CN110947003B - Gpr31抑制剂在制备治疗肾脏缺血再灌注损伤及相关疾病药物中的应用 - Google Patents
Gpr31抑制剂在制备治疗肾脏缺血再灌注损伤及相关疾病药物中的应用 Download PDFInfo
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Abstract
本发明的实验研究发现，随着组织缺血时间的延长，GPR31蛋白的表达量逐渐增加，与组织缺血再灌注损伤严重程度之间存在着正相关的关系。GPR31过表达会加重H/R处理引起的肝细胞、心肌细胞、肾脏细胞的活性。另外，肝细胞中GPR31表达的降低可抑制棕榈酸酯和油酸刺激导致的细胞脂质沉积。心肌细胞中GPR31过表达，会加重血管紧张素Ⅱ导致的心肌肥厚。因此，GPR31抑制剂可用于制备药物，所述药物用于治疗缺血再灌注损伤相关疾病；心肌肥厚及相关疾病；肝脏代谢疾病。
Description
本申请为2017年8月21日提交的，发明名称为“GPR31抑制剂在制药中的应用”的中国专利申请201710719318.9的分案申请。
技术领域
本发明属于生物医药技术领域，具体涉及GPR31抑制剂在制备药物中的用途，所述药物用于治疗缺血再灌注损伤和其相关疾病，心肌肥厚和其相关疾病，心脏的炎症性疾病，脂肪代谢异常及相关疾病等。
背景技术
G蛋白偶联受体(G protein-coupled receptor,GPCR)是一类具有7次跨膜结构的膜蛋白，有超过800个家族成员，是哺乳动物基因组中最大的一类膜蛋白。在人体中，GPCR广泛表达在心血管系统、免疫系统、神经系统等组织器官中，并参与生长发育和多种病理生理过程。由于其分布广泛、功能多样，GPCR家族分子被视为目前最有潜力的药物开发靶点，在FDA批准的上市药物中约20-30％以GPCR为作用靶标。
孤儿受体GPR31是GPCR家族分子，最早于1997年由Alessandra Zingoni等人发现(Zingoni,A.et al.Isolation and chromosomal localization of GPR31,a human geneencoding a putative G protein-coupled receptor.Genomics 42,519-523,doi:10.1006/geno.1997.4754(1997))。目前，对GPR31的研究主要集中在癌症领域，而对其在其他病理过程的功能尚不明确。GPR31蛋白包含319个氨基酸，分子量35KDa，在血小板、免疫细胞以及多种癌症细胞中高表达，包括膀胱癌细胞、乳腺癌细胞、慢性淋巴母细胞性白血病细胞等(Feinmark,S.J.et al.The Orphan Receptor GPR31 Is the Platelet and HUVECReceptor for 12(S)-HETE.Blood 114(2008).)。研究表明，GPR31在临床病人结肠癌组织中表达明显高于癌旁正常组织，且与癌细胞转移率呈正相关，与5年存活率呈负相关(Zou,Y.et al.[Expression and clinical significance of G protein-coupled receptor31in colorectal cancer tissue].Zhonghua Wei Chang Wai Ke Za Zhi 18,935-940(2015).)。GPR31可与RAS家族分子如KRAS、HRAS、NRAS等结合，并调控KRAS的细胞膜定位，从而参与癌症的发生发展(Fehrenbacher N,et al.The G protein–coupled receptorGPR31promotes membrane association of KRAS[J].J Cell Biol,2017:jcb.201609096.)。
缺血再灌注损伤(Ischemia-Reperfusion Injury，IRI)是1960年Jennings首先提出的概念，是指组织器官缺血后血液再灌注，不仅不能使组织器官功能恢复，反而加重组织器官的功能障碍和结构损伤。缺血再灌注损伤在许多重要器官包括心、肝、肺、肾、胃肠道等均可发生。
肝脏缺血再灌注损伤(Hepatic Ischemia Reperfusion Injury，HIRI)是肝脏外科手术中常见的病理过程，多见于休克、需要阻断肝脏血流的肝外科手术以及肝移植术等病理生理过程中。近年来，随着临床治疗技术的发展，肝移植、溶栓治疗以及肝门阻断术等手术的开展越来越多，尽管肝脏保护、外科技巧及术中监护不断改进，但缺血再灌注所致肝损伤依然是引起术后脏器无功能，移植失败甚至患者死亡的主要原因。肝脏经历缺血再灌注后，肝脏组织细胞发生一系列代谢、结构和功能的损伤，易诱发肝功能衰竭，是影响疾病预后、手术成功率和病人存活率的主要原因之一。
急性冠状动脉梗阻性疾病是目前心脑血管疾病的主要致死原因之一。尽管心脏搭桥术、介入及溶栓等治疗有了很大的进步，但急性心梗患者的死亡率依然较高，其中一个很重要的原因就是尚无有效办法抑制缺血心肌恢复血流时所引起的缺血再灌注损伤。冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术，冠状动脉旁路术，都会出现心肌再灌注损伤。心肌再灌注损伤其机制主要有自由基、钙超载、细胞粘附分子介导的中性粒细胞粘附、聚集、渗出，细胞凋亡，一氧化氮，补体系统，肾素-血管紧张素，核因子-κB等。
