CN117904104A - 一种基于Sort1基因的干扰RNA及其重组腺相关病毒在外周镇痛中的应用 - Google Patents
一种基于Sort1基因的干扰RNA及其重组腺相关病毒在外周镇痛中的应用 Download PDFInfo
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Abstract
一种基于Sort1基因的干扰RNA及其重组腺相关病毒在外周镇痛中的应用，并将它们应用在缓解神经病理性疼痛中。本发明通过设计特异性下调Sortilin表达的siRNA片段，为增加转染效果将RNA片段进行胆固醇修饰，通过DRG内注射的方式实现了Sortilin对外周感觉神经元上TREK1/2的表达水平的调控作用，从而实现缓解前驱糖尿病神经病理性疼痛，并再次基础上包装基于该干扰序列的重组腺相关病毒，发现外周感觉神经元内注射此类AAV病毒后仍可实现Sortilin对外周感觉神经元上TREK1/2的表达水平的调控作用，且作用时间更为持久、稳定。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及一种基于Sort1基因的干扰RNA及其重组腺相关病毒在外周镇痛中的应用。
背景技术
1、前驱糖尿病神经病理性疼痛的研究现状；
众所周知，疼痛是一种复杂的体验与经历，包括感觉识辨、情绪动机和认知评价三个主要组成部分。当疼痛加剧或机体受到炎症或各种损伤侵袭时，疼痛不再起到保护作用，逐渐发展为慢性痛，不仅可以造成感觉功能障碍(自发性疼痛，痛觉过敏和异常痛敏)，还会通过上行传导通路影响到诸多高级脑功能。神经病理性痛是临床治疗慢性痛的重点和难点问题之一。神经病理性痛(neuropathic pain)是由外周或中枢水平躯体感觉系统的损伤和疾病而直接造成的疼痛。其发病机制复杂，迁延不愈，可累及多神经系统，其中糖尿病神经病理性疼痛、外周神经损伤、带状疱疹后神经痛等难以根治的慢性疼痛多为累及外周神经系统的病理性疼痛。
前驱糖尿病神经病理性疼痛(Prediabetic neuropathic pain)是一种在糖尿病前驱期发生的神经病理性损伤和疼痛。前驱糖尿病的血糖值介于正常和高血糖之间(5.6-6.9mmol/L)的空腹血糖异常(Impaired fasting glucose，IFG)或糖耐量异常状态(Impaired glucose tolerance，IGT)。据流行病学统计，当前全球约3.74亿人罹患前驱糖尿病，预计至2045年患者人数会增加到5.48亿，约占全球人口的8.6％。而我国前驱糖尿病的患病率也从2008年的15.5％上升至2017年的35.2％，增涨迅速。随着前驱糖尿病概念的提出，大量研究显示非高血糖状态下患者即可罹患神经病变，约40-50％的前驱糖尿病患者会出现疼痛、自主运动紊乱样的神经病变。
临床研究已证实在糖尿病进展过程中，神经病变先于血糖改变。在功能研究上发现，这些外周神经病变可累及支配感觉和运动的神经纤维，神经电活动增强；在神经结构研究上，发现有髓鞘和无髓鞘神经纤维均发生损伤。在前驱糖尿病的迁延过程中，神经组织的功能紊乱往往会伴随着神经结构的进行性损伤而发生。许多研究显示在前驱糖尿病阶段即会出现周围神经神经纤维传导速度减慢，线粒体功能受损、氧化-硝化应激增加。而我们实验室的前期研究也发现，高能饮食的大鼠在尚未发展为糖尿病的前驱阶段即可出现痛觉敏化以及不同程度的神经损伤，特别是粗纤维脱髓鞘，薄髓鞘及无髓鞘神经纤维受损。但是由于该神经病变的诱因多样、发生机制复杂，目前尚未能对其发生机制加以较好的阐释，也缺少具有靶向性的干预方法。另外，糖尿病前驱期被认为是一个干预治疗的最佳窗口期，及时给予治疗可以更为有效的逆转病变，因此探索用于糖尿病前驱期的治疗药物和措施具有重要的医学价值。
2、神经病理性疼痛镇痛药物的研究现状；
