CN113667741A - 基于trim46基因的糖尿病视网膜病检测试剂盒 - Google Patents
基于trim46基因的糖尿病视网膜病检测试剂盒 Download PDFInfo
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Abstract
本发明属于糖尿病视网膜病技术领域,具体公开了一种基于TRIM46基因的糖尿病视网膜病检测试剂盒,包括特异性引物,序列分别为SEQ ID NO:1和SEQ ID NO:2。本发明探究TRIM46在DR形成过程中的作用,进一步开发基于TRIM46基因的糖尿病视网膜病检测试剂盒将会为糖尿病视网膜血管病变早期诊断和靶向治疗开辟新思路。对于完善我国防盲治盲、眼底病临床等眼科学科专业发展有着重要的促进作用和现实意义。
Description
技术领域
本发明属于糖尿病视网膜病技术领域,尤其涉及一种基于TRIM46基因的糖尿病视网膜病检测试剂盒。
背景技术
糖尿病视网膜病变(diabetic retinopathy,DR)在发达国家及地区人群中已经成为首位致盲性眼病,人数超过9300万。我国糖尿病患者总数已占据全球第一位,DR患病率达到37%,随着病程延长患病率还会逐步上升。
DR主要的病理特征是视网膜血管屏障功能破坏引起的渗透性增加及新生血管膜形成。过去20年的大量研究中涉及多种机制,主要包括:多元醇通路、蛋白激酶C通路(PKC)、糖基化终末产物(advanced glycation end products,AGEs)途径、血流量和血流动力学改变、肾素-血管紧张素途径、有丝分裂原激活蛋白激酶(MAPK)通路、血管生成途径以及与氧化损伤相关的途径。
本发明的发明人一直以来从糖基化终末产物(AGEs)的形成、线粒体活性氧(ROS)及泛素蛋白酶体通路(UPP)等机理方面进行了多项研究,包括之前发现泛素-蛋白酶体通路UPP通过影响线粒体活性氧ROS/PARP和NF-κB炎症因子通路参与糖尿病视网膜病变过程,高糖下HRECs、糖尿病大鼠视网膜组织以及增生性糖尿病视网膜病变视网膜纤维血管膜均发现UPP水平(Ub、E3、26S)、ROS/PARP通路及NF-κB通路及其下游因子的上调。但是糖尿病视网膜血管病变中泛素化水平上调如何调控NF-κB通路及其下游因子的分子机制尚不明确。
TRIM(Tripartite motif-containing)蛋白是一类具有保守的RING指、B-box及卷曲螺旋结构域的蛋白家族,TRIM参与调控细胞多种方面,包括细胞生存和死亡、细胞周期和分化、代谢状态、细胞膜修复、突触小泡的胞吐作用、衰老、干细胞多能性和红细胞的分化,同时控制病毒、细菌和真菌的感染。已有研究表明TRIM蛋白对多种感染所引起的炎症反应具有特定的免疫能力,在细胞免疫中发挥重要作用。
TRIM46属于Tripartite motif基因家族中的重要成员之一,目前关于TRIM46在疾病中的研究相对较少,现已发现TRIM46通过驱动平行微管阵列的形成来控制神经元极性和轴突的规范。此外还有报道TRIM46参与了小鼠和人类乳腺癌细胞的增殖和迁移。目前TRIM46在DR形成过程中是否发挥作用尚不清楚。
发明内容
本发明的目的在于提供一种基于TRIM46基因的糖尿病视网膜病检测试剂盒,以解决上述技术问题之一。
本发明提供的技术方案为:基于TRIM46基因的糖尿病视网膜病检测试剂盒,包括特异性引物,序列分别为SEQ ID NO:1和SEQ ID NO:2。
本技术方案的原理和有益效果在于:
本发明通过转录组测序(RNA-seq)分析10例增生性糖尿病视网膜血管病变患者的视网膜前纤维血管膜及10例非血管疾病引起的黄斑前膜组织,发现三基序蛋白(tripartitemotif,TRIM)家族多个成员表达异常,进一步扩大样本量及GEO数据(编号GSE60436)筛选确定TRIM46作为研究的靶基因。体外细胞实验发现TRIM46基因干扰可以阻断高糖诱导的内皮细胞的凋亡和细胞通透性,为了进一步研究TRIM46的调控机制,本发明筛选确定了与TRIM46相互作用的蛋白IκBα,推测TRIM46可能直接与IκBα相互结合,并对其泛素化修饰,调控NF-kB通路。
