【作者】 怀娟；

【导师】 岳寿伟；

【作者基本信息】 山东大学 ， 康复医学与理疗学， 2012， 博士

【摘要】 背景瞬时感受器电位离子通道香草素受体亚家族4(transient receptor potential vanilloid receptor4, TPRV4)是一种非选择性通透性阳离子通道,具有机械敏感性,其广泛分布于动物脑、背根神经节(dorsal root ganglion, DRG)、肾和肺等组织。TRPV4可被多种刺激激活,开放时引起细胞内Ca2+浓度的明显升高。TRPV4参与生物体伤害性感受的传导,在DRG神经元介导的疼痛传递过程中起重要作用。大鼠TRPV4全长为871个氨基酸(amino acid, aa),与其他TRP家族成员相似,TRPV4通道结构包括氨基端(N termini, Nt)、羧基端(C termini, Ct)两个胞质内片段及6个跨膜区(TM1-TM6),并在TM5和TM6之间形成一个袢环结构。目前认为,TRPV4的胞质内片段是TRPV4与其他蛋白相互作用的结构域。我们的前期研究提示,TRPV4与DRG损伤后的机械痛敏有关,但持续受压后DRG上TRPV4通道表达升高的机制尚待研究。对持续受压28天的大鼠DRG进行蛋白质组学分析,发现与细胞骨架有关的膜联蛋白A2的表达明显增高,而细胞骨架微管蛋白β5明显降低。因此推测细胞骨架(微管蛋白β5)及其相关蛋白(膜联蛋白A2)可能通过某种途径参与了机械性疼痛传导过程中TRPV4通道表达的调控。膜联蛋白(annexins)家族是一类Ca2+依赖性膜磷脂结合蛋白。膜联蛋白A2(annexin A2)是膜联蛋白家族成员,与内质网膜动力学、囊泡运输等有关。S100A10-膜联蛋白A2复合体可与TRPV5及TRPV6通道结合,调节通道活性。微管蛋白(35(tubulin β5)是微管蛋白的一个亚型。α/p微管蛋白异二聚体是微管的主要组成部分,微管作为重要的细胞骨架成分在许多关键性细胞活动中发挥重要作用。微管与神经元结构与功能有关,微管动力学的改变可以导致神经的病理学改变。微管蛋白二聚体及聚合的微管可以Ca2+依赖性的结合于TRPV1,调节通道功能。目前有研究表明细胞骨架及其相关蛋白,如膜联蛋白A2和微管蛋白p与细胞机械刺激传导过程关系密切,但其分子机制尚不明确。很多既往研究提示TRPV4与膜联蛋白A2及微管蛋白β5存在相互作用。本实验旨在通过基因工程技术构建重组真核表达载体,为下一步研究各蛋白的功能及相互作用提供必要的载体模型。目的通过基因工程学技术,克隆TRPV4及其N、C末端基因,构建标签表达载体。用TRPV4各段及膜联蛋白A2、微管蛋白β5标签质粒转染细胞,行western blot及细胞免疫荧光技术检测其蛋白表达及细胞内分布情况。方法以美国Duke大学W. Liedtke教授赠与的TRPV4cDNA全长质粒为模板,设计特异性引物,采用PCR、酶切、连接、转化、摇菌、质粒提取等步骤,构建TRPV4及其Nt, Ct带Myc标签表达载体。以本课题组制备的膜联蛋白A2及微管蛋白β5的FLAG标签质粒为模板,通过转化、摇菌扩增、质粒提取等步骤获得大量质粒备用。TRPV4全长及胞质内片段、膜联蛋白A2、微管蛋白β5重组质粒转染HEK293细胞后,采用western blot和细胞免疫荧光检测外源基因在转染细胞内的表达。结果1. TRPV4cDNA全长及氨基端、羧基端标签表达载体的成功构建PCR成功扩增TRPV4cDNA全长及胞质内氨基端、羧基端基因片段,产物经琼脂糖凝胶电泳,在相应位置出现DNA条带。重组质粒经PCR、酶切及测序鉴定。测序结果与Genebank基因序列完全一致,插入方向和读码框架正确,重组质粒构建正确。2. TRPV4全长及各段载体、膜联蛋白A2、微管蛋白β5在转染细胞内的稳定表达TRPV4、膜联蛋白A2、微管蛋白β5蛋白表达载体转染细胞蛋白样品,分别在相应98kD、39kD、55kD处出现单一蛋白条带,pcDNA3.1(+)空载体与pcDNA3.1-Myc,His A空载体对照组和阴性对照组无特异条带出现。重组质粒分别转染HEK293细胞48小时后,细胞免疫荧光检测到外源基因在HEK293细胞中有表达。结果提示,TRPV4主要位于细胞膜,胞质内也有少量分布,膜联蛋白A2在细胞膜和胞质内都有分布,微管蛋白β5也广泛分布于胞膜和胞质。转染pcDNA3.1(+)空载体组和阴性对照组的HEK293细胞内未检测到标记的蛋白荧光信号。结论本部分实验成功构建了TRPV4全长及氨基端、羧基端胞质内片段和膜联蛋白A2、微管蛋白p5表达载体,并通过转染使其在细胞内稳定表达。为后续在体外相对单纯的环境下观察TRPV4、膜联蛋白A2、微管蛋白β5在伤害性刺激传导的作用,及各蛋白间的相互作用研究打下基础,提供必要的载体模型。更多还原

