CXC趋化因子受体4在氧诱导视网膜病变发病中的作用及其机制

发布时间：2018-05-15 17:23

本文选题：CXC趋化因子受体4 + 氧诱导视网膜病变　；参考：《武汉大学》2012年博士论文

【摘要】：视网膜新生血管性疾病是世界上主要的致盲原因之一。此类疾病包括增殖性糖尿病视网膜病变(PDR)、早产儿视网膜病变(ROP)、视网膜中央静脉阻塞(CRVO)和年龄相关性黄斑变性(AMD)等。它们共同特征在于视网膜组织相对缺氧导致了病理性视网膜新生血管的形成。视网膜血管包含血管发生(vasculogenesis)和血管新生(neovascularization/angiogenesis)两种类型。血管发生是一个血管从无到有的过程,是由骨髓造血干细胞(HSCs)分化而来的内皮祖细胞(EPCs),组成相互连接的原始血管网,分化为内皮细胞并增殖而形成的血管。血管新生则多被视为一复杂的病理过程,例如视网膜受到缺血、缺氧等刺激后,原有血管基底膜降解,内皮细胞增殖、趋化迁移、形成新生血管管腔。大量研究表明,视网膜在受到缺血、缺氧损伤后能释放出诸多细胞因子如缺氧诱导因子-1(HIF-1)和血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)、血小板源性生长因子(PDGF)、胰岛素样生长因子(IGFs)、转化生长因子书(TGF-β)等。这些细胞生长因子促使血管内皮细胞有丝分裂和形成新生血管的管腔。SDF-1是一种趋化因子CXC亚家族成员,具有趋化活性,与其受体CXCR4构成了SDF-1/CXCR4轴。SDF-1与CXCR4结合作用启动下游信号通路,介导炎症反应、引导造血干细胞迁移及归巢、以及HIV感染等重要作用。SDF-1/CXCR4还参与了肿瘤、眼部组织新生血管等形成[1,2]。现SDF-1可以诱导内皮祖细胞到达视网膜缺血区域,参与视网膜新生血管发生[3]AMD3100(商品名为Plerixafor(?))是一种人工合成的CXCR4特异性拮抗剂,能与CXCR4有效结合,阻断SDF-1/CXCR4间信号转导,使CXCR4不发生激动,已证明可抑制肿瘤新生血管和抗HIV病毒的效果。然而,CXCR4在缺氧诱导的视网膜新生血管形成过程中是否起到作用,其作用机制如何等我们都还不得而知。为此,本课题建立OIR小鼠模型和培养并建立HUVECs缺氧细胞模型；使用CXCR4的特异性抑制剂AMD3100阻断SDF-1/CXCR4的信号转导,观察抑制CXCR4信号通路对OIR模型中视网膜新生血管形成的影响,并观察其对HUVEC的增殖和表达新生血管相关细胞因子的影响；探讨CXCR4在氧诱导视网膜新生血管生成中的作用及其可能的机制。第一部分氧诱导视网膜病变小鼠模型和缺氧人脐静脉内皮细胞模型的建立目的：采用Smith[4]介绍方法建立OIR小鼠模型；I型胶酶原消化法培养HUVECs并进行传代、鉴定,建立细胞缺氧模型。方法：取7日龄(P7)C57BL/6小鼠20只,随机分成正常组(正常氧环境下饲养至P17)和OIR组(P7开始每天置于氧浓度在75%±2%饲养至P12,然后取出置于常氧环境下饲养至P12)。两组小鼠在P17麻醉后摘取眼球、固定,每组5只小鼠(10只眼)行视网膜切片HE染色,计算视网膜切片中突破内界膜的血管内皮细胞核数；余5只小鼠(10只眼)采用FITC-Dextran荧光造影视网膜铺片,定性分析视网膜新生血管情况。无菌条件下取健康胎儿脐带一段,用D-Hank's液洗净脐静脉管腔,灌注I型胶原酶37℃消化20min后收集细胞,加入ECM内皮细胞培养基静置培养、传代,镜下观察细胞形态；vWF免疫荧光染色鉴定,绘制细胞生长曲线(MTT法检测)并计算不同浓度CoCl2缺氧造模后对细胞增殖的影响。结果：OIR模型的50张眼球切片中49张切片有突破内界膜的血管内皮细胞核,许多成簇出现,有些形成毛细血管腔,腔内可见红细胞,视网膜新生血管阳性率98%。正常组小鼠平均每张切片突破视网膜内界膜的血管内皮细胞核数为(0.20±0.447)个,OIR模型组平均每张切片的血管内皮细胞核数为(31.60±2.07)个,差异具有统计意义(t=-33.09,P0.01)荧光铺片显示视网膜大血管不规则扩张,走行迂曲,后极部大片无灌注区,周边部可见新生血管丛,伴荧光渗漏。原代培养的细胞为单层生长,初为圆形,后逐渐伸展呈长梭形,胞浆丰富,胞核清晰可见,细胞间有相互连接。vWF抗体免疫荧光染色鉴定示培养细胞的胞浆内含有大量黄绿色荧光颗粒,胞核内无荧光染色。所获内皮细胞消化、传代稳定,第2-3d生长速度加快,进入对数生长期；CoCl2缺氧造模(0μg/L,20μg/L,50μg/L,100μg/L,200μg/L和400μg/L)对细胞生存抑制随剂量增加而加重。结论：依照Smith方法可成功建立小鼠OIR模型,视网膜新生血管显著；酶原消化脐静脉法可稳定获得HUVECs来源,给予适量CoCl2模拟化学缺氧。第二部分CXCR4受体在氧诱导视网膜病变发病中的作用研究目的：探讨CXCR4在氧诱导视网膜病变发生发展的作用及可能的机制。方法：100只P7C57BL/6小鼠,随机分为5组(n=20)：正常组,OIR模型组,AMD3100大、小剂量玻璃体腔注射组、模型对照组。