肾脏也同样是高灌注器官，对缺血以及缺血再灌注均敏感。肾脏缺血再灌注损伤是缺血性急性肾功能衰竭的重要损伤环节，也是肾移植中影响移植肾早期功能恢复的制约因素。造成缺血再灌注损伤的相关因素很多，其中，缺血再灌注引起的炎症级联反应和活性氧氧化损伤是人们所关注的两个主要因素。目前对肾缺血再灌注损伤的防治策略主要包括：抑制白细胞激活及白细胞-内皮细胞相互作用，中和活性氧，抗内皮素。
缺血性脑损伤的机制至少也涉及到以下几个方面：兴奋性毒性、梗死周围去极化、炎症和凋亡。
心肌肥厚是心脏为适应各种刺激而产生的心肌细胞体积增大、重量增加。其病理变化包括心肌细胞肥大、心肌间质细胞增殖以及心脏细胞外基质改建等多方面的改变，即心肌重构。传统观点认为成熟心肌细胞终止分化，丧失有丝分裂能力，不能进入细胞周期；然而目前有证据显示在心脏发育及病理过程中存在心肌细胞的凋亡和增殖两种过程，以维持心脏功能的稳态水平。临床上造成心肌肥厚的疾病有许多，如原发性或继发性高血压、心肌梗死、瓣膜病、先天性心脏病等。虽然早期心肌肥厚有利于维持正常的心功能，但由于心肌肥厚本身也可增加心肌耗氧量，降低心肌顺应性，故长时间会导致心力衰竭，增加猝死发生率。目前研究认为，机械性与神经体液性因素诱导心肌肥厚，包括肾素-血管紧张素系统成分、儿茶酚胺类、胰岛素样生长因子、一氧化氮合成系统等。从细胞和分子水平上看，心肌细胞肥大的过程主要包括四个环节：刺激信号出现，跨膜信号传递，即刻早期反应基因激活，表达功能或结构蛋白质的基因向“胚胎型”表型转化。其中MAPK家族信号通路、Ca2+及其依赖的信号通路、磷脂酰肌醇-3激酶及其介导的信号通路、JAK/STAT途径都发现参与了心肌肥厚的信号传递，且各个通路之间又存在千丝万缕的联系，形成错综复杂的信号网络(戴文建等，心肌肥厚分子机制研究进展，心血管病学进展，2009年第30卷第1期，47-50)。
肝脏在机体脂肪代谢中占有重要地位，其参与脂质代谢过程中的多个重要环节，包括脂肪酸的摄取与合成，脂质的加工、贮存、氧化分解及输出。当肝脏获得脂肪酸的量超过其处理能力，造成脂质以甘油三酯的形式沉积于肝细胞内，导致肝细胞脂肪变性，成为单纯性肝脏脂肪变性、进而发展成为非酒精性脂肪性肝炎，部分患者可进展为肝纤维化、肝硬化、甚至肝癌(路然等，脂质代谢紊乱导致非酒精性脂肪性肝病的发病机制，临床肝胆病杂志，2015年第31卷第7期，1050-1054)。类似的情况，当血中游离脂肪酸水平增高或细胞内脂肪含量增多，超过脂肪组织的储存能力和各组织对游离脂肪酸的氧化能力，过多的游离脂肪酸以甘油三酯的形式沉积在胰岛素作用的靶组织如脂肪组织、肌肉和肝脏，就会造成胰岛素抵抗，从而引发一系列的代谢紊乱及相关疾病，如II型糖尿病、代谢综合征、心脑血管疾病等(陈金仲等，脂代谢紊乱与胰岛素抵抗的相关研究进展，中国实用医药，2008年第3卷第7期，147-149)。
发明内容
本发明的实验研究发现，随着组织缺血时间的延长，组织中GPR31蛋白的表达量逐渐增加，与组织缺血再灌注损伤严重程度之间存在着正相关的关系，表明GPR31有可能是一种组织或器官缺血再灌注损伤的新型调控因子。
GPR31过表达会加重缺氧和复氧处理引起的肝细胞、心肌细胞、肾脏细胞的活性降低，促进相应细胞的炎症反应，表明GPR31可促进肝脏、心和肾脏等脏器缺血再灌注损伤，以及这些脏器中发生的其他炎症反应的发生发展。
另外，肝细胞中GPR31表达的降低可抑制棕榈酸酯和油酸刺激导致的细胞脂质沉积，表明GPR31有望作为一个新型的调控肝细胞脂肪堆积的靶点，应用于脂肪代谢异常及相关疾病的治疗中。
心肌细胞中GPR31过表达，会加重血管紧张素Ⅱ导致的心肌肥厚，说明GPR31可促进心肌肥厚相关疾病的发生发展。
在此基础上，GPR31可以作为缺血再灌注损伤及其相关疾病、心肌肥厚及其相关疾病、心脏的炎症性疾病、脂肪代谢异常及其相关疾病的治疗靶点。
本发明的技术方案如下：
本发明的第一个方面是提供，GPR31抑制剂在制备药物中的用途，所述药物用于治疗缺血再灌注损伤和其相关疾病，心肌肥厚和其相关疾病，心脏的炎症性疾病，或脂肪代谢异常及相关疾病。
根据本发明，所述缺血再灌注损伤和其相关疾病选自肝脏缺血再灌注损伤及其相关疾病，心脏缺血再灌注损伤及其相关疾病，肾脏缺血再灌注损伤及其相关疾病，和/或脑缺血再灌注损伤及其相关疾病。所述缺血再灌注损伤可以是由器官移植、组织部分或全部切除、血管栓塞导致组织缺血等多种原因引发的。
肝脏缺血再灌注损伤及其相关疾病的引发因素包括但不限于：肝脏囊肿、肝脏移植、溶栓治疗、肝门阻断术。
心脏缺血再灌注损伤及其相关疾病的引发因素包括但不限于：心肌梗塞、心梗再通损伤、心脏移植、冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术，冠状动脉旁路术。
肾脏缺血再灌注损伤及其相关疾病的引发因素包括但不限于：肾脏移植、肾脏囊肿、肾脏血管手术。
脑缺血再灌注损伤及其相关疾病的引发因素包括但不限于：脑卒中、脑血管手术等。
优选所述缺血再灌注损伤和其相关疾病为肝脏缺血再灌注损伤，心脏缺血再灌注损伤，肾脏缺血再灌注损伤，和/或脑缺血再灌注损伤。
根据本发明，所述心肌肥厚及相关疾病包括但不限于：心肌肥厚、心力衰竭、心律失常、动脉栓塞、冠心病、心绞痛、心脏传导阻滞等。
造成心肌肥厚的疾病有许多，如原发性或继发性高血压、心肌梗死、瓣膜病、先天性心脏病等。
优选所述心肌肥厚及相关疾病为心肌肥厚、心力衰竭。
心脏的炎症性疾病包括但不限于：心肌炎、心内膜炎。
根据本发明，所述脂肪代谢异常及相关疾病包括但不限于：胰岛素抵抗、代谢综合征、非酒精性脂肪性肝病、肥胖、糖尿病、高血糖、高血脂症等。
非酒精性脂肪性肝病的疾病谱包括：单纯性肝脏脂肪变性、非酒精性脂肪性肝炎，肝纤维化、肝硬化、肝癌。