在过去的几十年里，虽然疼痛神经生物学的机制研究取得了很大的进展，但是在治疗慢性疼痛，特别是神经病理性疼痛的镇痛药物的研发上依然面临很大挑战(Woolf,2010)。疼痛治疗的主要目的是减轻患者的临床症状，使患者机体功能完全恢复，提高他们的生活质量。目前临床上治疗疼痛的方法很多，其中药物治疗是治疗疼痛最基本、最常用的方法，使用得当多数患者可以得到很好地镇痛效果。患者个体差异很大，因此要用药要根据个体化原则设计方案，按固定时间定时间隔给药，就可以取得很好地镇痛效果。目前临床上有90％的镇痛药都是以必利通等为代表的水杨酸类(非甾体抗炎药物)和可待因为代表的阿片类药物，其他还有抗抑郁药、抗惊厥药、神经安定药、以及NMDA受体拮抗剂、激素等(Schug and Goddard,2014)。尽管慢性疼痛在人群中患病率非常高，但是目前针对慢性疼痛的治疗策略非常有限，用于疼痛治疗的止痛药存在着各种不良反应。对于严重疼痛的患者来说，这些药物的治疗效果不佳，尤其是阿片类药物，它可以激动内源性阿片受体，通过抑制突触前递质释放，减少痛觉兴奋性冲动的递质释放，如P物质、乙酰胆碱。但是它的连续使用容易使患者对药物产生耐受性和成瘾性。非甾体抗炎药主要通过抑制环氧合酶，从而减少炎症局部前列腺的合成达到有效地镇痛效果，但是容易使病人产生严重的胃肠道反应。除了药物治疗，还有神经阻滞疗法、针刺镇痛疗法、物理和心理疗法辅助疗法等。神经阻滞是指在脑、脊神经、交感神经末梢内或者附近注入药物或者阻断神经传导功能。而对于针刺镇痛，张香桐教授定义其机制可能是“针刺镇痛是痛觉传入信号和穴位传入的信号在中枢神经系统内相互作用的结果”。它是对神经体液进行调节，是一种多通路、生理性调节综合过程。既往研究证明，中枢神经系统各级水平，如：脊髓、脑干、丘脑、和皮层都参与了针刺镇痛过程，同时，这些脑区通过针刺信号释放五羟色胺、吗啡等多种中枢递质。但是针刺治疗效果也是极其有限(Kiss,2015；Moore et al.,2015；Planjar-Prvan et al.,2004；Schug and Goddard,2014)。可以影响疼痛调节系统的药物成分依然是疼痛治疗的主要选择。
在临床中，传统的镇痛药物如阿片类仍然是中重度疼痛治疗的重要方案，虽然阿片类镇痛药可以缓解急性痛患者手术或术后疼痛，但对慢性神经病理性痛的治疗效果甚微，同时，阿片类物质可以使疼痛的敏感性发生改变(如痛敏)。NSAIDs(非甾体类抗炎药物)可以通过抑制PGE2的合成对炎性痛起到镇痛作用，进而减少外周和中枢敏化，但对长期慢性神经病理性疼痛的疗效不佳，并有明显的胃肠道和心血管系统的副作用。三环类抗抑郁药和抗惊厥药被应用于各种神经病理性痛的治疗。例如α2结合物普瑞巴林和加巴喷定可以作用于VGCCs而作为治疗神经病理性痛的有效药物。但同样因作用靶分子分布广泛而具有明显的副作用。近年一些作用于新近发现的分子靶点的药物也在进行临床试验，鞘内注射齐考诺肽可以结合孔形α亚单位阻断N型VGCC来减轻剧烈疼痛和慢性痛。TRPV1拮抗剂在临床II期试验中可以有效的减轻骨关节疼痛。
因此，开展疼痛的机制研究和探索疼痛治疗新策略，寻找更好更有效的镇痛分子，运用现代镇痛新药物、新技术为疼痛患者减轻疼痛，提高生活质量，仍然是疼痛医学领域亟待解决的关键问题。
3、疼痛治疗之基因疗法及干细胞在疼痛治疗中的应用；