除正常组外均建立OIR模型,后三组小鼠P12离开氧箱当天给予一次性玻璃体腔注射：AMD3100大剂量(100μg/μL)1μL和AMD3100小剂量(50μg/μL)1μL、模型对照组(无菌BSS)1μL。所有小鼠P17戊巴比妥麻醉,每组随机抽取5只用于视网膜石蜡切片HE染色和5只用于视网膜FITC-Dextran荧光造影铺片,计算突破视网膜内界膜内皮细胞数目并观察视网膜血管形态。抽取每组4只小鼠提取视网膜总RNA, RT-PCR半定量分析HIF-1α和VEGF mRNA表达；剩余每组6只依照western bolt方法检测视网膜HIF-1α和VEGF蛋白水平。结果：正常组(NOR)平均每张切片突破视网膜内界膜的内皮细胞核数为(0.01+/-0.12)个；缺氧小鼠模型(OIR)平均每张切片突破内界膜的内皮细胞核数为(30.33±1.51)个,与正常组有显著差异(t=-49.35,P0.01)；AMD3100大剂量(100μg/μL)治疗组平均每张切片突破内界膜的内皮细胞核数为(13.50±1.87)个,与正常组差异显著(t=-17.66,P0.01)；与OIR模型组有显著差异(t=17.17,P0.01)；AMD3100小剂量(50μg/μL)治疗组平均每张切片突破内界膜的内皮细胞核数为(20.83±1.72)个,与正常组有显著差异,(t=-29.68,P0.01),且与OIR模型组有显著差异,(t=-10.17,P0.01);模型对照组平均每张切片突破内界膜的内皮细胞核数为(27.33±2.95)个,与正常组有显著差异(t=-22.83,P0.01),与OIR模型组无显著差异(t=2.10,P0.05)。视网膜FITC荧光铺片显示正常组小鼠视网膜结构正常,未见显著新生血管；OIR模型组视网膜后极部存在大量无灌注区,大血管迂曲扩张、分支减少,网膜中周部有较多新生血管；AMD3100玻璃体腔大剂量注射组视网膜后极部无灌注区较OIR模型组的减少,周边部未见明显新生血管形成；AMD3100小剂量组的无灌注区较OIR模型组减少,新生血管不显；模型对照组后极部存在大量无灌注区,大血管迂曲扩张、分支减少,新生血管有荧光渗漏出现。玻璃体腔注射100μg/μL和50μg/μLAMD3100阻断CXCR4受体能抑制缺氧诱导的HIF-1α和VEGF mRNA和蛋白水平的上调,且100μg/mL AMD3100的抑制作用更强,与模型组表达的差异具有统计学意义。结论：玻璃体腔内注射AMD3100可以抑制OIR模型小鼠视网膜新生血管的形成,且可以使缺氧诱导的视网膜组织中的HIF-1α和VEGF mRNA和蛋白表达量下降,提示SDF-1/CXCR4信号在缺氧诱导的视网膜新生血管生成中的作用与视网膜组织中HIF-1α和VEGF蛋白表达的变化有关。第三部分CXCR4对缺氧条件下HUVECs的作用及其机制目的：探讨SDF-1/CXCR4信号对缺氧条件下培养的]HUVECs性状的改变及其可能机制。方法：MTT法检测AMD3100(50μmol/L,100μmol/L,200μmol/L,400μmol/L,800μmol/L), AMD3100(50μmol/L或100μmol/L)联合CoCl2(50μg/L或100μg/L)对HUVECs生存增殖率的影响；RT-PCR方法检测100μg/L CoCl2处理的HUVECs第0h,1h,2h,4h,6h,12h,24h HIF-1α,VEGF mRNA表达水平；western bolt方法检测100μg/L CoCl2处理HUVECs第0h,6h,12h和24h时HIF-1α和VEGF蛋白表达；AMD3100(50nM,100nM)预处理HUVECs1h后给予100μg/L CoCl2,在第24h检测HIF-1α和VEGF表达。结果：CXCR4受体信号对细胞的增殖存在影响,过量/AMD3100(800μmol/L)使HUVECs生存率降至正常细胞的80%。添加/AMD3100(50μmol/L或100μmol/L)加重了CoCl2(50μg/L或100μg/L)对细胞生成的抑制。在100μg/L CoCl2处理的0h-24h时间范围内,HIF-1α,VEGF mRNA在第6h-12h出现表达峰值,在24h表达水平下调；HIF-la, VEGF蛋白表达量在0h-24h间不断增加；AMD3100(50nM和100nM)预处理1h后100μg/L CoCl2诱导上调的HIF-1α和VEGF mRNA和蛋白出现下降,且100nM显示出更强的抑制作用,与缺氧模型组水平有显著差异性(P0.01)。结论：阻断细胞的CXCR4信号通路对缺氧HUVECs新生血管性改变具有抑制作用。全文总结阻断SDF-1/CXCR4信号通路有效抑制了模型小鼠缺氧诱导视网膜新生血管的发生,缺氧下HUVECs细胞的增殖以及HIF-lα、VEGF上调。缺氧环境使机体内皮细胞CXCR4受体敏感性上调,其下游信号活化,促新生血管细胞因子释放增加,影响视网膜微环境的变化,导致视网膜新生血管。
