AGEs-Cyr61信号通路在糖尿病小鼠激光诱导脉络膜新生血管生成中的作用及其机制

发布时间：2018-06-21 15:22

  本文选题：年龄相关性黄斑变性 + 脉络膜新生血管　； 参考：《第四军医大学》2017年博士论文

【摘要】：【研究背景】脉络膜新生血管(choroidalneovascularization,CNV)至少与40余种眼部疾病相关,常见于年龄相关性黄斑病变(age-relatedmaculardegeneration,AMD)、病理性近视黄斑变性、特发性CNV、眼组织胞浆菌病综合征以及眼外伤等,其中AMD是发达国家老年人视力丧失的首要原因。上述严重威胁视功能的脉络膜视网膜疾病具有共同的病理学特征,即CNV形成。因此,阐明CNV发生机理和临床防治策略已然成为近年来眼科学领域的研究热点和难点之一。目前,已证实CNV发生的危险因素主要包括年龄、吸烟、遗传及心血管疾病等[1],而糖尿病作为心血管疾病明确的诱因,其与CNV发生的相关性研究已逐渐受到学者们关注[2,3]。目前,临床流行病学调查是针对上述疾病开展的主要研究形式;然而,深入系统的基础理论研究尚缺乏,糖尿病影响CNV发生的分子机制仍不清楚。我们前期的实验研究发现,高血糖对CNV发生、发展具有十分重要的作用,主要证据包括:(1)糖尿病可加重链脲佐菌素(Streptozocin,STZ)诱导的糖尿病小鼠实验性CNV的严重程度;(2)给予糖尿病小鼠抗氧化剂治疗可明显缓解其氧化损伤程度,从而阻断与血管新生相关的细胞信号通路,导致下游细胞因子的分泌量减少,降低CNV生成风险;(3)高血糖增加RPE细胞氧化应激水平,进而上调VEGF的表达;(4)高血糖可通过上调RPE细胞中VEGF的表达促进骨髓来源的间充质干细胞趋化至CNV区域并参与血管发生,从而加剧CNV的严重程度。富半胱氨酸61(csteinrich61,Cyr61)作为一种十分重要的细胞基质调节因子,在细胞的增殖、粘附、侵袭与转移以及血管生成、炎症发生和组织重塑等重要生理、病理过程中发挥重要的调节作用,其是我们利用Agilent Mouse基因表达谱芯片对CNV小鼠及糖尿病条件下CNV小鼠RPE-脉络膜-巩膜复合体进行差异分析获得的基因。Cyr61及其介导的信号通路是否参与CNV的生成,其中具体的分子机制是什么?对于上述问题的回答必将为CNV的防治提供新的策略。【目的】探索AGEs-Cyr61信号通路对糖尿病小鼠激光诱导CNV形成的调节作用及具体分子机理,为CNV的临床防治开拓新的靶点奠定理论基础。【方法】一、Cyr61在糖尿病模型小鼠CNV生成中的表达状态分析(1)实验分组:将C57BL/6J小鼠随机分为三组:正常对照组、糖尿病组及糖尿病+氨基胍治疗组,每组20只;(2)动物模型:腹腔连续5 d注射STZ构建小鼠糖尿病模型,模型构建成功2 w后,应用532 nm倍频激光诱导CNV生成;激光光凝后14 d:(3)脉络膜铺片:将RPE-脉络膜-巩膜复合体铺展进行血管染色,通过脉络膜铺片血管染色,三维重建比较各组CNV体积;(4)组织病理学检查:通过HE染色观察各组CNV的高度和厚度差异;(5)组织免疫荧光染色:制备冰冻切片,抗体标记脉络膜组织,分析Cyr61与VEGF的表达定位情况;(6)ELISA实验:检测脉络膜组织中Cyr61和VEGF分泌量。二、Cyr61对糖尿病小鼠CNV生成的影响(1)实验分组:将C57BL/6J小鼠随机分为三组——正常对照组、糖尿病组及糖尿病+Cyr61单克隆抗体治疗组,每组20只;(2)动物模型:腹腔连续5 d注射STZ构建小鼠糖尿病模型,模型构建成功2 w后,应用532 nm倍频激光诱导CNV生成;激光光凝后14 d:(3)脉络膜铺片:将RPE-脉络膜-巩膜复合体铺展进行血管染色,通过脉络膜铺片血管染色,三维重建比较各组CNV体积;(4)组织病理学检查:通过HE染色观察各组CNV的高度和厚度差异;(5)组织免疫荧光染色:制备冰冻切片,抗体标记脉络膜组织,分析Cyr61与VEGF的表达定位情况;(6)ELISA实验:检测脉络膜组织中Cyr61和VEGF分泌量;(7)体外细胞行为学实验:建立RPE细胞与CEC细胞共培养体系,分别使用MTT、Transwell和管腔形成实验检测基因沉默RPE细胞中Cyr61表达后,CEC细胞活力、移行数量和管腔形成长度;三、AGEs对Cyr61的表达调控及其分子机制(1)实验分组:以RPE为细胞模型,分为六组——正常对照组、高糖处理组、10μg/m L BSA-AGEs处理组、50μg/mL BSA-AGEs处理组、100μg/mL BSA-AGEs处理组和sRAGE处理组;(2)Real-time PCR实验:给予各处理组作用后,观察Cyr61mRNA表达水平变化;(3)Western-blot:给予各处理组作用后,检测Cyr61、ERK1/2、p-ERK1/2、JNK、p-JNK、p38、p-p38、Stat 3h和p-Stat 3等蛋白表达水平变化;(4)双荧光素酶报告基因:转染pCMV-Stat质粒,分析Cyr61转录活性情况;(5)Ch IP实验:细胞经甲醛交联、超声破碎、免疫共沉淀和RT-PCR后,行琼脂糖凝胶电泳比较条带明暗程度。四、Cyr61对VEGF的表达调控及其分子机制(1)细胞模型:原代培养CEC细胞,将其作为后续实验研究对象;(2)RT-PCR实验:40 ng/mL重组Cyr61作用细胞0、30、60、90、120 min,观察VEGF mRNA表达水平变化;(3)Western-blot:给予各处理组作用后,检测VEGF、FAK、p-FAK、PI3K、p-PI3K、AKT、p-AKT、IKK、p-IKK、IκB、MMP2和MMP13等蛋白表达水平变化;(4)细胞免疫荧光:采用激光共聚焦显微镜,观察NF-κB细胞内定位情况变化。【结果】一、Cyr61在糖尿病模型小鼠CNV生成中的表达状态分析(1)AGEs抑制剂氨基胍可缓解糖尿病小鼠CNV生成的严重程度:光凝后14d,STZ诱导的糖尿病小鼠RPE层和Bruch膜出现破裂,RPE细胞有较明显增殖和迁移,新生血管增多,较正常小鼠CNV体积显著增大(P0.01),而给予氨基胍治疗,糖尿病小鼠CNV生成量明显减少,CNV的厚度和高度显著降低;(2)阻断AGEs形成可下调Cyr61和VEGF的表达:糖尿病小鼠CNV区域Cyr61和VEGF呈高表达状态,氨基胍不仅可抑制AGEs的形成,还可降低糖尿病小鼠眼部Cyr61及VEGF的分泌量。二、Cyr61对糖尿病小鼠CNV生成的影响(1)Cyr61单克隆抗体可降低糖尿病小鼠CNV生成的严重程度:光凝后14d,STZ诱导的糖尿病小鼠RPE层和Bruch膜出现破裂,RPE细胞有较明显增殖和迁移,新生血管增多,较正常小鼠CNV体积显著增大(P0.01),而给予Cyr61单克隆抗体治疗,糖尿病小鼠CNV生成量明显减少,CNV的厚度和高度显著降低;(2)特异性阻断Cyr61可减少Cyr61和VEGF的表达量:糖尿病小鼠CNV区域Cyr61和VEGF呈高表达状态,玻璃体腔注射Cyr61单克隆抗体可显著性降低糖尿病小鼠眼部Cyr61及VEGF的分泌量;(3)基因沉默Cyr61表达能够显著性抑制CEC细胞增生、移行和管腔形成:与AGEs处理组相比,腺病毒感染组CEC细胞增殖率下降32%(P0.01)、细胞迁移数量降低67%(P0.01)、管腔形成长度减少29%(P0.01)。