对于慢性疼痛尤其是神经病理性痛来说，现在的临床治疗只有部分起效，并且受到诸多随之产生的副作用的限制，或者有成瘾的潜在风险。高效的镇痛药物通常只具有短期的镇痛疗效。近年来，疼痛的神经生物学基础研究取得了巨大的进展。通过治疗基因转染使局部神经递质产生增加可能达到预期的治疗效果，并且避免了全身用药引起的其他通路同一受体的激活导致的副作用。在炎性痛或神经病理性痛动物模型的研究中，最常用的病毒载体通常基于重组腺病毒、腺伴随病毒、慢病毒属、单纯疱疹病毒来表达内源性阿片肽类(脑啡肽、内吗啡肽)、谷氨酸脱羧酶67或抗炎细胞因子(可溶性TNF受体，IL-4和IL-10)。以单纯疱疹病毒作为载体基因疗法利用该病毒的自然生物特性来将外源性基因转染至宿主细胞。利用表达靶基因siRNA的病毒载体可以敲除造成脊髓痛相关神经元激活或者敏化的分子的合成基因。非病毒基因转染表达IL-10可以减轻动物模型的慢性疼痛。人类第一例用于慢性疼痛的基因疗法是2008年12年在密歇根大学开始I期试验，试验构建了可以表达前脑啡肽原的非复制单纯疱疹病毒载体来治疗顽固性的癌性痛。
另外，干细胞应用于慢性疼痛治疗具有很好的前景。骨髓间充质干细胞可以产生抗炎细胞因子和生长因子。CD34细胞可以产生血管生长因子，并且在有些情况下可以直接分化为神经元。鞘内应用分别从胎盘和脐带血中分离出来的骨髓间充质干细胞和CD34细胞可以减轻人类脊髓损伤引起的神经病理性痛。
4、前驱糖尿病神经病理性疼痛的潜在治疗靶点；
Sortilin即分拣蛋白，属于Vacuolar protein sorting 10(Vps10)家族，在蛋白质运输和信号传递过程中发挥重要作用，是一种细胞内的蛋白转运体[17]。许多研究提示该蛋白家族与诸多神经退行性病变有关，包括Alzheimer's病、Parkinson's病、Ⅱ型糖尿病等。Sortilin可将反面高尔基网状结构(Trans Golgi Network,TGN)上修饰后的蛋白质运输至细胞膜表面、或分泌小泡、或溶酶体，从而介导物质的分泌与降解。Sortilin在神经系统广泛分布，约10％分布在细胞膜表面，90％位于细胞内，在调节神经元的发育、凋亡、物质转运、分泌等方面均发挥重要作用。而在病理情况下，发现脊髓损伤模型中Sortilin的表达增高；另外抑制Parkinson's病模型中Sortilin的表达能缓解多巴胺神经元的病理状况。在Ⅱ型糖尿病的研究中发现肝细胞中的Sortilin在脂质以及糖代谢过程中也发挥作用，另外它还介导胰岛素抵抗的发生(Shi and Kandror,2005；Strong et al.,2012)。但是目前在糖尿病神经病变，特别是前驱糖尿病神经病理性痛发生的过程中，Sortilin是否参于其中，发挥着什么样的调控作用尚未见有研究报道。而我们的研究发现前驱糖尿病神经病理疼痛动物高表达的Sortilin可以下调背根神经节(Dorsal root ganglion,DRG)神经元上双孔钾通道TREK1/2的表达水平，从而诱发痛觉敏化的发生(未发表结果)。
双孔钾通道TREK1和TREK2(统称TREK1/2)是一种机械门控的，可被花生四烯酸激活的钾通道；两者的基因序列约63％具有一致性，因此这两种通道也具有许多相似的功能特性，例如TREK1/2两种通道均在DRG神经元的中、小细胞上表达，均受脂质、酸性环境以及细胞肿胀等因素调控，可被不饱和脂肪酸激活对细胞产生保护作用；被PKA和PKC的磷酸化作用所抑制；它们对偏酸的PH敏感，细胞内、外的酸会抑制TREK1/2通道的开放(Honore,2007；Lesage et al.,2000)。另外发现TREK1/2通道在疼痛感知、抑郁以及炎症中也发挥着重要作用。例如TREK1－/－的动物出现机械刺激和热刺激的痛觉敏化(Alloui et al.,2006)，而TREK2的功能目前研究较少，但有实验证实激活DRG神经元上的TREK2通道可以减轻神经病理性疼痛(Lesage et al.,2000)；同样TREK2也参与维持静息膜电位水平，激活该通道可以降低神经细胞的兴奋性并减少神经递质的释放。然而在前驱糖尿病引发的脂质代谢紊乱以及酸性物质沉积时TREK1/2通道发生了什么样的变化，这在前驱糖尿病神经病理性痛的产生中发挥着什么样的调控作用？国内、外尚未见有研究报道。