[Abstract]:Retinal neovascular diseases are one of the major causes of blindness in the world. Such diseases include proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO), and age-related macular degeneration (AMD). Their common characteristics are that retinal tissue is histopathologically caused by relative hypoxia. The formation of the neovascularization of the retina.
The retinal vessels include two types of angiogenesis (vasculogenesis) and angiogenesis (neovascularization/angiogenesis). Angiogenesis is a process of vascular endothelial progenitor cells (EPCs) differentiated from bone marrow hematopoietic stem cells (HSCs), forming a interconnected primitive vascular network, differentiating into endothelial cells and proliferating. Angiogenesis is often seen as a complex pathological process, such as the retinal degeneration, endothelial cell proliferation, chemotaxis migration and formation of neovascular lumen after ischemia, hypoxia and other stimuli. A large number of studies show that the retina can release many cytokines such as hypoxia after injury to blood and hypoxia. Inducible factors -1 (HIF-1) and vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), insulin like growth factor (IGFs), transforming growth factor book (TGF- beta), etc.. These cell growth factors induce mitosis and formation of neovascularization in vascular endothelial cells The lumen.SDF-1 is a member of the chemokine CXC subfamily, which has chemotaxis, and its receptor CXCR4 constitutes a SDF-1/CXCR4 axis.SDF-1 and CXCR4 binding to start downstream signaling pathways, mediate inflammatory reactions, guide the migration and homing of hematopoietic stem cells, and HIV infection and other important roles.SDF-1/CXCR4 also participates in the tumor, and the eye tissue is new. [1,2]. SDF-1 can induce endothelial progenitor cells to reach the retinal ischemia area, and participate in retinal neovascularization [3]AMD3100 (commodity name Plerixafor (?)) is a synthetic CXCR4 specific antagonist. It can effectively combine with CXCR4, block the signal transduction between SDF-1/CXCR4 and make CXCR4 not exciting. It has been proved to be possible. Inhibition of neovascularization and anti HIV virus effect.