三、AGEs对Cyr61的表达调控及其分子机制(1)AGEs通过与受体RAGE结合调控Cyr61的表达:随着AGEs刺激浓度增加,Cyr61表达水平逐渐升高,而游离的RAGE可抑制上述正相关效应;(2)JNK信号通路介导AGEs对Cyr61的表达调控:AGEs可促进MAPK信号通路相关蛋白ERK1/2、JNK和p38磷酸化,但仅阻断JNK信号通路可下调Cyr61的表达;(3)AGEs通过JNK信号通路促使转录因子Stat 3活化:生物信息学分析发现Cyr61启动子区存在Stat 3结合位点,阻断JNK信号通路可显著性逆转AGEs致Stat 3磷酸化水平升高;(4)转录因子Stat 3增强Cyr61启动子区转录活性:转录因子Stat 3与Cyr61基因转录起始位点上游-1351至-1333 bp启动子区域结合,进而促进Cyr61的表达。四、Cyr61对VEGF的表达调控及其分子机制(1)Cyr61通过与整合素受体αVβ3结合调控VEGF表达:封闭整合素受体αVβ3可显著性下调VEGF的表达,而封闭整合素受体αVβ5和α5β1,未见VEGF表达量有变化;(2)FAK-PI3K信号通路参与VEGF的表达调控:在重组蛋白Cyr61的作用下,CEC细胞内FAK-PI3K/AKT信号通路被激活,且上述信号通路参与调控CEC细胞VEGF的表达;(3)Cyr61促进转录因子NF-kB细胞核转位:Cyr61刺激组6 h,染色主要集中于细胞核内,细胞浆中较少;与Cyr61刺激组比较,FAK抑制剂作用6 h时,细胞浆染色较细胞核内深;(4)Cyr61促进基质金属蛋白酶MMP2/MMP13的表达:与正常对照组相比,CEC细胞在Cyr61的作用下,MMP2、MMP13表达量显著升高(P0.01)。【结论】AGEs可通过与RPE细胞表面受体RAGE结合,诱导转录因子Stat 3活化,进而调控Cyr61的表达;Cyr61不仅可激活CEC细胞内整合素-PI3K/AKT信号,促进转录因子NF-κB核转位上调VEGF,还对MMP2/MMP13具有重要的调控作用;抑制AGEs的形成或特异性阻断Cyr61,能够显著性地抑制CEC细胞增生、移行和管腔形成,从而起到缓解糖尿病所致CNV严重程度的作用。综上所述,AGEs-Cyr61-VEGF信号通路参与了CNV生成过程的调控,此类研究在国内外尚未有文献报道,特异性阻断该通路可显著性地抑制CNV生成,这将为临床防治CNV性疾病提供新策略。
[Abstract]:[background] choroidalneovascularization (CNV) is associated with at least 40 kinds of eye diseases. It is common in age related macular lesions (age-relatedmaculardegeneration, AMD), pathological myopia macular degeneration, idiopathic CNV, ocular histoplasmosis syndrome, and ocular trauma, and AMD is a developed country. The primary cause of loss of visual acuity in the year. The above-mentioned serious threat of visual function of choroidal retinopathy has common pathological features, that is, CNV formation. Therefore, it is one of the hotspots and difficulties in the field of ophthalmology to clarify the mechanism of CNV and the strategy of clinical prevention and control. The main package of risk factors for the occurrence of CNV has been confirmed before. Including age, smoking, heredity and cardiovascular disease, [1], and diabetes as a definite inducement of cardiovascular disease, its correlation with CNV has gradually been paid attention to by scholars at present, and the clinical epidemiology investigation is the main research form for the above-mentioned diseases. However, the basic theoretical research of the system is still lacking, sugar is still lacking. The molecular mechanism of the occurrence of CNV in urine is still unclear. Our previous experimental study found that hyperglycemia has a very important role in the development of CNV. The main evidence includes: (1) diabetes can aggravate the severity of experimental CNV in diabetic mice induced by Streptozocin (STZ); (2) to give antioxidant to diabetic mice Agent therapy can significantly alleviate the degree of oxidative damage, thus blocking the cell signaling pathway associated with angiogenesis, reducing the secretion of downstream cytokines and reducing the risk of CNV generation; (3) hyperglycemia increases the oxidative stress level of RPE cells, and then up-regulated the expression of VEGF; (4) hyperglycemia can promote bone by up regulating the expression of VEGF in RPE cells. The marrow derived mesenchymal stem cells chemotaxis into the CNV region and participate in angiogenesis, which exacerbates the severity of CNV. Rich cysteine 61 (csteinrich61, Cyr61) is a very important cell matrix regulator in cell proliferation, adhesion, invasion and metastasis, blood Guan Shengcheng, inflammation and tissue