本发明探究TRIM46在DR形成过程中的作用,进一步开发基于TRIM46基因的糖尿病视网膜病检测试剂盒将会为糖尿病视网膜血管病变早期诊断和靶向治疗开辟新思路。对于完善我国防盲治盲、眼底病临床等眼科学科专业发展有着重要的促进作用和现实意义。
附图说明
图1为转录组测序Heatmap图;
图2为采用糖尿病视网膜血管病变GEO数据库(编号GSE60436)验证TRIM46表达的实验结果;
图3为高糖下HRCECs的TRIM46表达情况;
图4为采用流式细胞仪检测细胞凋亡率;
图5为采用电阻仪检测细胞跨膜TEER值Ω;
图6为采用CO-IP验证TRIM46和IκBα互作;
图7为TRIM46基因干扰抑制IκBα泛素化降解;
图8为TRIM46基因干扰对相关指标的影响。
具体实施方式
下面通过具体实施方式进一步详细说明:
实验一:TRIM46、IκBα及NF-κB通路分子在糖尿病患者视网膜病变中的差异表达及临床相关性分析
(1)RIM46在糖尿病视网膜病变组织中高表达
为了寻找糖尿病视网膜血管病变新的研究靶点,选取10例增生性糖尿病视网膜血管病变患者的视网膜前纤维血管膜,以及10例非血管疾病患者的黄斑前膜组织进行转录组测序(RNA-seq)分析。
增生性糖尿病视网膜血管病变患者和非血管疾病患者均就医于上海交通大学附属第一人民医院,入组患者均签署知情同意书,标本采集上海交通大学附属第一人民医院伦理委员会审议同意。
图1为转录组测序Heatmap图,由图1发现:三基序蛋白(TRIM)家族多个成员表达异常。
进一步扩大样本量,采用Q-PCR检测TRIM46的表达,采用糖尿病视网膜血管病变GEO数据库(编号GSE60436)验证TRIM46的表达。Q-PCR检测时所用到的引物是:
正向引物(SEQ ID NO:1):5′-GCCAAGGACATCGACGGG-3′;
反向引物(SEQ ID NO:2):5′-CGAACTGGTTACACGGGAAGG-3′。
β-actin forward:5′-TGGCATTGCCGACAGG-3′;
β-actin reverse:5′-GCATTTGCGGTGGACG-3′。
图2为采用糖尿病视网膜血管病变GEO数据库(编号GSE60436)验证TRIM46表达的实验结果;由图2可以得出,与Control组和Normal组相比,TRIM46在糖尿病视网膜病变组高表达。
(2)高糖下HRCECs的TRIM46表达增加
使用25mmol/L的高糖分别处理人视网膜微血管内皮细胞(HRCECs)12h、24h和48h后,采用Q-PCR和Western blot检测TRIM46的表达。
图3为高糖下HRCECs的TRIM46表达情况,如图3所示,高糖处理组TRIM46表达显著升高。
(3)发现TRIM46基因干扰抑制高糖诱导的细胞凋亡和通透性
构建TRIM46基因干预和空载预处理的人视网膜微血管内皮细胞HRCECs,使用25mmol/L的高糖处理后,采用流式细胞仪检测细胞凋亡率,采用电阻仪检测细胞跨膜TEER值Ω。
图4为采用流式细胞仪检测细胞凋亡率,图5为采用电阻仪检测细胞跨膜TEER值Ω。
由图4可以发现,TRIM46基因可以阻断高糖诱导的细胞凋亡;由图5可以发现,TRIM46基因可以阻断高糖诱导的细胞通透性。
(4)TRIM46和IκBα存在互作,TRIM46基因干扰可以抑制IκBα的泛素化降解
为了深入研究TRIM46在高糖引起细胞凋亡中的分子机制,本发明克隆融合FLAG标签的TRIM46过表达慢病毒载体和空载慢病毒感染人视网膜微血管内皮细胞HRCECs,用FLAG抗体进行免疫共沉淀(Co-IP)实验,纯化bait的TRIM46蛋白质的相互作用复合物进行SDS-PAGE电泳,切割差异显著的条带酶解后提取肽段液相色谱-质谱(LC-MS)测试分析,筛选与TRIM46相互作用的IκBα蛋白,并采用免疫共沉淀(Co-IP)验证TRIM46和IκBα存在互作。
图6为采用CO-IP验证TRIM46和IκBα互作;图7为TRIM46基因干扰抑制IκBα泛素化降解。
(5)TRIM46基因干扰促进IκBα蛋白的表达而对IκBα基因无影响
