CN118518875A - 组织蛋白酶d或其抑制剂或抗体在糖尿病性视网膜病变中的应用 - Google Patents
组织蛋白酶d或其抑制剂或抗体在糖尿病性视网膜病变中的应用 Download PDFInfo
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Abstract
组织蛋白酶D或其抑制剂或抗体在糖尿病性视网膜病变中的应用，属于基因的功能与应用领域，通过检测糖尿病视网膜病变患者眼房水中CTSD的表达，并以单核细胞过表达CTSD小鼠及单核细胞敲除CTSD小鼠为实验对象。结果提示随着糖尿病患者出现视网膜病变，眼内CTSD表达逐渐增加，说明其参加了糖尿病视网膜病变的发生发展；单核细胞过表达CTSD小鼠视网膜微血管呈囊泡转运为途径的渗漏现象，单核细胞敲除CTSD糖尿病小鼠比野生糖尿病小鼠视网膜渗漏现象好转。针对CTSD上述功能，提供CTSD作为血视网膜屏障药物靶标在研制糖尿病视网膜病变中的应用。
Description
技术领域
本发明属于基因的功能与应用领域，特别涉及一种组织蛋白酶D作为药物靶标在筛选治疗糖尿病性视网膜病变中的应用，以及组织蛋白酶D的抑制剂或抗体在制备治疗糖尿病性视网膜病变药物中的应用。
背景技术
糖尿病(Diabetes mellitus,DM)是目前影响人类健康的主要代谢性疾病。截止2017年，世界范围内有8.8％的人口患DM，且患病人数逐年增长，国际糖尿病联合会预计2040年全球患糖尿病人数将达到6.42亿。据统计，35％-49％的糖尿病患者伴有糖尿病视网膜病变(Diabetic retinopathy,DR)。DR是影响糖尿病人群视功能的主要致盲原因，以视网膜微血管异常渗漏和血视网膜屏障破坏为主要特征，抑制视网膜微血管异常改变为DR防治的重要措施。
目前在临床上，针对DR的治疗主要集中在抗VEGF眼内注射、视网膜激光光凝、玻璃体手术等。由于DR早期视功能改变不明显，且易被忽视，以上手段均主要应用于已影响视功能的DR患者，或临床随访轻、中度非增殖期DR直到出现增殖期改变时。因此，对于轻、中度非增殖期DR目前暂无特异的、有效的延缓其进展的方法，只能通过控制血糖、应用口服药等途径控制DR病情变化，待病程进展严重时才予以眼科相关治疗。对于增殖期DR或糖尿病性黄斑水肿者，抗VEGF药物需周期性多次眼内注射，增加患者的经济负担和感染风险，且有复发可能，视网膜激光光凝则以破坏周边视网膜为代价，使患者出现夜盲或视野缩小等。以上治疗均针对已出现视网膜明显异常的患者，但对于早期DR目前除定期随访外尚无有效的预防手段。因此，探索DR发生早期的生物治疗靶点对于延缓糖尿病性视网膜血管损伤进展或与现有药物联合应用等均有重要的应用意义。
经查阅文献资料，糖尿病性视网膜血管渗透性增加与高糖血症引起的糖基化终末产物、多元醇等异常物质引起视网膜局部氧化应激、炎症、缺氧相关。以上改变可引起视网膜血管内皮生长因子(VEGF)、单核细胞趋化因子(MCP-1)、白介素6(IL-6)等因子的表达异常升高，导致视网膜内皮细胞连接松解、周细胞丢失、白细胞粘附等现象，引起血视网膜屏障功能的破坏，血浆中物质渗漏入视网膜实质中引起视网膜结构的改变与视觉功能的异常。
血视网膜屏障(Blood-retinal barrier,BRB)是维持视网膜局部微环境稳态的重要结构，其核心组分为视网膜血管内皮细胞之间通过紧密连接构成的单层内皮细胞，能够有效的阻止血液中的物质渗入到视网膜的实质侧。当内皮细胞失去了屏障功能，BRB则遭到破坏。传统意义上的BRB损伤主要集中于内皮细胞间的紧密连接崩解中，此时视网膜血管渗漏主要经由内皮细胞旁途径。调研文献，在DR发生发展的早期即可出现视网膜血管渗漏现象，但此时内皮细胞的紧密连接尚未发生明显损害，提示存在除紧密连接以外的其他的途径参与BRB损害中。