我们现在的研究证实：在前驱糖尿病神经病理性痛大鼠的DRG神经元上TREK1/2通道表达下调，且由这两个通道介导的钾电流也明显减小。通过基因干预手段下调正常动物DRG神经元TREK1/2通道的表达后，动物会出现明显的机械刺激和热刺激痛觉敏化(未发表结果)。然而这种钾通道的表达和功能的下调，是什么样的分子、细胞机制介导的？也有研究对其潜在机制进行探讨，发现疼痛过程中许多信号通路也参与其中，例如ERK/JNK、P38等。但是这些信号通路多作用广泛，不适合作为一种治疗疼痛的靶点进行干预，因此新的靶点分子的探索和研究迫在眉睫。2010年Jean Mazella等人在关于抑郁症发生机制的动物研究中发现，在大鼠的海马组织中Sortilin分子的胞外短肽Spadin可以与细胞膜上的Sortilin-TREK1通道复合体结合(结合部位具体在Sortilin上还是TREK上未知)，使该复合体发生内化，继而被溶酶体所降解，致使神经元钾离子通道表达减少，钾电流变小，兴奋性增高(Nykjaer and Willnow,2012；Mazella et al.,2010)；但是目前尚缺乏关于Sortilin-TREK2复合体的研究。另外，在外周感觉神经元上的Sortilin是否可以对TREK1以及TREK2进行调节，从而参与痛敏的发生尚未见文献报道。
在我们的研究发现在大鼠DRG神经元上，Sortilin不仅与TREK1通道蛋白存在相互作用，Sortilin与TREK2也存在相互蛋白互作。而通过基因调控的手段上调正常动物DRG神经元上Sortilin的表达时，而神经元上的TREK1和TREK2的表达水平相应下调，动物出现明显痛觉过敏反应。此外通过给予前驱糖尿病神经病理性疼痛大鼠DRG内注射特异性的Sortilin小干扰RNA(siRNA)后，DRG神经元上的TREK1和TREK2的表达水平升高，动物的痛觉敏化明显缓解(未发表结果)。因此我们推断：在外周神经系统中Sortilin与TREK1/2存在蛋白互作，Sortilin可以通过调控TREK1/2的表达水平来影响疼痛的发生。因此Sortilin可能是通过干预外周神经系统缓解疼痛的潜在分子靶点。
目前临床上治疗疼痛的方法很多，但长期治疗效果不佳。随着各种新技术的出现，也为我们的疼痛治疗带来了曙光，例如基因治疗的发展迅速，越来越多的临床试验将其用于各种治疗，小干扰RNA作用机制的独特性，这类分子能够用来靶向几乎所有疾病相关的兴趣基因。但siRNA疗法的研发时间与小分子、单克隆抗体等相比要短得多。随着用于罕见疾病治疗的siRNA疗法商业化，截至目前，许多siRNA疗法已经进入临床Ⅲ期实验。此外，FDA也批准的基于AAV的疗法分别用于治疗遗传性视网膜疾病和脊髓性肌萎缩症。AAV正处于基因治疗的前沿，临床认可的产品正相继出现，使用AAV载体的基因治疗具有精准靶向，并实现低剂量的体内疗效的优势，因此也具有更为广阔的应用前景。
目前前驱糖尿病神经病理性疼痛的发生机制和治疗仍然是医学研究中亟待解决的问题之一。
发明内容
为了克服上述现有技术的不足，本发明的目的是提供一种基于Sort1基因的干扰RNA及其重组腺相关病毒在外周镇痛中的应用，通过设计特异性下调Sortilin表达的siRNA片段，为增加转染效果将RNA片段进行胆固醇修饰，通过DRG内注射的方式实现了Sortilin对外周感觉神经元上TREK1/2的表达水平的调控作用，从而实现缓解前驱糖尿病神经病理性疼痛，并再次基础上包装基于该干扰序列的重组腺相关病毒，发现外周感觉神经元内注射此类AAV病毒后仍可实现Sortilin对外周感觉神经元上TREK1/2的表达水平的调控作用，且作用时间更为持久、稳定。
为了实现上述目的，本发明采用的技术方案是：
一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)，其特征在于，AAV病毒中shRNA序列：