However, it is not known whether CXCR4 plays a role in the process of hypoxia induced retinal neovascularization, and the mechanism of its action is not known. For this reason, the OIR mouse model was established and the HUVECs hypoxic cell model was developed and the CXCR4 specific inhibitor AMD3100 was used to block the signal transduction of SDF-1/CXCR4 and to observe the inhibition. The effect of CXCR4 signaling pathway on the formation of retinal neovascularization in the OIR model and its effect on the proliferation of HUVEC and the expression of cytokines related to neovascularization, and the role of CXCR4 in the formation of retinal neovascularization in oxygen induced by oxygen and its possible mechanism are also discussed.
The first part is the establishment of oxygen induced retinopathy model and hypoxia human umbilical vein endothelial cell model.
Objective: to establish a OIR mouse model by Smith[4] method, and to cultivate HUVECs with I type enzyme digestion method and carry out the passage, identify and establish the cell hypoxia model. Methods: 20 mice of 7 day old (P7) C57BL/6 mice were randomly divided into normal group (normal oxygen environment, P17) and OIR group (P7 starting at 75% + 2% to P12, at the beginning of P7. Then, the two groups of mice were taken to P12). The two groups of mice were taken eyeball after P17 anesthesia, fixed and 5 mice in each group (10 eyes) were stained with retina slices. The number of vascular endothelial nuclei breaking through the inner boundary membrane in the retinal section was calculated. The remaining 5 mice (10 eyes) were analyzed by FITC-Dextran fluorescein retina paving, qualitative analysis. The condition of retinal neovascularization. Take a healthy fetal umbilical cord under aseptic condition, wash the umbilical vein lumen with D-Hank's liquid, collect I collagenase at 37 C to digest 20min, collect cells, add ECM endothelial cell culture medium, observe cell morphology under microscope, vWF immunofluorescence staining identification, draw cell growth curve (MTT method test). Results: the effects of different concentrations of CoCl2 on cell proliferation were calculated. Results: 49 slices of 50 slice of the eyeball of the OIR model had the vascular endothelial nuclei breaking through the inner boundary membrane, many clusters appeared, some formed capillary cavity, red cells were visible in the cavity, and the positive rate of retinal neovascularization in the normal group of 98%. mice was each cut. The number of vascular endothelial nuclei of the inner boundary membrane of the retina was (0.20 + 0.447). The average number of vascular endothelial nuclei of each slice in OIR model group was (31.60 + 2.07). The difference was statistically significant (t=-33.09, P0.01), which showed irregular expansion of the large retinal vessels in the retina. The primary cultured cells were single layer, and the primary cells were round, and the cells were long shuttle, rich in cytoplasm and clear in the nucleus. There were interconnected.VWF antibody immunofluorescence staining to identify a large number of yellow green fluorescent particles in the cytoplasm of the cultured cells and no fluorescent staining in the nucleus. The cells were digested, the passages were stable, the growth rate of 2-3D accelerated and entered the logarithmic growth period; the CoCl2 hypoxia model (0 mu g/L, 20 mu g/L, 50 mu g/L, 100 mu g/L, 200 mu g/L and 400 mu g/L) aggravated the cell survival inhibition with the dose increase. Conclusion: the mouse OIR model can be established by the Smith method, the retinal neovascularization is significant; the zymogen digestion navel static Pulse method can stabilize the source of HUVECs and give appropriate CoCl2 to simulate chemical hypoxia.
The second part is the role of CXCR4 receptor in the pathogenesis of oxygen induced retinopathy.
