糖尿病视网膜病变中SP1对Robo4的调节机制及作用研究

发布时间：2018-05-16 09:13

本文选题：SP1 + Robo4　；参考：《吉林大学》2017年博士论文

【摘要】：糖尿病视网膜病变(diabetic retinopathy,DR)为糖尿病严重的微血管并发症之一,其发病机制极为复杂,目前研究表明,视网膜在持续高糖环境的刺激下,可发生氧化应激、炎症反应、神经退行性改变和表观遗传学修饰异常等变化,它们之间相互作用并启动各自相关的信号通路,共同导致视网膜微血管病变的发生。根据视网膜血管病理改变的不同,可将DR的病程分为非增殖期和增殖期两个阶段。在DR非增殖期,病变主要表现为视网膜微血管内皮细胞结构损伤甚至丢失、周细胞凋亡、微血管细胞间紧密连接松弛、通透性增加等,进而引起血-视网膜屏障的破坏。在DR增殖期,大面积的毛细血管闭塞使视网膜局部严重缺氧,引起众多生长因子和炎症介质的上调,进而导致视网膜新生血管的形成。因此保护视网膜微血管的完整性、抑制视网膜新生血管形成是预防和治疗DR的关键。Roundabout 4(Robo4)是2002年被发现的Robo家族新成员,可特异性表达于血管内皮细胞的表面,具有调节内皮细胞通透性、影响新生血管形成等功能。近期研究发现,Robo4的表达变化与DR的发生发展密切相关。在体外低氧条件培养的人视网膜血管内皮细胞(human retinal endothelial cells,HREC)中,随着低氧作用时间的延长,细胞内Robo4表达逐渐升高,而抑制Robo4的表达可有效改善低氧诱导的HREC增殖和迁移能力的异常,且在体内糖尿病动物模型中,发现伴随DR的发生和病程延长,Robo4在视网膜内的表达水平逐渐增加。由此提示,Robo4在DR发生发展过程中可能发挥重要的作用,但与其相关的具体作用机制尚不清楚。在有关Robo4表达调控机制的研究中,利用基因启动子预测软件分析Robo4上游的启动子区域,发现其中存在多个specificity protein 1(SP1)的结合位点,且研究表明SP1作为常见的转录因子,可调控下游多个靶基因的转录而影响DR的发生发展,由此推测Robo4可能为SP1的另一靶向因子,在DR发病中,SP1通过调控Robo4的转录活性而影响视网膜微血管病变的发生。本研究于体内检测增殖性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)患者纤维血管膜中SP1与Robo4的表达情况,并利用高糖条件培养HREC,以模拟DR的体内环境,探讨此时SP1与Robo4的表达水平及二者间的转录调控机制,并进一步探讨该机制在高糖条件下对HREC功能的影响。方法1.检测SP1与Robo4在PDR患者纤维血管膜中的表达情况收集临床中手术获取的PDR患者的纤维血管膜,对其进行HE染色,观察膜组织的病理结构,采用免疫组织化学染色法检测SP1与Robo4蛋白在纤维血管膜中的表达水平,并利用双重免疫荧光标记法检测二者表达的共定位情况。2.检测SP1与Robo4在体外高糖培养的HREC中的表达情况体外用高糖培养基培养HREC,利用RT-q PCR和Western blot法检测伴随高糖作用时间的延长,SP1与Robo4在HREC中m RNA转录和蛋白表达水平的变化。3.检测向HREC中分别转染SP1、Robo4小干扰RNA(small interfering RNA,si RNA)后SP1与Robo4的表达变化向体外高糖培养的HREC中转染SP1 si RNA以抑制细胞内SP1的表达水平,利用RT-q PCR法和Western blot法检测Robo4 m RNA转录水平和蛋白表达量的变化,同样方法转染Robo4 si RNA,观察其是否对SP1的表达水平亦产生影响。4.检测SP1与Robo4在HREC中的转录调控机制采用染色质免疫共沉淀技术(Ch IP)分别检测HREC在正常和高糖两种培养条件下SP1蛋白与Robo4启动子区的结合活性,并在Ch IP实验结果的基础上,利用荧光素酶报告基因系统(Luciferase Assay System)检测SP1蛋白与Robo4基因间的确切结合位点。5.检测SP1调控Robo4对HREC功能的影响向高糖条件培养的HREC中转染si RNA以抑制SP1与Robo4的表达水平,并通过Transwell法、细胞划痕法、单层细胞通透性实验和细胞管腔形成实验分别观察SP1/Robo4表达变化对HREC迁移、单层HREC通透性和管腔形成能力的影响。结果1.PDR患者纤维血管膜的病理组织结构主要分为四种类型:以增殖细胞为主要成分的膜组织、以新生血管居多的膜组织、新生血管破裂后红细胞大量溢出的血管膜与血管退化后以致密纤维组织为主要成分的纤维膜,SP1与Robo4在不同类型纤维血管膜中的表达均异常增高,且二者的表达共定位于纤维血管膜的微血管内皮细胞中;2.在体外高糖培养的HREC中,SP1与Robo4的m RNA转录与蛋白表达水平随高糖作用时间的延长呈同步性稳定增加;3.在高糖培养的HREC中,转染SP1 si RNA后,SP1的表达水平受到明显抑制,同时Robo4的m RNA和蛋白水平也显著降低,相反细胞内转染Robo4 si RNA仅能有效抑制Robo4的表达水平,对SP1的表达无显著影响;4.Ch IP结果显示在正常培养的HREC中,Robo4启动子-2400/-2700bp区域可与转录因子SP1相结合,而在高糖培养的HREC中,启动子-2400/-2700bp与-1800/-2100bp区域均与SP1有结合活性,Luciferase结果显示,HREC在正常条件下,SP1可与Robo4启动子区-2452/-2448bp位点结合调控Robo4的转录水平,而在高糖条件下,SP1可作用于Robo4启动子区-2452/-2448bp和-1912/-1908bp两个结合位点,进而增强Robo4的转录活性;5.高糖条件可引起HREC的迁移能力增强、单层HREC通透性增加和管腔形成能力下降,向细胞内转染SP1 si RNA或Robo4 si RNA后,HREC的迁移能力有所下降,单层HREC通透性显著降低,且HREC的管腔形成能力得到明显改善。结论本实验研究SP1与Robo4在DR发生发展中的作用及调控机制,发现SP1与Robo4在PDR患者的纤维血管膜内表达异常增高,提示二者可能参与纤维血管膜的形成,同时在体外高糖培养的HREC中,SP1与Robo4表达呈同步性上升,且下调SP1的表达可明显抑制高糖诱导的Robo4表达升高,说明SP1对Robo4表达存在一定的调控作用。进一步研究SP1与Robo4间的转录调控机制,发现高糖条件可诱导HREC中SP1过表达,并通过与Robo4启动子区-2452/-2448bp和-1912/-1908bp两个结合位点相互作用,从而增强Robo4的转录活性,提高Robo4的表达水平。且SP1/Robo4通路与HREC的功能密切相关,抑制SP1/Robo4的表达水平可显著改善高糖诱导的HREC功能异常,说明SP1/Robo4通路在DR微血管病变的发生中可能发挥一定的促进作用。
[Abstract]:Diabetic retinopathy (diabetic retinopathy, DR) is one of the severe microvascular complications of diabetes. Its pathogenesis is very complex. The current research shows that the retina can occur oxidative stress, inflammatory response, neurodegenerative changes and epigenetic modification, with the stimulation of continuous high glucose environment. According to the pathological changes of retinal vessels, the course of DR can be divided into two stages of non proliferative and proliferative stages. In the non proliferation stage of DR, the lesion is mainly caused by the damage or even loss of the retinal microvascular endothelial cell structure, and the pericytes withering. In DR proliferation period, large area of capillary occlusion causes severe hypoxia in the retina, causing