近年来，以囊泡转运(Transcytosis)为主要特征的穿内皮细胞渗漏途径在血脑屏障和血视网膜屏障中引起了广泛关注。囊泡转运为血浆中物质经由内皮细胞以囊泡内吞外吐的形式实现向视网膜实质转运的过程，通常囊泡直径约50-100nm，以窝蛋白1构成内皮细胞特异表达形成囊泡的主要物质，可介导以囊泡转运为特征的BRB损害。因此，探究以囊泡转运为特征的早期糖尿病性视网膜渗漏对DR的早期防治起着关键作用。
组织蛋白酶D(Cathepsin D,CTSD)由单核-巨噬细胞产生，可参与细胞蛋白质的降解过程中，并参与癌症和阿尔兹海默等神经退行性疾病的发生发展中。近年来，人们发现CTSD存在一种可分泌到细胞外的CTSD前体形式，这种分泌形式使其可作用于周围细胞并产生影响。申请人首次在DR患者眼房水中检测CTSD的表达，发现DR患者眼内CTSD与对照组相比呈现显著高表达；在小鼠单核细胞过表达CTSD后，透射电镜观察内皮细胞紧密连接完好，却出现以囊泡转运为特征的视网膜渗漏现象；小鼠单核细胞敲除CTSD后诱导糖尿病模型，视网膜的血管渗漏现象减弱，提示CTSD具有抑制早期糖尿病性视网膜渗漏的潜力，针对CTSD为药物靶点制作抑制剂或抗体可为DR患者早期的预防或治疗起到关键作用。
发明内容
针对上述早期DR的临床治疗干预手段有限等问题，本发明的首要目的为提供CTSD作为药物靶标在筛选治疗早期糖尿病视网膜病变的药物中的应用。
本发明的另一目的为提供一种CTSD的抑制剂或抗体在制备治疗糖尿病视网膜病变药物中的应用。
一种治疗糖尿病视网膜病变的药物，包含CTSD的抑制剂或抗体。
所述的CTSD抑制剂为CTSD基因的RNA干扰载体、siRNA及其他抑制CTSD表达的抑制剂中的任一种。
所述的CTSD的抗体为单克隆抗体、多克隆抗体以及其他能够中和CTSD表达的抗体中的任一种。
本发明的目的通过以下技术方案实现：
本发明利用ELISA法检测对照组、糖尿病患者不伴视网膜病变、糖尿病视网膜病变患者眼房水中CTSD的表达，结果提示CTSD的表达呈梯度上升，差异具有统计学意义。CTSD在糖尿病患者尚未发生视网膜病变时已经呈现分泌增多现象，提示在视网膜病变早期CTSD已经对眼部微环境产生影响。伴有视网膜病变组比单纯糖尿病组CTSD表达显著增高，提示CTSD具有恶化糖尿病视网膜病变的作用。以上结果为CTSD在糖尿病视网膜病变的新靶点提供了临床基础。
本发明以单核细胞过表达CTSD转基因小鼠为实验对象，利用FITC-葡聚糖检测视网膜渗漏，结果提示过表达CTSD可增加小鼠视网膜血管渗漏现象；透射电镜观察视网膜内皮细胞紧密连接与对照组相比无明显改变，而内皮细胞囊泡显著增多，提示CTSD是通过增加内皮细胞囊泡转运引起视网膜渗漏现象。相反的，对单核细胞敲除CTSD小鼠做链脲霉素诱导糖尿病模型中观察视网膜渗漏现象，结果提示CTSD敲除减弱了视网膜血管渗漏情况。
所述CTSD在糖尿病视网膜病变中的功能，主要体现为其在糖尿病患者早期即在眼内液中表达增高，且可破坏血视网膜屏障的作用，特别是CTSD可恶化糖尿病视网膜病变的作用。
针对CTSD的上述功能，提供一种CTSD作为药物靶标在筛选治疗早期糖尿病视网膜病变的药物中应用。
针对CTSD的上述功能，提供一种CTSD的抑制剂或抗体在制备治疗糖尿病视网膜病变药物中的应用。
所述CTSD的抑制剂为CTSD的RNA干扰载体、siRNA以及其它能够抑制CTSD表达抑制剂中的任一种。
所述CTSD的抗体为单克隆抗体、多克隆抗体以及其他能够中和CTSD表达的抗体中的任一种。
所述的CTSD的抑制剂或抗体治疗方案优选为玻璃体腔注射。
一种治疗糖尿病视网膜病变的药物，包含CTSD的抑制剂或抗体。