GGACATGGTCTTCATGCATGTTTCAAGAGAACATGCATGAAGACCATGTCC。
一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)的培育过程，其特征在于，包括以下步骤：
筛选作用的RNA序列、构建质粒、包装AAV病毒以及病毒纯化，得到AAV病毒中shRNA序列；
(1)病毒载体构建
通过PCR扩增或者限制性核酸内切酶酶切的方法获得目的基因，再通过同源重组或者T4 DNA连接的方法组装，将外源片段构建入目的载体，转化E.coli Stbl3感受态细胞，用菌落PCR对转化子进行筛选，阳性克隆的进行测序，经序列比对确定所需要的阳性克隆。
(2)载体构建信息
质粒信息：
目的基因片段和载体的获取：
扩增引物
载体酶切结果：
载体酶切大小：282bp/7112bp
电泳图见图6：
电泳图说明：
2#：载体酶切回收7116bp；
1#：Plus IIDNA Marker(条带自上而下依次为：8kb、5kb、3kb、2kb、1kb、750bp、500bp、250bp、100bp)；
重组质粒构建：
PCR产物插入线性化表达载体
将线性化载体与片段按比例混合，在T4DNA连接酶作用下，实现片段与载体的连接。
所述的通过基因库筛选构建作用于Sortilin蛋白的干扰RNA片段，筛选4个序列：如下：
1)5’-GAAAGUCAGUGGAAAGUAAdTdT-3’，5’-UUACUUUCCACUG ACUUUCdTdT-3’；
2)5’-AGUUGGUUGUUAUCCAGAAdTdT-3’，5’-UUCUGGAUAACAACCAACUdTdT-3’；
3)5’-GCUGAUAAGGAUACAACAAdTdT-3’，5’-UUGUUGUAUCCUUAUCAGCdTdT-3’；
4)5’-GUUCAU GCAUGUAGAUGAAdTdT-3’，5’-UUCAU CUACAUGCAUGAACdTdT-3’。
所述的一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)应用于缓解前驱糖尿病性神经病理性疼痛治疗。
所述的一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)应用于制备神经病理性疼痛的靶点药物。
本发明的有益效果为：
动物疼痛行为学验证：高脂饮食喂养60-80天的大鼠，尽管血糖仍然在正常范围内，但动物会出现胰岛素抵抗()。并伴有明显的机械刺激和热刺激痛觉敏化。给模型动物背根神经节内注射作用于Sortilin的小干扰RNA，3-5天后动物的机械刺激疼痛和热刺激疼痛均明显减轻，背根神经节上Sortilin蛋白表达下降，但TREK1和TREK2通道蛋白表达上调。同样给予该RNA片段的重组AAV病毒进行DRG注射，4周后，动物的机械刺激疼痛和热刺激疼痛均明显减轻，背根神经节上Sortilin蛋白表达下降，TREK1和TREK2通道蛋白表达上调。
因此Sortilin的小干扰RNA及其重组AAV病毒均可有效地缓解前驱糖尿病模型动物的神经病理性疼痛，为神经病理性疼痛的治疗提供了新的靶点药物。
附图说明
图1为构建含有SORT1基因的干扰RNA的质粒；
图2为在外周DRG神经元上，Sortilin与TREK1和TREK2共表达，且存在蛋白相互作用；
图3为DRG内注射小干扰RNA-SORT调控Sortilin蛋白的表达，前驱糖尿病神经病理性疼痛动物的机械刺激和热刺激痛觉敏化明显缓解，且TREK1和TREK2的表达明显增高；
图4为DRG内注射rAAV-SORT1调控Sortilin蛋白的表达后，TREK1和TREK2的表达明显增高，且前驱糖尿病神经病理性疼痛动物的机械刺激和热刺激痛觉敏化明显缓解；
