本文選題：年齡相關(guān)性黃斑變性 + 脈絡(luò)膜新生血管　； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：【研究背景】脈絡(luò)膜新生血管(choroidalneovascularization,CNV)至少與40余種眼部疾病相關(guān),常見于年齡相關(guān)性黃斑病變(age-relatedmaculardegeneration,AMD)、病理性近視黃斑變性、特發(fā)性CNV、眼組織胞漿菌病綜合征以及眼外傷等,其中AMD是發(fā)達國家老年人視力喪失的首要原因。上述嚴(yán)重威脅視功能的脈絡(luò)膜視網(wǎng)膜疾病具有共同的病理學(xué)特征,即CNV形成。因此,闡明CNV發(fā)生機理和臨床防治策略已然成為近年來眼科學(xué)領(lǐng)域的研究熱點和難點之一。目前,已證實CNV發(fā)生的危險因素主要包括年齡、吸煙、遺傳及心血管疾病等[1],而糖尿病作為心血管疾病明確的誘因,其與CNV發(fā)生的相關(guān)性研究已逐漸受到學(xué)者們關(guān)注[2,3]。目前,臨床流行病學(xué)調(diào)查是針對上述疾病開展的主要研究形式;然而,深入系統(tǒng)的基礎(chǔ)理論研究尚缺乏,糖尿病影響CNV發(fā)生的分子機制仍不清楚。我們前期的實驗研究發(fā)現(xiàn),高血糖對CNV發(fā)生、發(fā)展具有十分重要的作用,主要證據(jù)包括:(1)糖尿病可加重鏈脲佐菌素(Streptozocin,STZ)誘導(dǎo)的糖尿病小鼠實驗性CNV的嚴(yán)重程度;(2)給予糖尿病小鼠抗氧化劑治療可明顯緩解其氧化損傷程度,從而阻斷與血管新生相關(guān)的細(xì)胞信號通路,導(dǎo)致下游細(xì)胞因子的分泌量減少,降低CNV生成風(fēng)險;(3)高血糖增加RPE細(xì)胞氧化應(yīng)激水平,進而上調(diào)VEGF的表達;(4)高血糖可通過上調(diào)RPE細(xì)胞中VEGF的表達促進骨髓來源的間充質(zhì)干細(xì)胞趨化至CNV區(qū)域并參與血管發(fā)生,從而加劇CNV的嚴(yán)重程度。富半胱氨酸61(csteinrich61,Cyr61)作為一種十分重要的細(xì)胞基質(zhì)調(diào)節(jié)因子,在細(xì)胞的增殖、粘附、侵襲與轉(zhuǎn)移以及血管生成、炎癥發(fā)生和組織重塑等重要生理、病理過程中發(fā)揮重要的調(diào)節(jié)作用,其是我們利用Agilent Mouse基因表達譜芯片對CNV小鼠及糖尿病條件下CNV小鼠RPE-脈絡(luò)膜-鞏膜復(fù)合體進行差異分析獲得的基因。Cyr61及其介導(dǎo)的信號通路是否參與CNV的生成,其中具體的分子機制是什么?對于上述問題的回答必將為CNV的防治提供新的策略�！灸康摹刻剿鰽GEs-Cyr61信號通路對糖尿病小鼠激光誘導(dǎo)CNV形成的調(diào)節(jié)作用及具體分子機理,為CNV的臨床防治開拓新的靶點奠定理論基礎(chǔ)。【方法】一、Cyr61在糖尿病模型小鼠CNV生成中的表達狀態(tài)分析(1)實驗分組:將C57BL/6J小鼠隨機分為三組:正常對照組、糖尿病組及糖尿病+氨基胍治療組,每組20只;(2)動物模型:腹腔連續(xù)5 d注射STZ構(gòu)建小鼠糖尿病模型,模型構(gòu)建成功2 w后,應(yīng)用532 nm倍頻激光誘導(dǎo)CNV生成;激光光凝后14 d:(3)脈絡(luò)膜鋪片:將RPE-脈絡(luò)膜-鞏膜復(fù)合體鋪展進行血管染色,通過脈絡(luò)膜鋪片血管染色,三維重建比較各組CNV體積;(4)組織病理學(xué)檢查:通過HE染色觀察各組CNV的高度和厚度差異;(5)組織免疫熒光染色:制備冰凍切片,抗體標(biāo)記脈絡(luò)膜組織,分析Cyr61與VEGF的表達定位情況;(6)ELISA實驗:檢測脈絡(luò)膜組織中Cyr61和VEGF分泌量。