本发明经过研究结果提示，单核细胞CTSD过表达可引起糖尿病视网膜病变早期的以囊泡转运为途径的视网膜渗漏现象，证明CTSD在糖尿病视网膜病变中具有重要的恶化作用。抑制CTSD表达可以改善糖尿病视网膜病变早期的血视网膜屏障破坏及视网膜微血管渗漏现象。以上结果为CTSD在糖尿病视网膜病变早期药物开发和应用提供了理论依据。
本发明相比现有技术具有如下优点：
1.本发明发现CTSD基因的新功能，即CTSD可通过内皮细胞囊泡转运增加糖尿病早期视网膜微血管渗漏现象。
2.CTSD基因具有能够恶化糖尿病视网膜病变的作用，为研制糖尿病视网膜病变的药物提供有效生物靶点。
附图说明
图1.糖尿病患者眼房水中CTSD表达呈升高改变。
图2.单核细胞过表达人源CTSD转基因小鼠(hCTSDhigh)呈现明显视网膜渗漏现象。
A为野生型(WT)小鼠与hCTSDhigh转基因小鼠构建示意图；
B为WT和hCTSDhigh转基因小鼠基因鉴定结果图；
C为WT和hCTSDhigh转基因小鼠FITC-葡聚糖(40kDa)视网膜渗漏；
D为WT和hCTSDhigh转基因小鼠FITC-葡聚糖(40kDa)渗漏入血管外荧光强度统计图；
E为WT和hCTSDhigh转基因小鼠应用透射电镜观察血管内皮内囊泡；
F为WT和hCTSDhigh转基因小鼠视网膜内皮细胞囊泡计数统计图观察视网膜内皮细胞囊泡；
G为WT和hCTSDhigh转基因小鼠应用透射电镜观察血管内皮间的紧密连接；
H为WT和hCTSDhigh转基因小鼠视网膜血管内皮间紧密连接间隙宽度统计。
图3.单核细胞敲除CTSD后抑制糖尿病小鼠视网膜渗漏。
A为单核细胞敲除CTSD(CTSDmKO)小鼠构建示意图；
B为CTSDmKO小鼠基因鉴定结果图；
C为CTSDmKO小鼠诱导糖尿病后FITC-葡聚糖渗漏情况；
D为CTSDmKO小鼠诱导糖尿病后FITC-葡聚糖渗漏入血管外荧光强度统计图。
具体实施方式
以下结合实例及附图对本发明做进一步阐述，但本发明的实施方式不限于此。
实施例1
糖尿病患者眼内液中CTSD表达显著升高
为求证CTSD在糖尿病及伴有视网膜病变者眼内的表达，我们收集了单纯白内障作为对照组、白内障同时患有糖尿病者或伴有糖尿病视网膜病变者术中眼房水，排除患有风湿系统疾病、心血管疾病等全身伴随疾病。
通过ELISA法检测各组人群眼内液房水中CTSD的表达，以下所用试剂均包含在ELISA试剂盒中(Abcam，ab119586)，具体步骤如下：
1)将眼房水在样本稀释液中以1:200比例稀释，同时配置不同浓度的蛋白标准品156、312、625、1250、2500pg/ml；
2)将100μl样品稀释液(空白对照)、蛋白标准品、已稀释的样品加入预包被抗CTSD抗体的ELISA孔板中，封膜后37℃孵育1.5小时；
3)弃去样品，加入生物素化的抗CTSD抗体，封膜后37℃孵育1小时；
4)每孔加入300μl PBS摇床清洗1分钟，重复3次；
5)每孔加入100μl抗生物素蛋白混合物溶液，封膜后37℃孵育0.5小时；
6)弃去孔内液体，每孔加入300μl PBS摇床清洗1分钟，重复5次；
7)每孔加入90μl TMB显色液37℃孵育5分钟；
8)每孔加入100μl TMB终止液；
9)终止反应后立即酶标仪在450nm处测量吸光度；
10)每孔度数减去空白对照即为实际吸光度值，根据蛋白标准品计算标准曲线计算所测样品浓度，再将结果乘以200稀释比例即为房水中CTSD的表达含量。
结果提示糖尿病人群在尚未出现糖尿病性视网膜病变时CTSD在眼内的表达已经显著升高，在伴有糖尿病视网膜病变人群中，CTSD的表达进一步升高(图1)。提示CTSD在糖尿病患者尚未发生明显视网膜改变时即已对眼内微环境产生一定的影响，参与糖尿病患者视网膜的早期损伤中，并且加剧了糖尿病视网膜病变损伤，具有恶化糖尿病视网膜病变的作用。
实施例2
小鼠单核细胞过表达CTSD后视网膜渗漏增加