图5为DRG内注射小干扰RNA或rAAV的位置及药物作用机理的示意图。
图6为电泳图。
具体实施方式
以下结合附图及实施例对本发明进一步叙述，但本发明不局限于以下实施例。
一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)，通过基因库筛选构建作用于Sortilin蛋白的干扰RNA片段，筛选4个序列：1).5’-GAAAGUCAGUGGAAAGUAAdTdT-3’，5’-UUACUUUCCACUG ACUUUCdTdT-3’；2).5’-AGUUGGUUGUUAUCCAGAAdTdT-3’，5’-UUCUGGAUAA CAACCAACUdTdT-3’；3).5’-GCUGAUAAGGAUACA ACAAdTdT-3’，5’-UUGUUGUAUCCUUAUCAGCdTdT-3’；4).5’-GUUCAUGCAUGUAGAUGAAdTdT-3’，5’-UUCAU CUACAUGCAUGAACdTdT-3’。
基于上述研究，构建包含有该干扰RNA片段的质粒(如图1所示)，之后进行包装筛选。
外周感觉神经元Sortilin和TREK1、TREK2共表达。如图2所示，(A)通过免疫荧光双标研究发现，前驱糖尿病神经病理性疼痛组及对照组动物的DRG神经元上Sortilin(红色)和TREK1(绿色)共表达，且病变组DRG上Sortilin的荧光强度明显增强，提示Sortilin表达增高，而中间图版中为Sortilin(红色)和TREK2(绿色)共表达。右图统计分析发现80％以上的Sortilin阳性表达神经元同时共标记TREK1和TREK2。(B)免疫共沉淀研究再次验证，发现Sortilin蛋白分别与TREK1、TREK2蛋白之间存在直接相互作用。
Sortilin蛋白的小干扰RNA注射可以增高TREK1和TREK2的表达并缓解疼痛。如图3所示，(A)前驱糖尿病神经病理性疼痛动物L3-L5 DRG内注射Sortilin蛋白的小干扰RNA(10μl/DRG)，5天后灌流取材并进行免疫荧光双标染色，Sortilin为红色，TREK1和TREK2为绿色荧光。(B)平均荧光强度分析统计发现小干扰RNA注射的DRG神经元上Sortilin表达明显下调(P<0.01)，而TREK1和TREK2的表达显著增高(P<0.01)。(C)L3-L5 DRG内注射Sortilin蛋白的小干扰RNA(10μl/DRG)，5天后DRG组织匀浆并进行Western Blot实验，发现小干扰RNA注射的DRG神经元的Sortilin蛋白表达明显下调(P<0.05)，而TREK1和TREK2的表达显著增高(P<0.05)。(E)前驱糖尿病神经病理性疼痛动物L3-L5 DRG内注射Sortilin蛋白的小干扰RNA(10μl)，第3天动物的机械刺激痛觉敏化开始出现缓解(P<0.05)，第5-7天，机械刺激足缩阈值的升高达到峰值水平(P<0.01)。而动物的热刺激足缩潜伏期(PWTL)从注射后第5天开始明显增高(P<0.01)。
基于Sortilin蛋白的重组AAV注射也可以增高TREK1和TREK2的表达并缓解疼痛。如图4所示，(A)动物DRG内注射示意图，对动物L3-L5的DRG进行rAAV注射。(B)rAAV注射4周后，前驱糖尿病神经病理性疼痛动物机械刺激痛觉敏化开始出现缓解(P<0.05)，动物的热刺激足缩潜伏期(PWTL)也明显缩短(P<0.05)。(C)L3-L5 DRG内注射下调Sortilin蛋白表达的rAAV(1μl/DRG)，4周后DRG组织匀浆并进行Western Blot实验，发现rAAV-SORT1注射的DRG神经元的TREK1(P<0.05)和TREK2(P<0.01)的表达显著增高。