【Abstract】 BackgroundTransient receptor potential vanilloid receptor4(TPRV4) is a kind of non-selective cation channels. It is mechanosensitive and widely expressed in the brain, DRG, kidney, and lungs. TRPV4could response to many kinds of stimuli, and cause an obvious increase of intracellular Ca2+. TRPV4plays a role in nociception and mechanical allodynia. The full length of rat TRPV4protein is871aa. Like the other TRP channels, TRPV4possesses six transmembrane domains and two flanking tails in the N-and C-termini. The intracellular terminal domains are considered to be essential in the regulation of channel function and in channel assembly.Our previous studies suggested that TRPV4contributes to mechanical allodynia after CCD. In CCD model, we found an increased expression and sensibilized function of TRPV4channel, with an increased expression of annexin A2and decreased expression of tubulin β5. We supposed that annexin A2and tubulin β5played a role in the regulation of TRPV4channel expression in the transduction of nociceptive stimuli.Annexin A2is a Ca2+-dependent membrane-binding protein. Annexin A2is associated with endosomal membrane dynamics and vesicular trafficking, and participates in fibrinolysis, blood clotting and angiogenesis. The S100A10-annexin A2complex could bind to TRPV5and TRPV6and regulate the channel activities. Tubulin β5is a subtype of β tubulin. Microtubules consisting of α/β tubulin heterodimers are important part of cytoskeleton and play important roles in key cellular events. Microtubules are related to the neuronal structure and function, and changes in microtubule dynamics can induce pathological neurological conditions. Tubulin dimers, as well as polymerized microtubules, can bind to TRPV1in a Ca2+sensitive manner. There were evidences that annexin A2and tubulin β5acted in the mechanotransduction, but the molecular mechnisms were still unknown.The aim of this part is to construct recombinant eukaryotic expression vectors through gene engineering techniques, and to provide necessary vector models for the further studies on the functions and interactions of TRPV4, annexin A2and tubulin β5.ObjectiveTo obtain TRPV4, TRPV4-Nt and TRPV4-Ct genes and construct epitope-tagged eukaryotic expression vectors with gene engineering techniques.To observe the expressions of TRPV4, annexin A2and tubulin β5in the transfected HEK293cells.MethodsThe full-length TRPV4cDNA were provided as a present. Specific primer pairs were designed to construct TRPV4, and its Nt, Ct epitope-tagged expression vectors, following PCR, restriction endonuclease digestion, ligation, transformation and plasmid extraction. The recombinant plasmids of TRPV4, annexin A2and tubulin β5were transfected into HEK293cells.48hours after transfection, western blot and immunofluorescence were used to detect the expression and localization of the proteins in the transfected cells in vitro.Results1. Construction of eukaryotic expression vectors for rat TRPV4and its N&C termini.The size of PCR amplified product of TRPV4and its N&C termini were both consistent with the expected fragments. The recombinant plasmids were verified with PCR, restriction endonuclease digestion and automated nucleotide sequencing.2. Expression of eukaryotic expression vectors of TRPV4, annexin A2and tubulin β5in transfected HEK293cells.TRPV4, annexin A2and tubulin (35plasmids transfected cell protein samples were detected single protein bands at98kD、39kD、55kD respectively. There were none specific bands in the there control groups. Immunofluorescence was used to obserbe the localization of the exogenous proteins. TRPV4protein was detected on the cell membranes, as well as in the cytoplasm. Annexin A2distributed extensively in the cell membranes and cytoplasm. Tubulin beta5was present on both the cell membrane and cytoplasm. FLAG or Myc protein was not detected in pcDNA3.1(+) transfected and un-transfected HEK293cells.ConclusionsWe successfully constructed the eukaryotic expression vectors pcDNA3.1-TRPV4-Myc, His, pcDNA3.1-TRPV4-Nt-Myc, His, pcDNA3.1-TRPV4-Ct-Myc, His, pcDNA3.1(+)-Anxa2-FLAG, and pcDNA3.1(+)-Tubb5-FLAG, which could effectively expressed in HEK293cells. This would facilitate the following invitro study of the molecular mechanisms of TRPV4, annexin A2and tubulin β5in the nociceptive pain conduction and their interactions.更多还原