1.实验动物：选取8-10周龄、C57BL/6J野生型小鼠(WT，购自北京维通利华实验动物技术有限公司)、单核细胞过表达CTSD转基因小鼠(hCTSDhigh，由中国医科大学生命科学学院赵伟东教授实验室构建)为实验对象。
2.小鼠基因鉴定：转基因小鼠通过剪尾提取基因组DNA，经过PCR鉴定，检测其转基因情况。PCR鉴定引物信息为：上游引物5’-CACCTACTCAGACAATGCGAT-3’(SEQ ID NO.1)，下游引物5’-ACTGTGTTTGACCGTGACAAC-3’(SEQ ID NO.2)，PCR步骤如下：
1)配置鼠尾裂解液100mM Tris-HCl，pH 8.8，5mM EDTA，pH 8.0，0.2％ SDS，200mMNaCl，在裂解前加入蛋白酶K(储存液20mg/ml)，使蛋白酶K终浓度在100μg/ml；
2)每个鼠尾加入500μl鼠尾裂解液中55℃颠倒摇匀12小时，次日14000rpm离心5分钟，取上清1:10稀释在灭菌水中，得到DNA模版；
3)每个样本加入无菌DEPC水10.5μl，Premix TaqTM(TAKARA，RR901A)12.5μl，10mM上游引物0.5μl，10mM下游引物0.5μl，DNA模版1μl；
4)PCR程序：94℃5分钟，94℃20秒53℃30秒72℃30秒，共35个循环，72℃10分钟；
5)琼脂糖凝胶电泳：0.8g琼脂糖加入80ml TAE溶液中，微波加热至完全溶解后加入EB替代物3.5μl，混匀后倒入模具中冷却成胶。每孔加入7μl PCR产物，140V电泳20分钟，紫外凝胶成像。
3.通过FITC-葡聚糖检测小鼠视网膜渗漏情况，具体步骤如下：
1)将FITC-葡聚糖(40kDa，Sigma，FD40S)与PBS配制50mg/ml工作液；
2)将1)中配置好的葡聚糖溶液通过鼠尾静脉注入小鼠体内，每只小鼠注射100μl，注射后的小鼠放置于鼠盒中自由活动30分钟，鼠盒下放置37℃热垫以利于葡聚糖在小鼠体内循环；
3)二氧化碳安乐死小鼠，立即将眼球取材，于4℃PBS中去除角膜和晶状体，将视网膜平铺在载玻片上，50％甘油封片；
4)基恩士荧光显微镜488nm激发光下拍摄视网膜葡聚糖渗漏的情况；
5)ImageJ分析统计渗漏入血管外的FITC荧光强度。
4.透射电镜观察小鼠视网膜内皮细胞，具体步骤如下：
1)二氧化碳安乐死小鼠后，将眼球取材，3％戊二醛4℃固定12小时，剪取视网膜组织1mm×1mm×1mm，PBS漂洗3次，每次15分钟。
2)后固定：1％的锇酸·0.1M磷酸缓冲液PB(pH7.4)室温(20℃)固定2小时。PBS(pH7.4)漂洗3次，每次15分钟。
3)脱水：组织依次入50％-70％-80％-90％-95％-100％-100％酒精-100％丙酮-100％丙酮上行脱水，每次15分钟。
4)渗透：丙酮︰812包埋剂＝1︰1 2-4小时，丙酮︰812包埋剂＝2︰1渗透12小时，纯812包埋剂5-8小时，将纯812包埋剂倒入包埋板，将样品插入包埋板后37℃烤箱12小时。
5)包埋：60℃烤箱聚合48小时。
6)切片：超薄切片机切片60-80nm超薄切片。
7)染色：铀铅双染色(2％醋酸铀饱和酒精溶液，枸橼酸铅，各染色15分钟)，切片室温干燥12小时。
8)透射电子显微镜下观察，采集图像分析，观察小鼠视网膜内皮细胞内的囊泡情况及紧密连接。
5.结果分析：hCTSDhigh小鼠的表达由单核细胞CD68启动子驱动(图2A)，并通过琼脂糖凝胶电泳鉴定于263bp处过表达基因条带(图2B)。FITC-葡聚糖在hCTSDhigh小鼠视网膜渗漏比对照野生型小鼠明显增多(图2C，2D)，透射电镜观察小鼠内皮细胞内囊泡及内皮细胞间紧密连接情况，我们发现单核细胞过表达CTSD后视网膜内皮细胞内囊泡数量明显增多(图2E，2F)，而内皮细胞间的紧密连接间隙未发生明显变化(图2G，2H)。提示CTSD引起的视网膜渗漏是通过穿内皮细胞的囊泡转运而非紧密连接松解后的细胞旁途径。