remodeling. In the process, it plays an important role in the use of the Agilent Mouse gene expression chip to analyze the difference of the gene.Cyr61 and the mediated signaling pathway of the RPE- choroidal sclera complex in the CNV mice and the diabetic CNV mice. What are the specific molecular mechanisms? The answers to the questions will certainly provide a new strategy for the prevention and control of CNV. [Objective] to explore the regulatory role of AGEs-Cyr61 signaling pathway and the specific molecular mechanism of laser induced CNV formation in diabetic mice, and to lay a theoretical foundation for the development of new targets for the clinical prevention and control of CNV. [method] 1, Cyr61 expression in the formation of CNV in diabetic mice. State analysis (1) experimental group: C57BL/6J mice were randomly divided into three groups: normal control group, diabetes group and diabetes + aminoguanidine treatment group, 20 rats in each group; (2) animal model: 5 d intraperitoneal continuous injection of STZ to construct a diabetic model of mice, after the model construction was 2 W, 532 nm frequency doubling laser induced CNV generation, and 14 d: (3) veins after laser photocoagulation. Membrane sheet: staining RPE- choroidal and scleral complex, stained with blood vessel of choroid membrane, and comparing CNV volume with three-dimensional reconstruction. (4) histopathological examination: the height and thickness difference of CNV in each group was observed by HE staining; (5) tissue immunofluorescence staining: preparation of frozen section, antibody labelled choroidal tissue, and analysis of Cyr6 1 and VEGF expression localization; (6) ELISA experiment: detect the secretion of Cyr61 and VEGF in choroid tissue. Two, Cyr61 on the formation of CNV in diabetic mice (1) experimental grouping: C57BL/6J mice were randomly divided into three groups: normal control group, diabetes group and diabetes + Cyr61 monoclonal antibody treatment group, 20 mice in each group; (2) animal model: abdominal cavity After 5 d injection of STZ to construct a diabetic model of diabetic mice, after the construction of the model was 2 W, CNV was induced by 532 nm frequency doubling laser; 14 d: (3) choroidal spread after laser photocoagulation: vascular dyeing of RPE- choroidal scleral complex, vascular dyeing through choroidal patch, and three-dimensional reconstruction to compare each group of CNV volume; (4) histopathological examination HE staining was used to observe the height and thickness difference of CNV in each group; (5) tissue immunofluorescence staining: preparation of frozen section, antibody labelled choroidal tissue, analysis of expression and localization of Cyr61 and VEGF; (6) ELISA experiment: detecting the secretion of Cyr61 and VEGF in choroid tissue; (7) in vitro cell behavior test: co culture of RPE cells and CEC cells System, MTT, Transwell and lumen formation were used to test the expression of Cyr61 in RPE cells with gene silencing, CEC cell vitality, migration number and length of lumen formation; three, AGEs on Cyr61 expression regulation and molecular mechanism (1) experimental grouping: RPE as the cell model, divided into six groups: normal control group, high sugar treatment