a large number of growth factors and inflammatory mediators to rise, resulting in the formation of retinal neovascularization, thus protecting retinal microvessels. The integrity of the inhibition of retinal neovascularization is the key.Roundabout 4 (Robo4) for the prevention and treatment of DR (Robo4), a new member of the Robo family found in 2002, which is specifically expressed on the surface of vascular endothelial cells, regulating endothelial cell permeability and affecting neovascularization. Recent studies have found that the expression changes of Robo4 and DR In human retinal vascular endothelial cells (human retinal endothelial cells, HREC) in vitro hypoxia conditions, the expression of Robo4 in cells increases gradually with the prolongation of hypoxia, and the inhibition of the expression of Robo4 can effectively improve the proliferation and migration of hypoxia induced HREC, and in vivo In the animal model of diabetes, the expression level of Robo4 in the retina is gradually increased with the occurrence and duration of DR. Thus, it is suggested that Robo4 may play an important role in the development of DR, but its specific mechanism is not clear. In the study of the regulatory mechanism of Robo4 expression, the gene promoter is used. The prediction software analyses the promoter region of the upstream of Robo4. It is found that there are multiple specificity protein 1 (SP1) binding sites, and the study shows that SP1 as a common transcription factor can regulate the transcription of multiple target genes in the downstream and affect the development of DR. Thus, it is presumed that Robo4 may be another target factor of SP1, and in DR pathogenesis, SP1 passes. Regulation of the transcriptional activity of Robo4 affects the occurrence of retinal microvascular lesions. In this study, the expression of SP1 and Robo4 in the fibrous vascular membrane of proliferative diabetic retinopathy (PDR) patients was detected in this study, and HREC was cultured with high glucose conditions to simulate the internal environment of DR, and to explore SP1 and Ro at this time. The expression level of bo4 and the transcriptional regulation mechanism between the two and the effect of the mechanism on the HREC function under high glucose. Method 1. to detect the expression of SP1 and Robo4 in the fibrous membrane of patients with PDR and to collect the fibrous membrane of the patients of PDR in the clinical operation, and to stain them by HE staining and to observe the pathological structure of the membrane tissue. The expression of the expression of SP1 and Robo4 protein in the fibrous vascular membrane was detected by immunohistochemical staining, and the co localization of the two were detected by double immunofluorescence..2. was used to detect the expression of SP1 and Robo4 in HREC in high glucose culture in vitro, and HREC was cultured with Gao Tangpei culture medium in vitro, and RT-q PCR and Western blot method were used. The change of M RNA transcriptional and protein expression levels between SP1 and Robo4 in HREC was detected by.3., and SP1 was transfected into HREC, and Robo4 small interference RNA (small interfering) was transfected to the high glucose culture in vitro. RT-q PCR and Western blot were used to detect the changes of Robo4 m RNA transcriptional level and protein expression. The same method transfected Robo4 Si RNA, and observed whether the expression level of SP1 was also affected by the transcription regulation mechanism of.4. detection and chromatin immunoprecipitation. The binding activity of SP1 protein and Robo4 promoter in two culture conditions, and on the basis of Ch IP experimental results, using the luciferase reporter gene system (Luciferase Assay System) to detect the exact binding site of SP1 protein