优选所述脂肪代谢异常及相关疾病为非酒精性脂肪性肝病、肥胖、高血脂症、胰岛素抵抗，更优选为：单纯性肝脏脂肪变性、非酒精性脂肪性肝炎、肥胖、高脂血症。
根据本发明，GPR31抑制剂可以是抑制GPR31蛋白质活性或蛋白质水平的抑制剂、或者抑制GPR31的mRNA水平的抑制剂。所述抑制活性可以是可逆的或不可逆的。
抑制GPR31蛋白质活性或蛋白质水平的抑制剂包括但不限于GPR31的抗体、抑制GPR31蛋白质活性或蛋白质水平的蛋白质、多肽、酶、天然化合物、合成化合物、有机物、无机物。所述抑制GPR31蛋白质活性或蛋白质水平的抑制剂是指可以结合GPR31但在结合时不产生生物应答的物质。所述抑制剂可以阻断、抑制或减弱由激动剂介导的应答，并可与激动剂竞争结合GPR31。
抑制GPR31的mRNA水平的抑制剂可以是其反义核酸序列、siRNA、miRNA、shRNA、dsRNA，或者其它能够抑制GPR31的mRNA水平的蛋白质、多肽、酶、化合物。
根据本发明，所述抗体包括但不限于单克隆抗体、合成抗体、多克隆抗体、多特异性抗体、人抗体、人源化抗体、嵌合抗体、单链Fv(scFv)(包括双特异性scFv)、单链抗体、Fab片段、F(ab')片段、二硫键连接的Fv(sdFv)和任何上述的表位结合片段。特别地，用于本发明的抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性部分。用于本发明的免疫球蛋白分子可以是免疫球蛋白分子的任何类型(例如，IgG、IgE、IgM、IgD、IgA和IgY)、类别(例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。优选地，抗体是人或人源化单克隆抗体。如本文所用，“人”抗体包括具有人免疫球蛋白的氨基酸序列的抗体，并且包括从人免疫球蛋白文库或从由人基因表达抗体的小鼠或其它动物分离的抗体。
在本发明的一个实施方式中，所述抑制剂为GPR31的mRNA的shRNA，其干扰的靶向序列为CACTCTCCTGCCTTCAGTTTG。
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG-3’。
根据本发明，所述药物进一步包含药学上可接受的辅料。
所述药学上可接受的辅料是制药领域中常用或已知的各种辅料，包括但不限于：稀释剂、粘合剂、抗氧化剂、pH调节剂、防腐剂、润滑剂、崩解剂等。
所述稀释剂例如：乳糖、淀粉、纤维素衍生物、无机钙盐、山梨醇等。所述粘合剂例如：淀粉、明胶、羧甲基纤维素钠、聚乙烯吡咯烷酮等。所述抗氧化剂例如：维生素E、亚硫酸氢钠、亚硫酸钠、丁羟基茴香醚等。所述pH调节剂例如：盐酸、氢氧化钠、柠檬酸、酒石酸、Tris、乙酸、磷酸二氢钠、磷酸氢二钠等。所述防腐剂例如：对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、间甲酚、苯扎氯铵等。所述润滑剂例如：硬脂酸镁、微粉硅胶、滑石粉等。所述崩解剂例如：淀粉、甲基纤维素、黄原胶、交联羧甲基纤维素钠等。
本发明药物的剂型可以是口服剂的形式，例如片剂、胶囊、丸剂、粉剂、颗粒剂、悬浮剂、糖浆剂等；也可以是注射给药的剂型，例如注射液、粉针剂等，通过静脉内、腹膜内、皮下或肌肉内的途径。所有使用的剂型形式都是药学领域普通技术人员所熟知的。
本发明的药物可以本领域已知的途径施用于受试者，包括但不限于口服，胃肠外，皮下，肌内，静脉内，腹腔内，肝内，心肌内，肾内，阴道，直肠，颊，舌下，鼻内，透皮方式等。
施用的剂量将取决于接受者的年龄、健康和体重，联用药物的种类，治疗频率，给药途径等。药物可以单一日剂量施用，或者总日剂量以每天两次，三次或四次的分开剂量施用。剂量可以施用一次或多次，施药时间可以单日至几个月或更长时间。
根据本发明，所述药物还可以与其他能改善或抑制缺血再灌注损伤相关疾病的药物联合使用。
根据本发明，所述药物还可以与其他能改善或抑制心肌肥厚及相关疾病的药物联合使用。
根据本发明，所述药物还可以与其他能改善或抑制心脏的炎症性疾病的药物联合使用。
根据本发明，所述药物还可以与其他能改善或抑制脂肪代谢异常的药物联合使用。
本发明的第二个方面是提供，表达靶向于GPR31的mRNA的shRNA的载体在制备药物中的应用，所述药物用于治疗缺血再灌注损伤和其相关疾病，心肌肥厚和其相关疾病，心脏的炎症性疾病，或脂肪代谢异常及相关疾病。
所述疾病的定义同前。
所述shRNA干扰的靶向序列为CACTCTCCTGCCTTCAGTTTG。
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG-3’。
所述载体可以是表达载体。表达载体中可以包含与上述shRNA序列可操作地连接的启动子和转录终止序列。
所述表达载体可以是真核细胞表达载体。
所述真核细胞表达载体可以是质粒表达载体或病毒表达载体。
所述质粒表达载体可以是但不限于pcDNA3.1+/-，pcDNA4/HisMax B，pSecTag2 A，pVAX1，pBudCE4.1，pTracer CMV2，pcDNA3.1(-)/myc-His A，pcDNA6-Myc/His B，pCEP4，pIRES，pIRESneo，pIRES hyg3，pCMV-myc，pCMV-HA，pIRES-puro3，pIRES-neo3，pCAGGS，pSilencer1.0，pSilencer2.1-U6 hygro，pSilencer3.1-H1 hygro，pSilencer3.1-H1 neo，pSilencer4.1-CMV neo。