二、Cyr61對糖尿病小鼠CNV生成的影響(1)實驗分組:將C57BL/6J小鼠隨機分為三組——正常對照組、糖尿病組及糖尿病+Cyr61單克隆抗體治療組,每組20只;(2)動物模型:腹腔連續(xù)5 d注射STZ構(gòu)建小鼠糖尿病模型,模型構(gòu)建成功2 w后,應(yīng)用532 nm倍頻激光誘導(dǎo)CNV生成;激光光凝后14 d:(3)脈絡(luò)膜鋪片:將RPE-脈絡(luò)膜-鞏膜復(fù)合體鋪展進行血管染色,通過脈絡(luò)膜鋪片血管染色,三維重建比較各組CNV體積;(4)組織病理學(xué)檢查:通過HE染色觀察各組CNV的高度和厚度差異;(5)組織免疫熒光染色:制備冰凍切片,抗體標(biāo)記脈絡(luò)膜組織,分析Cyr61與VEGF的表達定位情況;(6)ELISA實驗:檢測脈絡(luò)膜組織中Cyr61和VEGF分泌量;(7)體外細(xì)胞行為學(xué)實驗:建立RPE細(xì)胞與CEC細(xì)胞共培養(yǎng)體系,分別使用MTT、Transwell和管腔形成實驗檢測基因沉默RPE細(xì)胞中Cyr61表達后,CEC細(xì)胞活力、移行數(shù)量和管腔形成長度;三、AGEs對Cyr61的表達調(diào)控及其分子機制(1)實驗分組:以RPE為細(xì)胞模型,分為六組——正常對照組、高糖處理組、10μg/m L BSA-AGEs處理組、50μg/mL BSA-AGEs處理組、100μg/mL BSA-AGEs處理組和sRAGE處理組;(2)Real-time PCR實驗:給予各處理組作用后,觀察Cyr61mRNA表達水平變化;(3)Western-blot:給予各處理組作用后,檢測Cyr61、ERK1/2、p-ERK1/2、JNK、p-JNK、p38、p-p38、Stat 3h和p-Stat 3等蛋白表達水平變化;(4)雙熒光素酶報告基因:轉(zhuǎn)染pCMV-Stat質(zhì)粒,分析Cyr61轉(zhuǎn)錄活性情況;(5)Ch IP實驗:細(xì)胞經(jīng)甲醛交聯(lián)、超聲破碎、免疫共沉淀和RT-PCR后,行瓊脂糖凝膠電泳比較條帶明暗程度。四、Cyr61對VEGF的表達調(diào)控及其分子機制(1)細(xì)胞模型:原代培養(yǎng)CEC細(xì)胞,將其作為后續(xù)實驗研究對象;(2)RT-PCR實驗:40 ng/mL重組Cyr61作用細(xì)胞0、30、60、90、120 min,觀察VEGF mRNA表達水平變化;(3)Western-blot:給予各處理組作用后,檢測VEGF、FAK、p-FAK、PI3K、p-PI3K、AKT、p-AKT、IKK、p-IKK、IκB、MMP2和MMP13等蛋白表達水平變化;(4)細(xì)胞免疫熒光:采用激光共聚焦顯微鏡,觀察NF-κB細(xì)胞內(nèi)定位情況變化�！窘Y(jié)果】一、Cyr61在糖尿病模型小鼠CNV生成中的表達狀態(tài)分析(1)AGEs抑制劑氨基胍可緩解糖尿病小鼠CNV生成的嚴(yán)重程度:光凝后14d,STZ誘導(dǎo)的糖尿病小鼠RPE層和Bruch膜出現(xiàn)破裂,RPE細(xì)胞有較明顯增殖和遷移,新生血管增多,較正常小鼠CNV體積顯著增大(P0.01),而給予氨基胍治療,糖尿病小鼠CNV生成量明顯減少,CNV的厚度和高度顯著降低;(2)阻斷AGEs形成可下調(diào)Cyr61和VEGF的表達:糖尿病小鼠CNV區(qū)域Cyr61和VEGF呈高表達狀態(tài),氨基胍不僅可抑制AGEs的形成,還可降低糖尿病小鼠眼部Cyr61及VEGF的分泌量。