and Robo4 gene,.5. SP1 regulation of Robo4 on the effect of Robo4 on the high sugar condition Si RNA was used to inhibit the expression of SP1 and Robo4, and the effects of SP1/Robo4 expression on HREC migration, monolayer HREC permeability and lumen formation ability were observed by Transwell, cell scratch, monolayer permeability and cell cavity formation. Results the pathological histological structure of the fibrous membrane of 1.PDR patients was mainly divided into two parts. The four types: the membrane tissue of the proliferating cell as the main component, the membrane tissue of the neovascularization, the large spillover of the red blood cells after the rupture of the neovascularization and the fibrous membrane of the dense fibrous tissue after the degeneration of the blood vessels. The expression of SP1 and Robo4 in the different types of fibrous vascular membrane are all abnormally increased and the expression of the two 2. in the microvascular endothelial cells in the fibrous vascular membrane; 2. in the high sugar cultured HREC in vitro, the m RNA transcription and protein expression level of SP1 and Robo4 increased synchronously with the prolongation of the time of high glucose; 3. in the high sugar culture HREC, the expression level of SP1 was obviously inhibited after transfecting SP1 Si RNA, and Robo4 M The protein level also decreased significantly. On the contrary, the transfection of Robo4 Si RNA only effectively inhibited the expression of Robo4, and had no significant effect on the expression of SP1. The 4.Ch IP results showed that the Robo4 promoter -2400/-2700bp region could be combined with the SP1 of the transcription factor in the normal cultured HREC. Both /-2100bp region and SP1 have binding activity. Luciferase results show that under normal conditions, SP1 can regulate the transcriptional level of Robo4 with the -2452/-2448bp locus of Robo4 promoter region in normal conditions, but in high glucose conditions, SP1 can act on the two binding sites of -2452/-2448bp and -1912/ Robo4 in the promoter region of Robo4, and then increase the transcriptional activity of Robo4; 5 High glucose conditions could increase the migration ability of HREC, increase the permeability of single layer HREC and decrease the formation ability of the lumen. After transfecting SP1 Si RNA or Robo4 Si RNA into the cells, the migration ability of HREC decreased, the permeability of single-layer HREC decreased significantly, and the capacity of the HREC lumen formation was obviously improved. The role of SP1 and Robo4 in the fibrous vascular membrane of PDR patients was found to be abnormal, suggesting that the two may participate in the formation of fibrous vascular membrane, while the expression of SP1 and Robo4 increased synchronously in HREC in high glucose culture in vitro, and the expression of SP1 could inhibit the elevation of Robo4 expression induced by high glucose. It is suggested that SP1 has a certain regulatory effect on the expression of Robo4, and further studies the transcriptional regulation mechanism between SP1 and Robo4. It is found that high sugar conditions can induce SP1 overexpression in HREC and interact with the two binding sites of -2452/-2448bp and -1912/-1908bp in the promoter region of Robo4, thus strengthening the transcriptional activity of Robo4 and improving the Robo4 expression level. And the SP1/Robo4 pathway is closely related to the function of HREC. Inhibition of the expression level of SP1/Robo4 can significantly improve the abnormal HREC function induced by high glucose, which indicates that the SP1/Robo4 pathway may play a certain role in the pathogenesis of DR microvascular lesions.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R774.1

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