所述病毒表达载体可以为慢病毒载体、腺病毒载体、腺相关病毒表达载体或其他类型病毒载体，包括但不限于pLKO.1，pLVX-IRES-ZsGreen1，pCDH-EF1-Luc2-T2A-tdTomato，pCDH-MSCV-MCS-EF1-Puro，pCDH-MSCV-MCS-EF1-copGFP，pLVX-ZsGreen1-C1，pAdEasy-1，pShuttle-CMV，pShuttle，pAdTrack，pAdTrack-CMV，pShuttle-IRES-hrGFP-1，pShuttle-IRES-hrGFP-2，pShuttle-CMV-lacZ，pShuttle-CMV-EGFP-C，pXC1，pBHGE3，pAAV-MCS，pAAV-RC，pHelper，pAAV-LacZ，优选pLKO.1载体。
本发明的第三个方面是提供，含有靶向于GPR31的mRNA的shRNA的慢病毒载体在制备药物中的应用，所述药物用于治疗缺血再灌注损伤和其相关疾病，心肌肥厚和其相关疾病，心脏的炎症性疾病，或脂肪代谢异常及相关疾病。
所述疾病的定义同前。
所述shRNA干扰的靶向序列为CACTCTCCTGCCTTCAGTTTG或其他可干扰GPR31表达的靶向序列。
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG-3’。
优选所述慢病毒载体为pLKO.1载体。
能用于制备所述第二和第三个方面所述的药物的药用载体，可以是本领域中常规使用的注射液载体，如等渗的NaCl溶液，等渗的葡萄糖溶液，或等渗的含有缓冲体系的溶液，如PBS溶液等。也可以根据制剂需要，选择性的添加防止慢病毒发生物理或化学变化而失活的保护剂，例如二价阳离子盐或表面活性剂等。
附图说明
图1：不同缺血时间下，肝脏组织中GPR31蛋白表达量的western-blot检测图。图中GAPDH为对照标准品。
图2：L02细胞经GFP及GPR31过表达慢病毒转染后GPR31蛋白表达情况鉴定图。图中GAPDH为对照标准品。
图3：L02细胞分别在过表达和正常表达GPR31的情况下，缺氧和复氧处理后，LDH释放检测结果统计图(n.s.代表P≥0.05，**代表P<0.01)。
图4：L02细胞分别在过表达和正常表达GPR31的情况下，缺氧和复氧处理后，炎症因子Il-6、Tnf-α，趋化因子Cxcl2的mRNA含量RT-PCR检测结果统计图(*代表0.01≤P<0.05，**代表P<0.01)。
图5：H9C2细胞经GFP及GPR31过表达慢病毒转染后GPR31蛋白表达情况鉴定图。
图6：H9C2细胞分别在过表达和正常表达GPR31的情况下，缺氧和复氧处理后，细胞活性的结果统计图(n.s.代表P≥0.05，*代表0.01≤P<0.05，**代表P<0.01)。
图7：HK2细胞经GFP及GPR31过表达慢病毒转染后GPR31蛋白表达情况鉴定图。
图8：HK2细胞分别在过表达和正常表达GPR31的情况下，缺氧和复氧处理后，细胞LDH释放检测的结果统计图(n.s.代表P≥0.05，**代表P<0.01)。
图9：L02细胞经shRNA及shGPR31慢病毒转染后GPR31基因mRNA含量鉴定图(**代表P<0.01)。
图10：L02细胞分别在低表达和正常表达GPR31的情况下，用棕榈酸酯(PA)及油酸(OA)(PA 0.5mM+OA 1mM)刺激后，肝细胞油红O染色的镜检图。“PA+OA”代表棕榈酸酯及油酸刺激组。
图11A：H9C2细胞分别在过表达和正常表达GPR31的情况下，用血管紧张素Ⅱ处理后，细胞镜检图。
图11B：H9C2细胞分别在过表达和正常表达GPR31的情况下，用血管紧张素Ⅱ处理后，细胞表面积的统计图(n.s.代表P≥0.05，**代表P<0.01)。
图11C：H9C2细胞分别在过表达和正常表达GPR31的情况下，用血管紧张素Ⅱ处理后，细胞肥大标志基因Anp及Myh7的mRNA表达量的RT-PCR检测结果统计图(n.s.代表P≥0.05，**代表P<0.01)。
具体实施方式
以下结合实施例对本发明做进一步描述。需要说明的是，实施例不能作为对本发明保护范围的限制，本领域的技术人员理解，任何在本发明基础上所作的改进和变化都在本发明的保护范围之内。
以下实施例所用化学试剂都是常规试剂，均可商购获得。未做特殊说明的实验方法都是采用本领域已知的常规方法。
在以下实施例中所采用的动物模型及各研究指标的检测方法：
实验动物：选用8-10周龄、体重在24g-27g、背景为雄性C57BL/6品系的野生型小鼠(购自北京华阜康生物科技股份有限公司)为实验对象。
动物饲养:所有实验小鼠均饲养在武汉大学SPF级实验动物中心。饲养条件：室温在22-24℃之间，湿度在40-70％之间，明暗交替照明时间为12h，自由饮水摄食。
HEK293T，人胚肾细胞，购自中国科学院细胞库，目录号GNHu43。
L02，人肝细胞系，购自中国科学院细胞库，目录号GNHu6。
H9C2，大鼠心肌细胞，购自中国科学院细胞库，目录号GNR5。
HK2，人肾近曲小管细胞，购自中国科学院细胞库，目录号SCSP-511。
细胞均培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中。培养环境：37℃，5％CO2。
小鼠肝缺血再灌注(ischemia/reperfusion，I/R)损伤模型构建：
1)手术前12h给小鼠禁食，可自由饮水。
2)手术前用3％戊巴比妥钠成功麻醉小鼠后，将其平卧固定肢体，用剃毛器将小鼠腹部术区毛刮净，用10％碘酒和75％乙醇对术区消毒。
3)取腹正中切口进腹，暴露肝脏左、中叶之肝蒂。