对上述研究的给药部位及药物的作用机理进行汇总。如图5所示：注射小干扰RNA及其rAAV于外周神经系统L3-L5的DRG中，发现下调DRG上Sortilin的表达可缓解痛觉敏化。在前驱糖尿病神经病理性疼痛状态下，外周DRG神经元上双孔钾通道TREK1及TREK2的表达下调、神经元兴奋性增高，出现显著的神经病理性疼痛表现。由于Sortilin与TREK1及TREK2通道相互作用，并负调控其表达，因此在DRG注射Sortilin的小干扰RNA或rAAV下调Sortilin蛋白的表达后,DRG上TREK1及TREK2的表达水平上调，使神经元的兴奋性水平降低并缓解神经病理性疼痛。
Claims (5)
1.一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)，其特征在于，AAV病毒中shRNA序列：
GGACATGGTCTTCATGCATGTTTCAAGAGAACATGCATGAAGACCATGTCC。
2.根据权利要求1所述的一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)的培育过程，其特征在于，包括以下步骤：
筛选作用的RNA序列、构建质粒、包装AAV病毒以及病毒纯化，得到AAV病毒中shRNA序列；
(1)病毒载体构建；
通过PCR扩增或者限制性核酸内切酶酶切的方法获得目的基因，再通过同源重组或者T4 DNA连接的方法组装，将外源片段构建入目的载体，转化E.coli Stbl3感受态细胞，用菌落PCR对转化子进行筛选，阳性克隆的进行测序，经序列比对确定所需要的阳性克隆；
(2)载体构建信息；
质粒信息：
基因名称：Sort1，GenBank ID：83576；
目的基因片段和载体的获取，载体名称：rAAV-U6-shRNA2(Sort1)-CMV-mCherry-SV40polyA
扩增引物；
ID：2251-F；
Seq：caccGGACATGGTCTTCATGCATGTTTCAAGAGAACATGCATGAAGACCATGTCC；
ID：2251-R；
Seq：aaaaGGACATGGTCTTCATGCATGTTCTCTTGAAACATGCATGAAGACCATGTCC；
载体酶切结果：
载体酶切大小：282bp/7112bp
电泳图说明：
2#：载体酶切回收7116bp；
1#：Plus IIDNA Marker(条带自上而下依次为：8kb、5kb、3kb、2kb、1kb、750bp、500bp、250bp、100bp)；
重组质粒构建：
PCR产物插入线性化表达载体，将线性化载体与片段按比例混合，在T4DNA连接酶作用下，实现片段与载体的连接。
3.根据权利要求2所述的培育方法，其特征在于，所述的通过基因库筛选构建作用于Sortilin蛋白的干扰RNA片段，筛选4个序列：如下：
1)5’-GAAAGUCAGUGGAAAGUAAdTdT-3’，5’-UUACUUUCCACUG ACUUUCdTdT-3’；
2)5’-AGUUGGUUGUUAUCCAGAAdTdT-3’，5’
-UUCUGGAUAACAACCAACUdTdT-3’；
3)5’-GCUGAUAAGGAUACAACAAdTdT-3’，5’
-UUGUUGUAUCCUUAUCAGCdTdT-3’；
4)5’-GUUCAU GCAUGUAGAUGAAdTdT-3’，5’-UUCAU
CUACAUGCAUGAACdTdT-3’。
4.一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)应用于缓解前驱糖尿病性神经病理性疼痛治疗。
5.一种针对SORT1基因的干扰RNA序列组以及其重组腺相关病毒(AAV)应用于制备神经病理性疼痛的靶点药物。
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