实施例3
小鼠单核细胞敲除CTSD后减弱了糖尿病视网膜渗漏现象
1.实验动物：选取8-10周龄、C57BL/6J野生型小鼠(WT，购自北京维通利华实验动物技术有限公司)、CTSDflox/flox小鼠、Lyz2-Cre小鼠(购自赛业生物科技有限公司)为研究对象。
将CTSDflox/flox小鼠、Lyz2-Cre小鼠进行杂交，得到CTSDflox/+；Lyz2-Cre小鼠即为单核细胞特异性敲除CTSD基因的小鼠(CTSDmKO)。相对应的，CTSDflox/flox小鼠即为CTSDmKO小鼠的对照组。
2.小鼠基因鉴定：转基因小鼠通过剪尾提取基因组DNA，经过PCR鉴定，检测其转基因情况。PCR鉴定CTSD loxp引物信息为：上游引物5’-GCATTGCTTGGTATTTGCAAAAGG-3’(SEQID NO.3)，下游引物5’-TATAGGTCCAGTAACTCTCTCCCA-3’(SEQ ID NO.4)。PCR鉴定Lyz2-Cre引物信息为：上游引物5’-CTTGGGCTGCCAGAATTTCTC-3’(SEQ ID NO.5)，下游引物5’-CCCAGAAATGCCAGATTACG-3’(SEQ ID NO.6)，其余步骤同实施例2。
3.诱导糖尿病小鼠模型，具体操作如下：
将链脲霉素(STZ)溶解在0.01M柠檬酸盐缓冲液(pH 4.5)中，对空腹4小时的小鼠进行腹腔注射(40mg/kg体重)，对照应用0.01M柠檬酸盐缓冲液(pH 4.5)缓冲液，连续注射5日诱导糖尿病小鼠模型。2周后取鼠尾血检测血糖高于288mg/dL者视为建模成功，取造模成功后2个月小鼠为实验对象。
4.通过FITC-葡聚糖检测小鼠视网膜渗漏情况，具体步骤同实施例2。
5.结果分析：单核细胞特异性敲除CTSD小鼠由Lyz2-Cre和CTSDflox/flox小鼠杂交产生(图3A)，通过琼脂糖凝胶电泳观察到CTSDflox/+(402/293bp)条带和Lyz2-Cre(721bp)条带(图3B)。对CTSDflox/flox小鼠和CTSDmKO小鼠进行STZ或对照溶液腹腔注射诱导糖尿病后进行FITC-葡聚糖视网膜渗漏实验。结果提示糖尿病的CTSDflox/flox-STZ小鼠出现明显的视网膜微血管渗漏现象，而单核细胞敲除CTSD后的CTSDmKO-STZ视网膜渗漏明显减轻(图3C)，血管外荧光强度定量统计CTSDmKO-STZ小鼠视网膜渗漏显著少于CTSDflox/flox-STZ小鼠(图3D)。
以上所述为本发明较佳的实施方式，但本发明的实施方式不受上述实施例的限制，其它未背离本发明的神经实质与原理下所做的改变、简化、组合、替代等均应视为等效置换，都包含在本发明的保护范围内。
Claims (6)
1.一种组织蛋白酶D作为药物靶标在筛选治疗早期糖尿病视网膜病变的药物中应用。
2.一种组织蛋白酶D的抑制剂或抗体在制备治疗糖尿病视网膜病变药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述组织蛋白酶D的抑制剂为组织蛋白酶D的RNA干扰载体、siRNA以及其它能够抑制组织蛋白酶D表达抑制剂中的任一种。
4.根据权利要求2所述的应用，其特征在于，所述组织蛋白酶D的抗体为单克隆抗体、多克隆抗体以及其他能够中和组织蛋白酶D表达的抗体中的任一种。
5.根据权利要求2所述的应用，其特征在于，所述的组织蛋白酶D的抑制剂或抗体治疗方案为玻璃体腔注射。
6.一种治疗糖尿病视网膜病变的药物，其特征在于，包含组织蛋白酶D的抑制剂或抗体。
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