4)用无创血管夹夹闭中叶和左叶的门静脉和肝动脉，使约70％的肝脏缺血，0.5min后，与非阻断的右叶相比，可见阻断叶变白，说明阻断成功，维持缺血60min(Sham组的小鼠与手术组小鼠平行操作，但不进行血流阻断)。
5)缺血60min后去除血管夹，恢复缺血的肝脏血流，关闭腹腔，手术后小鼠置单独饲养，观察。
取材：于术后1h取假手术组(Sham)以及缺血再灌注组小鼠，3％戊巴比妥钠过量麻醉，取缺血区肝组织，立即放入液氮中30min以上，之后保存于-80℃冰箱中，用于RT-PCR及Western blot分析。
RT-PCR
1)细胞中RNA的提取
①收集细胞并用PBS缓冲液洗涤2次，完成后加入1ml TRizol，用加样器吹打均匀，吸入1.5ml离心管中，漩涡混匀器震荡30s，室温静置5min，使核蛋白完全从核酸上解离。
②4℃12000r/min离心5min，取上清液，加氯仿200μl，漩涡混匀器震荡30s，冰盒上静置10min；
③4℃12000r/min离心15min，取上清液，加入0.5ml异丙醇，充分混匀，冰盒上静置10min，使RNA充分沉淀；
④4℃12000r/min离心15min，弃去上清，加入1ml预冷的75％乙醇，漩涡混匀器震荡30s洗涤RNA沉淀；
⑤4℃12000r/min离心5min，弃去上清液，沉淀快速风干。提取RNA加适量的DEPC去离子水溶解。
2)反转录
使用Transcriptor First Strand cDNA Synthesis Kit(04896866001,Roche,Basel,Switzerland)反转录试剂盒，根据试剂盒说明书进行反转录实验。
Western blot
1)组织蛋白提取
①向于干冰中预冷的EP管中放入3-4颗钢珠，并加入称重定量之后的组织样本。
②向裂解液中加入PMSF，混匀后加至样品中，迅速摇匀。
③于-80℃预冷研磨仪适配器中研磨样品，研磨参数设置为30Hz/s，90s。
④研磨结束后，冰上放置10min，取出钢珠。
⑤超声裂解仪裂解样本(5KHz/次，每次1s，间隔1s，重复10次)，超声完成后冰上放置10min。
⑥样本放入4℃预冷的离心机中，12000rpm/min离心30min。
⑦吸取上清转移到新的EP管中，4℃，14000rpm/min离心10min。
⑧吸取上清转移到新的EP管中继续离心，4℃，14000rpm/min离心5min。
⑨准确吸取清液并利用BCA Protein Assay Kit(PierecTM，23225)进行蛋白定量。
2)细胞中蛋白提取
细胞加入裂解液，裂解完成后离心取上清，运用BCA Protein Assay Kit定量收集蛋白样品。
3)上样与电泳
配置好电泳凝胶，并在电泳槽内加入电泳液。把蛋白样品上样到SDS-PAGE胶加样孔内，点样完成后开始电泳。
4)转膜
①配制转膜液，于4℃预冷。
②PVDF膜使用前在甲醇中浸泡15s后放入转膜液中备用。
③取出凝胶板中的凝胶，用转膜液洗涤凝胶，将PVDF膜覆盖其上。
④转膜电压设为250V，电流设为0.2A，转移1.5h。
⑤转移结束后，取出PVDF膜。
5)封闭
把蛋白膜放置到预先准备好的TBST中，洗去膜上的转膜液。蛋白膜放入封闭液中，在摇床上缓慢摇动，室温封闭1-4h。
6)一抗孵育
①用TBST洗涤蛋白膜3次，每次5min。
②封口机将薄膜封入杂交袋中，加上一抗，封口。
③将杂交袋放入4℃摇床中，过夜。
7)二抗孵育
①将薄膜取出用TBST洗涤3次，每次5min，回收一抗。
②将膜放入对应的加有二抗的二抗稀释液中，避光孵育1h。
8)蛋白检测
孵育后用TBST洗涤3次，每次5min。利用Bio-Rad Chemi Doc XRS+凝胶成像系统检测目的条带。
GPR31过表达质粒构建：
1)PCR扩增GPR31基因，引物为：
正向：5’-ACACCGGCGGCCACGCGTATGCCATTCCCAAACTGCTC-3’；
反向：5’-GGAGGTACCTCCGGATCCTTACTTATCGTCGTCATCCTTG-3’；
2)PCR产物进行琼脂糖凝胶电泳，随后使用DNA凝胶回收试剂盒(天根)进行DNA片段的回收；
3)将所得DNA产物和限制性内切核酸酶FastDigest restriction enzymes(Thermo)、
buffer或者
Green buffer、ddH2O混合均匀(50μl体系)，置于37℃条件下反应。使用
AxyPrepTMPCR Clean-Up Kit(Axygen)回收酶切产物。
4)使用
PCR一步定向克隆试剂盒(Novoprotein)，按照试剂盒说明书进行重组反应；
5)制作大肠杆菌感受态细胞，将上述连接产物进行转化实验，涂板，置于37℃培养箱，过夜培养；
6)从37℃培养箱中取出过夜培养的平板，挑克隆摇菌，并检测菌落PCR阳性克隆。
7)将PCR鉴定为阳性的菌液吸取5-10μl接种至5ml LB(含抗性)培养基中，在220rpm，37℃摇床中过夜培养。
8)取出过夜培养的菌液，对已经混浊的菌液进行质粒提取(天根质粒DNA小提试剂盒)。
9)提取后的质粒可直接用于GPR31瞬转或构建慢病毒稳转细胞系。
GPR31干扰质粒构建
1)GPR31靶向干扰序列为CACTCTCCTGCCTTCAGTTTG，设计适合pLKO.1载体的寡聚核苷酸；正向寡聚核苷酸:5’CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG 3’；反向寡聚核苷酸:5’AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG 3’；阴性对照siRNA序列为：CAACAAGATGAAGAGCACCAA；