group, 10 u g/m L BSA-. AGEs treatment group, 50 g/mL BSA-AGEs treatment group, 100 g/mL BSA-AGEs treatment group and sRAGE treatment group; (2) Real-time PCR experiment: after giving various treatment groups, the changes of Cyr61mRNA expression level were observed. (3) Western-blot: was given to each treatment group. (4) double luciferase reporter gene: transfection of pCMV-Stat plasmid and analysis of Cyr61 transcriptional activity; (5) Ch IP experiment: cells via formaldehyde crosslinking, ultrasonic breakage, immunoprecipitation and RT-PCR, agarose gel electrophoresis was used to compare the degree of light and shade with agarose gel electrophoresis. Four, Cyr61 on the regulation of VEGF and its molecular mechanism (1) cell model: primary culture CE C cells as a follow-up study object; (2) RT-PCR experiment: 40 ng/mL recombinant Cyr61 cell 0,30,60,90120 min, observe the change of VEGF mRNA expression level; (3) Western-blot: give the changes of protein expression level of VEGF, FAK, p-FAK, reject, etc. Immunofluorescence: a laser confocal microscope was used to observe the changes in the localization of NF- kappa B cells. [results] an analysis of the expression status of Cyr61 in the CNV production of diabetic mice (1) the AGEs inhibitor aminoguanidine could relieve the severity of CNV generation in diabetic mice: the RPE layer and Bruch membrane induced by STZ induced by 14d, STZ induced diabetic mice were broken. RPE cell proliferation and migration, neovascularization, increased CNV volume in normal mice (P0.01), and aminoguanidine treatment, CNV production in diabetic mice decreased significantly, the thickness and height of CNV decreased significantly. (2) blocking AGEs formation can reduce the expression of Cyr61 and VEGF: the Cyr61 and VEGF in the CNV region of diabetic mice is high table Aminoguanidine can not only inhibit the formation of AGEs, but also reduce the secretion of Cyr61 and VEGF in diabetic mice. Two, Cyr61 affects the formation of CNV in diabetic mice (1) Cyr61 monoclonal antibodies can reduce the severity of CNV generation in diabetic mice: 14d after photocoagulation, RPE layer and Bruch membrane in STZ induced diabetic mice, RPE finer Cell proliferation and migration, neovascularization, increased CNV volume in normal mice (P0.01), and Cyr61 monoclonal antibody treatment, CNV production in diabetic mice decreased significantly, CNV thickness and height decreased significantly; (2) specific blocking Cyr61 can reduce the expression of Cyr61 and VEGF: Cyr61 and VEGF in the CNV region of diabetic mice The expression of Cyr61 monoclonal antibody could significantly reduce the secretion of Cyr61 and VEGF in the eye of diabetic mice; (3) the expression of gene silencing Cyr61 could significantly inhibit the proliferation, migration and lumen formation of CEC cells: compared with the AGEs treatment group, the proliferation rate of CEC cells in the adenovirus infected group decreased by 32% (P0.01) and the cell migration number. The volume decreased by 67% (P0.01), the length of the cavity formation was reduced by 29% (P0.01). Three, the expression regulation of Cyr61 and its molecular mechanism (1) AGEs regulated the expression of Cyr61 by binding to the receptor RAGE: with