构建TRIM46基因干预和空载预处理的人视网膜微血管内皮细胞HRCECs,使用25mmol/L的高糖处理后,Q-PCR和Wesrern blot检测TRIM46、IκBα及细胞通透性的紧密连接蛋白Occludin、ZO-1的表达。
图8为TRIM46基因干扰对相关指标的影响;由图8可以发现:TRIM46基因干扰促进IκBα蛋白的表达而对IκBα基因无影响,TRIM46基因干扰促进Occludin和zo-1的表达。推测TRIM46可能直接与IκBα相互结合,并对其泛素化修饰,调控NF-kB通路。
实施例
基于TRIM46基因的糖尿病视网膜病检测试剂盒,包括特异性引物,序列分别为SEQID NO:1和SEQ ID NO:2。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
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<110> 上海市第一人民医院
<120> 基于TRIM46基因的糖尿病视网膜病检测试剂盒
<160> 2
<170> Patentln version 3.5
<210> 1
<211> 18
<212> DNA
<213> homo sapins
<400> 1
gccaaggaca tcgacggg 18
<210> 2
<211> 21
<212> DNA
<213> homo sapins
<400> 2
cgaactggtt acacgggaag g 21
Claims (1)
1.基于TRIM46基因的糖尿病视网膜病检测试剂盒,其特征在于,包括特异性引物,序列分别为SEQ ID NO:1和SEQ ID NO:2。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
CN109609614A (zh) * | 2017-09-30 | 2019-04-12 | 首都医科大学附属北京胸科医院 | 检测trim2、trim4、trim32和/或trim46基因或蛋白产品的用途 |
WO2020180146A1 (ko) * | 2019-03-07 | 2020-09-10 | (주)레티마크 | 당뇨망막병증 진단용 복합 마커 및 이의 용도 |
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2021
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
CN109609614A (zh) * | 2017-09-30 | 2019-04-12 | 首都医科大学附属北京胸科医院 | 检测trim2、trim4、trim32和/或trim46基因或蛋白产品的用途 |
WO2020180146A1 (ko) * | 2019-03-07 | 2020-09-10 | (주)레티마크 | 당뇨망막병증 진단용 복합 마커 및 이의 용도 |
Non-Patent Citations (3)
Title |
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CHEN W等: "Homo sapiens glutathione peroxidase 4 (GPX4), transcript variant 1, mRNA,NM_002085.5", 《GENBANK》 * |
JANGHAM JUNG等: "Trim46 contributes to the midbrain development via Sonic Hedgehog signaling pathway in zebrafish embryos", 《ANIMAL CELLS AND SYSTEMS》 * |
靳亚西: "利用实时细胞分析技术检测前列腺与胰腺癌细胞药物敏感性及Trim46基因低表达对胰腺癌细胞Capan-2的增殖和迁移能力的抑制", 《中国优秀博硕士学位论文全文数据库(硕士)》 * |
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