2)将上述两条寡聚核苷酸分半加无菌水溶解，终浓度为100mM，进行融合；
3)根据“GPR31表达质粒构建”步骤进行酶切反应、酶切产物的回收、连接反应、转化、挑单克隆和测序、提取质粒；
4)所得质粒可用于慢病毒介导的GPR31敲低细胞系构建。
慢病毒载体构建和包装
1)用胰酶消化并记数293T细胞，按1×106个293T/孔传至6孔板中。
2)细胞汇合度至80％时开始转染。
3)取1.5ml灭菌EP管，加入2个包装质粒(pSpax和pMD2G)和过表达或干扰质粒各1μg溶于100μl的无血清培养基。轻柔混匀，室温孵育5min。
4)取1.5ml灭菌EP管，取3μl PEI(1.6μg/μl)溶于100μl无血清培养基中。轻柔混匀，室温孵育5min。
5)将DNA溶液和PEI溶液轻柔混匀。室温孵育15min。
6)将上述DNA-PEI混合液，逐滴加入6孔板中。
7)转染6h后，换新鲜培养基。
8)转染后48-72h收获含病毒的上清。3000rpm离心10min，去除沉淀，并用0.45μm的滤膜过滤。
9)过滤后的病毒可立即用于感染或-80℃贮存。
细胞缺氧复氧(H/R)：
1)细胞培养至对数期，预温PBS洗2次，弃去。
2)将细胞分成正常对照组和H/R实验组，对照组换完全培养基，放37℃，5％CO2培养，实验组换无糖无血清的DMEM培养基，并放O2/CO2细胞培养系统的培养箱内(37℃，5％CO2，1％O2)缺氧培养，缺氧不同时间后，实验组换完全培养基复氧培养。
3)复氧至预定的复氧培养时间后，弃去上清，用PBS洗2次，收集细胞保存在-80℃备用。
LDH释放及细胞活性(cell viability)检测：
使用LDH细胞毒性比色测试试剂盒(G1782,Promega,Madison,WI,USA)检测LDH的释放量。使用非放射性CCK-8试剂盒(CK04；Dojindo,Kumamoto,Japan)检测细胞活性。按照说明书进行相关检测。
油红O染色：
1)样品组和对照组用1×PBS洗涤2次，加入300μl 3％多聚甲醛固定20min；
2)加入1×PBS洗涤2次后，加入60％异丙醇漂洗10s；
3)加入1×PBS洗涤2次，通风橱吹干；
4)每孔500μl加入油红O染色1h；
5)加入1×PBS洗涤2次，60％异丙醇进行分选，再加入1×PBS洗2次；镜检，拍照。
心肌细胞免疫荧光染色：
1)细胞用4％多聚甲醛固定；
2)PBS洗涤3次，每次5min；
3)加入0.2％Triton，摇床上摇5min；
4)PBS洗涤3次，每次5min；
5)滴加8％羊血清，于湿盒中摇床室温孵育60min，用于封闭；
6)弃去封闭液勿洗，滴加一抗(Anti-α-Actinin，Millipore，#05-384)(1％羊血清1:100稀释)，37℃2h或4℃过夜；
7)弃去一抗，PBS洗4次，每次5min；
8)加入30μl稀释后的二抗(
488donkey anti-mouse IgG，Invitrogen，A21202，用PBS按1:200比例稀释)，37℃孵育60min；
9)弃去二抗，PBS洗5次，每次5min；
10)用SlowFade Gold antifade reagent with DAPI封片，于荧光镜下观察拍照。
实施例1GPR31在缺血不同时间肝脏组织中表达变化
C57小鼠随机分为6组，分别为Sham组及手术组(分为5个不同时间点：缺血5min、10min、20min、40min、60min)，取手术组小鼠以及Sham组小鼠肝脏组织，Western blot检测各组肝脏组织中GPR31蛋白含量变化情况(3次独立重复实验)。其中WB所用一抗为：Anti-GPCR GPR31 antibody(ab75579；Abcam)，二抗为：Peroxidase AffiniPure goat anti-rabbit-IgG(H+L)(#111-035-003；Jackson Laboratory)。以GAPDH为表达量检测的对照标准品。
结果如图1所示，Sham组WB结果几乎不可见GPR31条带，手术组随着缺血时间的延长，GPR31蛋白条带越来越明显。这一结果表明，GPR31蛋白的表达情况与肝脏缺血再灌注损伤严重程度之间存在着正相关的关系。
实施例2GPR31过表达对H/R处理诱导的L02细胞损伤及炎症反应的影响
L02细胞分为4组：GFP对照组、GPR31对照组、GFP H/R组、GPR31H/R组。贴壁L02细胞分别瞬转对应质粒，24h后进行H/R处理(缺氧6h，复氧6h)。质粒转染完成后，提取细胞总蛋白，Western blot检测GPR31的过表达情况(3次独立重复实验，每次3个重复)。H/R处理完成后检测培养基中LDH的释放量(每组6个重复)，以评价GPR31过表达对H/R诱导的肝细胞损伤的影响；提取RNA进行RT-PCR分析(2次独立重复实验，每次3个重复)，检测炎症相关细胞因子及趋化因子mRNA含量变化，以评价GPR31过表达对H/R诱导的肝细胞炎症反应的影响。LDH释放检测结果以GFP对照组值为1，计算其余各组与其的比值。
RT-RCR所用引物序列如下：
| 基因 | 正向引物 | 反向引物 |
| Il6 | TCTGGATTCAATGAGGAGACTTG | GTTGGGTCAGGGGTGGTTAT |
| Tnfα | TACTCCCAGGTCCTCTTCAAGG | TTGATGGCAGAGAGGAGGTTG |
| Cxcl2 | GCGCCCAAACCGAAGTCATA | TTTCTTAACCATGGGCGATGC |
GPR31过表达WB检测结果如图2所示，相比于GFP组，GPR31过表达组蛋白条带显著增强，即L02细胞中GPR31过表达显著。