the increase of AGEs stimulation concentration, the Cyr61 expression level increased gradually, while the free RAGE could inhibit the above positive correlation effect; (2) JNK signaling pathway mediated AGEs against. Expression regulation: AGEs can promote the phosphorylation of MAPK signaling pathway related proteins ERK1/2, JNK and p38, but only blocking JNK signaling pathway can downregulate the expression of Cyr61; (3) AGEs activates the activation of the transcription factor Stat 3 through the JNK signaling pathway: bioinformatics analysis found that Cyr61 promoter region is stored at the Stat 3 binding site and blocking the signaling pathway can be significantly reversed. The conversion of AGEs to Stat 3 phosphorylation level increased; (4) transcription factor Stat 3 enhanced the transcriptional activity of Cyr61 promoter region: transcription factor Stat 3 and Cyr61 gene transcription start site upstream -1351 to -1333 BP promoter region binding, and then promote the expression of Cyr61. Four, Cyr61 on the expression of VEGF and its molecular mechanism (1) through the integrin receptor alpha V beta 3 combined with the regulation of VEGF expression: blocking integrin receptor alpha V beta 3 significantly downregulates the expression of VEGF, while blocking integrin receptor alpha V beta 5 and alpha 5 beta 1 and no VEGF expression changes; (2) FAK-PI3K signaling pathway participates in the regulation of VEGF expression: the FAK-PI3K/AKT signaling pathway within CEC cells is activated under the action of recombinant protein Cyr61, and the above signal The pathway participates in the regulation of the expression of CEC cell VEGF; (3) Cyr61 promotes the transcription factor NF-kB nuclear transposition: Cyr61 stimulation group is 6 h, and the staining is mainly concentrated in the nucleus and in the cytoplasm. Compared with the Cyr61 stimulation group, the cytoplasmic staining is deeper than the nucleus when the action of FAK inhibitor is 6 h; (4) Cyr61 promotes the MMP2/MMP13 expression of matrix metalloproteinase: Compared with the normal control group, the expression of MMP2 and MMP13 in CEC cells increased significantly (P0.01) under the action of Cyr61. [Conclusion] AGEs can induce the activation of the transcription factor Stat 3 by binding to the RPE cell surface receptor RAGE, and then regulate the expression of Cyr61. Cyr61 can not only activate the signal of the integrin in the CEC cell, but also promote the transcription factor kappa nuclear transfer. The up regulation of VEGF also plays an important role in the regulation of MMP2/MMP13, and the inhibition of the formation of AGEs or the specific blocking of Cyr61 can significantly inhibit the proliferation, migration and formation of CEC cells, thus alleviating the CNV severity caused by diabetes. In summary, the AGEs-Cyr61-VEGF signaling pathway is involved in the regulation of CNV production. No specific studies have been reported at home and abroad. Specific blocking of this pathway can significantly inhibit the generation of CNV, which will provide a new strategy for clinical prevention and treatment of CNV diseases.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R774.5
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