo dai chang ji zhi 。3.TRPV1yu Na+tong dao dian liu de fa yo bian hua tong guo quan xi bao mo pian qian ji shu jian ce TRPV1+/+、TRPV1+/-、TRPV1-/-xiao shu DRGshen jing yuan xi bao zhong de dian liu biao da fa yo qing kuang 。jie guo xian shi ,2Wling TRPV1+/-xiao shu DRGshen jing yuan xi bao de feng zhi dian liu yao wei 2300 pA,fan zhuai dian wei zai 30 mV,ji huo dian wei zai 50 mV。TRPV1+/+xiao shu 4Wde dian liu feng zhi dian liu zai yao 4000 pA,fan zhuai dian wei zai 18 mV,ji huo dian wei zai 60 mV。TRPV1+/+xiao shu 5Wde dian liu feng zhi dian liu zai yao 2400 pA,fan zhuai dian wei zai 25 mV,ji huo dian wei zai 60 mV。TRPV1+/+xiao shu 4Whe 5Wxiang bi ,5Wxiao shu de feng zhi dian liu bian xiao ,shi huo jian man 。TRPV1san chong ji yin xing xiao shu 2Wdian liu bi jiao ,TRPV1ji yin qiao chu hou ,feng zhi dian liu jian xiao ,Na+tong dao yi zhi ji huo ,yan huan shi huo 。jie lun :1.jie shi le lian xu 43℃re ci ji ji TRPV1ji yin qiao chu hou jiang di xiao shu de ji chu dai xie ,ju dui you hai ci ji de gan zhi neng li jiang di 。ji zhong 1W-2Wwei ji guan jian shi ji 。2.jie shi le bu lian xu 43℃re ci ji ying xiang xiao shu ti chang he ti chong de sheng chang ,dui you hai ci ji de gan zhi neng li jiang di 。ji zhong 1W-2Wwei ji guan jian shi ji 。3.jie shi le lian xu 43℃re ci ji ji Nav1.6ji yin qiao chu hou ,xiao shu ti chang bu shou ying xiang ,er ti chong jian qing ,dui teng tong de gan zhi geng wei min gan 。4.jie shi le TRPV1yu Nav1.6ji yin qiao chu hou ying xiang Na+tong dao he TRPtong dao de biao da ,tui ce liang chong tong dao cun zai yi chong hu zuo dai chang ji zhi ,Na+tong dao bu jin ke yi jie dao dong zuo dian wei de chuan bo ,ye ke yi xiang ying re 。
论文参考文献
论文详细介绍
论文作者分别是来自延安大学的郭欢，发表于刊物延安大学2019-07-19论文，是一篇关于通道论文,通道论文,背根神经节论文,发育论文,疼痛论文,延安大学2019-07-19论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自延安大学2019-07-19论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：通道论文; 背根神经节论文; 发育论文; 疼痛论文; 延安大学2019-07-19论文;