LDH释放检测结果如图3所示，GPR31对照组LDH释放与GFP对照组相比无显著差异。当进行H/R处理后，LDH的释放量显著增加，且GPR31过表达组LDH的释放量的增加程度显著高于GFP组。这一结果表明，GPR31过表达加重H/R处理引起的肝细胞损伤及肝细胞毒性。
炎症因子及趋化因子mRNA检测，以actin为对照标准品，结果如图4所示，当进行H/R处理后，GPR31过表达组的炎症因子Il-6、Tnf-α，趋化因子Cxcl2的mRNA含量均显著增加。这一结果表明，GPR31过表达加重H/R处理引起的肝细胞炎症反应。
实施例3GPR31过表达对H/R处理诱导的H9C2细胞活性的影响
H9C2细胞分为4组：GFP对照组、GPR31对照组、GFP H/R组、GPR31 H/R组。对应的重组慢病毒病毒液分别感染培养的H9C2细胞，24h后进行H/R处理(缺氧1h，复氧6h)。质粒转染完成后，提取细胞总蛋白，Western blot检测GPR31的过表达情况(3次独立重复实验)。H/R完成后检测细胞活性(每组6个重复)。以GFP对照组检测结果为1，计算其余各组相比于该组的比值。
WB检测结果如图5所示，相比于GFP组，GPR31过表达组蛋白条带显著增强，即H9C2细胞中GPR31过表达显著。
细胞活性检测结果如图6所示，GPR31对照组细胞活性相比于GFP对照组无显著差异。当进行H/R处理后，细胞活性相比于对照组显著降低。而当GPR31过表达后，GPR31 H/R组细胞活性的降低程度显著大于GFP H/R组。这一结果表明GPR31过表达可显著促进H/R诱导的心肌细胞损伤，降低心肌细胞的活性。
实施例4GPR31过表达对H/R处理后HK2细胞损伤的影响
HK2细胞分为4组：GFP对照组、GPR31对照组、GFP H/R组、GPR31 H/R组。对应的慢病毒病毒液分别感染培养的HK2细胞，24h后进行H/R处理(缺氧3h，复氧24h)，质粒转染完成后提取细胞总蛋白，Western blot检测检测GPR31的过表达情况(3次独立重复实验)。H/R完成后检测培养基中LDH的释放量(每组6个重复)，以评价GPR31过表达对H/R诱导的肾细胞损伤的影响。以GFP对照组LDH释放检测结果为1，计算其余各组相比于该组的比值。
HK2细胞中GPR31过表达检测结果如图7所示，相比于GFP组，GPR31过表达组细胞种GPR31蛋白含量显著增加。
肾脏细胞LDH释放量检测结果如图8所示，GPR31对照组LDH释放与GFP对照组相比无显著差异。当进行H/R处理后，LDH的释放量显著增加，且GPR31过表达组LDH的释放量的增加程度显著高于GFP组。这一结果表明，与GPR31在肝脏、心肌细胞中作用相同，GPR31的过表可加重H/R处理引起的肾脏细胞损伤。
实施例5GPR31敲低对肝细胞脂肪堆积的影响
L02细胞分为4组：shRNA对照组、shGPR31对照组、shRNA实验组、shGPR31实验组。贴壁L02细胞分别瞬转对应质粒，24h后向两个实验组中加入棕榈酸酯(PA)及油酸(OA)(PA0.5mM+OA 1mM)刺激，对照组中加入同等量的BSA，12h后进行油红O染色。
RT-PCR所用引物序列如下：
| 基因 | 正向引物 | 反向引物 |
| GPR31 | TCAACCTGCTGTCTCCTCAG | GTGCTTCCTGCCAGATGATG |
质粒转染完成后，提取细胞RNA，利用RT-PCR检测GPR31基因mRNA含量(进行3次独立重复实验，每次2次重复)，结果如图9所示。GPR31敲低组(shGPR31)GPR31基因的mRNA含量较shRNA对照组显著降低。
油红O染色结果如图10所示，对照组细胞无明显红色，而当加入PA+OA刺激后，被油红O染红的细胞相比于对照组显著增多，且GPR31敲低组(shGPR31实验组)着色面积的增加程度较shRNA实验组小。该结果说明，GPR31表达的降低可抑制PA刺激的L02细胞脂质沉积。
实施例6GPR31过表达对心肌肥厚的影响
H9C2细胞分为4组：GFP对照组、GPR31对照组、GFP AngⅡ组、GPR31 AngⅡ组。对应的慢病毒病毒液分别感染培养的H9C2细胞，24h后，用1μM血管紧张素Ⅱ(AngⅡ)或PBS(对照组)刺激48h，然后进行免疫荧光试验。慢病毒转染完成后，提取细胞总蛋白，Western blot检测H9C2细胞种GPR31蛋白的表达情况。AngⅡ刺激完成后收获细胞进行RT-PCR分析(2次独立重复实验，每次3个技术重复)检测肥厚标志基因Anp及Myh7的mRNA含量。细胞表面积统计结果以GFP对照组值为1，计算其余组相对于该组的比值。
RT-RCR所用引物序列如下：
| 基因 | 正向引物 | 反向引物 |
| Anp | ACCTGCTAGACCACCTGGAG | CCTTGGCTGTTATCTTCGGTACCGG |
| Myh7 | CCGAGTCCCAGGTCAACAA | CTTCACGGGCACCCTTGGA |
H9C2细胞中GPR31蛋白表达情况检测结果同实施例3。
H9C2细胞肥大及心肌肥厚标志物表达情况检测结果如图11所示，当用AngⅡ处理后，相比于PBS对照组，细胞表面积显著增大，且GPR31过表达组细胞增大程度显著大于GFPAngⅡ组(图11A、B)；与上述结果一致，mRNA分析显示，细胞经AngⅡ处理后GPR31过表达组细胞肥大标志基因Anp及Myh7的上调程度显著高于对照组(图11C)。
序列表
<110> 武汉大学
<120> GPR31抑制剂在制备治疗肾脏缺血再灌注损伤及相关疾病药物中的应用
<160> 18