二、Cyr61對糖尿病小鼠CNV生成的影響(1)Cyr61單克隆抗體可降低糖尿病小鼠CNV生成的嚴(yán)重程度:光凝后14d,STZ誘導(dǎo)的糖尿病小鼠RPE層和Bruch膜出現(xiàn)破裂,RPE細(xì)胞有較明顯增殖和遷移,新生血管增多,較正常小鼠CNV體積顯著增大(P0.01),而給予Cyr61單克隆抗體治療,糖尿病小鼠CNV生成量明顯減少,CNV的厚度和高度顯著降低;(2)特異性阻斷Cyr61可減少Cyr61和VEGF的表達量:糖尿病小鼠CNV區(qū)域Cyr61和VEGF呈高表達狀態(tài),玻璃體腔注射Cyr61單克隆抗體可顯著性降低糖尿病小鼠眼部Cyr61及VEGF的分泌量;(3)基因沉默Cyr61表達能夠顯著性抑制CEC細(xì)胞增生、移行和管腔形成:與AGEs處理組相比,腺病毒感染組CEC細(xì)胞增殖率下降32%(P0.01)、細(xì)胞遷移數(shù)量降低67%(P0.01)、管腔形成長度減少29%(P0.01)。三、AGEs對Cyr61的表達調(diào)控及其分子機制(1)AGEs通過與受體RAGE結(jié)合調(diào)控Cyr61的表達:隨著AGEs刺激濃度增加,Cyr61表達水平逐漸升高,而游離的RAGE可抑制上述正相關(guān)效應(yīng);(2)JNK信號通路介導(dǎo)AGEs對Cyr61的表達調(diào)控:AGEs可促進MAPK信號通路相關(guān)蛋白ERK1/2、JNK和p38磷酸化,但僅阻斷JNK信號通路可下調(diào)Cyr61的表達;(3)AGEs通過JNK信號通路促使轉(zhuǎn)錄因子Stat 3活化:生物信息學(xué)分析發(fā)現(xiàn)Cyr61啟動子區(qū)存在Stat 3結(jié)合位點,阻斷JNK信號通路可顯著性逆轉(zhuǎn)AGEs致Stat 3磷酸化水平升高;(4)轉(zhuǎn)錄因子Stat 3增強Cyr61啟動子區(qū)轉(zhuǎn)錄活性:轉(zhuǎn)錄因子Stat 3與Cyr61基因轉(zhuǎn)錄起始位點上游-1351至-1333 bp啟動子區(qū)域結(jié)合,進而促進Cyr61的表達。四、Cyr61對VEGF的表達調(diào)控及其分子機制(1)Cyr61通過與整合素受體αVβ3結(jié)合調(diào)控VEGF表達:封閉整合素受體αVβ3可顯著性下調(diào)VEGF的表達,而封閉整合素受體αVβ5和α5β1,未見VEGF表達量有變化;(2)FAK-PI3K信號通路參與VEGF的表達調(diào)控:在重組蛋白Cyr61的作用下,CEC細(xì)胞內(nèi)FAK-PI3K/AKT信號通路被激活,且上述信號通路參與調(diào)控CEC細(xì)胞VEGF的表達;(3)Cyr61促進轉(zhuǎn)錄因子NF-kB細(xì)胞核轉(zhuǎn)位:Cyr61刺激組6 h,染色主要集中于細(xì)胞核內(nèi),細(xì)胞漿中較少;與Cyr61刺激組比較,FAK抑制劑作用6 h時,細(xì)胞漿染色較細(xì)胞核內(nèi)深;(4)Cyr61促進基質(zhì)金屬蛋白酶MMP2/MMP13的表達:與正常對照組相比,CEC細(xì)胞在Cyr61的作用下,MMP2、MMP13表達量顯著升高(P0.01)。【結(jié)論】AGEs可通過與RPE細(xì)胞表面受體RAGE結(jié)合,誘導(dǎo)轉(zhuǎn)錄因子Stat 3活化,進而調(diào)控Cyr61的表達;Cyr61不僅可激活CEC細(xì)胞內(nèi)整合素-PI3K/AKT信號,促進轉(zhuǎn)錄因子NF-κB核轉(zhuǎn)位上調(diào)VEGF,還對MMP2/MMP13具有重要的調(diào)控作用;抑制AGEs的形成或特異性阻斷Cyr61,能夠顯著性地抑制CEC細(xì)胞增生、移行和管腔形成,從而起到緩解糖尿病所致CNV嚴(yán)重程度的作用。綜上所述,AGEs-Cyr61-VEGF信號通路參與了CNV生成過程的調(diào)控,此類研究在國內(nèi)外尚未有文獻報道,特異性阻斷該通路可顯著性地抑制CNV生成,這將為臨床防治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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R774.5
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本文編號：2049229