Objective: To explore the role and possible mechanism of CXCR4 in the development of oxygen induced retinopathy. Methods: 100 P7C57BL/6 mice were randomly divided into 5 groups (n=20): normal group, OIR model group, AMD3100 large, low dose intravitreal injection group, model control group. The OIR model was established except the normal group, and the later three groups of mice P12 left oxygen box was given the same day Injection of one time vitreous cavity: AMD3100 large dose (100 mu g/ L) 1 L and AMD3100 small dose (50 mu g/ mu L) 1 mu L, model control group (aseptic BSS) 1 u L. all mice P17 pentobarbital anaesthesia, each group randomly selected 5 for retinal paraffin section HE staining and 5 use of retina fluorescein radiography, to calculate the breakthrough retina The number of endothelial cells in the membrane and the retinal vascular morphology were observed. 4 mice in each group were extracted to extract the total retinal RNA, RT-PCR semi quantitative analysis of HIF-1 A and VEGF mRNA expression. The remaining 6 rats were detected the retinal HIF-1 alpha and VEGF protein levels according to the Western bolt method. Results: the average section of the normal group (NOR) broke through the inner boundary membrane of the retina. The number of endothelial nuclei was (0.01+/-0.12), and the average number of endothelial nuclei in the average segment of the hypoxic mouse model (OIR) was (30.33 + 1.51), which was significantly different from the normal group (t=-49.35, P0.01). The number of endothelial nuclei of each section of the AMD3100 large dose (100 mu g/ mu L) group was (13.50 + 1.87), and the number of endothelial nuclei was (13.50 + 1.87) per slice. The difference of the normal group was significant (t=-17.66, P0.01), and there was significant difference with the OIR model group (t=17.17, P0.01), and the number of endothelial nuclei of the average AMD3100 small dose (50 mu g/ mu L) in the treatment group was (20.83 + 1.72), which was significantly different from the normal group, (t= -29.68, P0.01), and there was a significant difference from the OIR model group. In the control group, the average number of endothelial nuclei of the inner boundary membrane was (27.33 + 2.95), which was significantly different from the normal group (t=-22.83, P0.01). There was no significant difference from the OIR model group (t=2.10, P0.05). Retina FITC fluorescent display showed that the normal group of retina was normal in the normal group and no significant neovascularization was found in the normal group, and the posterior retinal pole in the OIR model group was in the posterior pole of the retina. There was a large number of non perfusion areas in the Department. The large blood vessels were tortuous and dilated, the branches were reduced. There were more neovascularization in the periphery of the omentum. The non perfusion area of the posterior retinal pole in the large dose injection group of AMD3100 vitreous cavity was less than that in the OIR model group, and there was no obvious neovascularization in the peripheral part, and the non perfusion area of the small dose group of AMD3100 was less than that of the OIR model group. There was a large number of non perfusion areas in the posterior polar part of the model control group. The large vessels were circuitous and dilated, the branches decreased, and the new blood vessels had fluorescence leakage. The blocking of CXCR4 receptors by intravitreal injection of 100 g/ mu L and 50 u g/ mu LAMD3100 could inhibit the up-regulation of HIF-1 alpha and VEGF mRNA and protein levels induced by hypoxia, and the inhibition of 100 u g/mL AMD3100 The difference between the model group and the model group has statistical significance. Conclusion: Intravitreal injection of AMD3100 can inhibit the formation of retinal neovascularization in OIR model mice, and can reduce the expression of HIF-1 alpha and VEGF mRNA and protein in the retinal tissue induced by hypoxia, and suggest the new SDF-1/CXCR4 signal in the hypoxia induced retina. The role of angiogenesis is related to the expression of HIF-1 alpha and VEGF protein in retina.
The third part is the effect and mechanism of CXCR4 on HUVECs under anoxia condition.
Objective: To investigate the changes and possible mechanisms of the]HUVECs character of SDF-1/CXCR4 signals under hypoxia conditions. Methods: MTT method was used to detect the effect of AMD3100 (50 mu mol/L, 100 mu mol/L, 200 mu mol/L, 400 mu mol/L, 800 micron), AMD3100 (50 mu mol/L or 100 micron) on the survival and proliferation rate. HUVECs 0h, 1H, 2h, 4h, 6h, 12h, 24h HIF-1 alpha were measured by 100 g/L CoCl2. The 4 receptor signal has an effect on the proliferation of cells. Excessive /AMD3100 (800 mu mol/L) reduces the HUVECs survival rate to the normal cell 80%. adding /AMD3100 (50 mu mol/L or 100 mu mol/L) to increase the inhibition of CoCl2 (50 u g/L or 100 mu g/L) to cell formation. Peak value decreased at 24h expression level; HIF-la, VEGF protein expression increased continuously in 0h-24h; AMD3100 (50nM and 100nM) pretreated 1H 100 u g/L CoCl2 induced HIF-1 alpha and VEGF proteins and proteins decreased, and showed a stronger inhibitory effect, and there was a significant difference with the level of the hypoxia model group. Conclusion: blocking. The CXCR4 signaling pathway can inhibit the neovascularization of hypoxic HUVECs.
A summary of the full text
Blocking the SDF-1/CXCR4 signal pathway effectively inhibits the occurrence of retinal neovascularization in the model mice, the proliferation of HUVECs cells under hypoxia and the up regulation of HIF-l alpha and VEGF. The hypoxia environment makes the CXCR4 receptor sensitivity up up, the downstream signal activation, the increase of the release of the neovascularization factor, and the influence of the retinal microring. Changes in the territory of the retina lead to the retinal neovascularization.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

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