<170> SIPOSequenceListing 1.0
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<212> DNA
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cactctcctg ccttcagttt g 21
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<212> DNA
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Claims (20)
1.GPR31抑制剂在制备药物中的用途，所述药物用于治疗肾脏缺血再灌注损伤；
所述抑制剂为GPR31的mRNA的shRNA，其干扰的靶向序列为CACTCTCCTGCCTTCAGTTTG。
2.如权利要求1所述的用途，其特征在于，所述shRNA序列为：正向寡聚核苷酸: 5’-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG -3’; 反向寡聚核苷酸:5’-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG- 3’。
3.如权利要求1或2所述的用途，其特征在于，所述肾脏缺血再灌注损伤的引发因素选自肾脏移植、肾脏囊肿和/或肾脏血管手术。
4.如权利要求1或2所述的用途，所述药物进一步包含药学上可接受的辅料。
5.表达靶向于GPR31的mRNA的shRNA的载体在制备药物中的应用，所述药物用于治疗肾脏缺血再灌注损伤；
所述shRNA干扰的靶向序列为CACTCTCCTGCCTTCAGTTTG。
6.如权利要求5所述的应用，其特征在于，所述shRNA序列为：正向寡聚核苷酸: 5’-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG- 3’; 反向寡聚核苷酸:5’-AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG- 3’。
7.如权利要求5或6所述的应用，其特征在于，所述肾脏缺血再灌注损伤引发因素选自肾脏移植、肾脏囊肿和/或肾脏血管手术。
8.如权利要求5或6所述的应用，所述载体是表达载体，包含与所述shRNA序列可操作地连接的启动子和转录终止序列。
9.如权利要求8所述的应用，其特征在于，所述表达载体是真核细胞表达载体。
10.如权利要求9所述的应用，其特征在于，所述真核细胞表达载体是质粒表达载体或病毒表达载体。
11.如权利要求10所述的应用，其特征在于，所述质粒表达载体选自pcDNA3.1+/-，pcDNA4/HisMax B，pSecTag2 A，pVAX1，pBudCE4.1，pTracer CMV2，pcDNA3.1（-）/myc-His A，pcDNA6-Myc/His B，pCEP4， pIRES，pIRESneo，pIRES hyg3，pCMV-myc，pCMV-HA，pIRES-puro3，pIRES-neo3，pCAGGS，pSilencer1.0，pSilencer2.1-U6 hygro，pSilencer3.1-H1hygro，pSilencer3.1-H1 neo，pSilencer4.1-CMV neo。
12.如权利要求10所述的应用，其特征在于，所述病毒表达载体选自慢病毒载体、腺病毒载体、腺相关病毒表达载体。
13.如权利要求12所述的应用，其特征在于，所述病毒表达载体选自pLKO.1， pLVX-IRES-ZsGreen1，pCDH-EF1-Luc2-T2A-tdTomato，pCDH-MSCV-MCS-EF1-Puro，pCDH-MSCV-MCS-EF1-copGFP，pLVX-ZsGreen1-C1，pAdEasy-1，pShuttle-CMV，pShuttle，pAdTrack，pAdTrack-CMV，pShuttle-IRES-hrGFP-1，pShuttle-IRES-hrGFP-2，pShuttle-CMV-lacZ，pShuttle-CMV-EGFP-C，pXC1， pBHGE3，pAAV-MCS，pAAV-RC，pHelper或pAAV-LacZ。
14.含有靶向于GPR31的mRNA的shRNA的慢病毒载体在制备药物中的应用，所述药物用于治疗肾脏缺血再灌注损伤；
所述shRNA干扰的靶向序列为CACTCTCCTGCCTTCAGTTTG。
15.如权利要求14所述的应用，其特征在于，所述shRNA序列为：正向寡聚核苷酸: 5’CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG 3’; 反向寡聚核苷酸:5’AATTCAAAAACACTCTCCTGCCTTCAGTTTG CTCGAGCAAACTGAAGGCAGGAGAGTG 3’。
16.如权利要求14或15所述的应用，其特征在于，所述肾脏缺血再灌注损伤的引发因素选自肾脏移植、肾脏囊肿和/或肾脏血管手术。
17.如权利要求14或15所述的应用，所述慢病毒载体为pLKO.1载体。
18.如权利要求5或6或14或15所述的应用，所述药物进一步含有药用载体。
19.如权利要求18所述的应用，其特征在于，所述药用载体是注射液载体。
20.如权利要求19所述的应用，其特征在于，所述注射液载体选自：等渗的NaCl溶液，等渗的葡萄糖溶液，等渗的含有缓冲体系的溶液。
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