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
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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,P<0.01)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,P<0.01)；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,P<0.01)；与OIR模型组有显著差异(t=17.17,P<0.01)；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,P<0.01),且与OIR模型组有显著差异,(t=-10.17,P<0.01);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,P<0.01),与OIR模型组无显著差异(t=2.10,P>0.05)。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(P<0.01)。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。
The retinal neovascularization is a main cause of blindnesses worldwide. Retinal neovascular diseases include proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO) and age related macular degeneration (AMD). They feature in pathological retinal neovascularization induced by relative hypoxia.
Formation of retinal vessels involves in vasculogenesis and angiogenesis/neovascularization. Vasculogenesis refers to primary vascular network formed by endothelial progenitor cells, which generated from bone marrow hematopoietic stem cells, and differentiated into mature endothelial cells to form vessels. This is how blood vessels are developed originally. Angiogenesis are always considered as pathological processes. As in retina, when it subjected to hypoxia insults, vascular basement membrane degradated, endothelial cells sprout out from previous existed blood vessels, proliferate and form vascular lumens. In vivo and in vitro studies showed, retinas suffered from hypoxia or ischemia insults can release numerous cellular factors such as HIF-1alpha, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nsulin-like growth factor (IGF-1), transformation growth factor books (TGF-beta), etc. These factors effect on vascular endothelial cells, accelerate mitosis of endothelial cell, which lead to angiogenesis.
SDF-1belongs to the CXC chemokine sub-family, possessing cellular chemotactic unctions. SDF-1and receptor CXCR4constitute the SDF-1/CXCR4axis. When SDF-1and CXCR4combine together the downstream signals were activated, it could mediate nflammation, HIV infection and homing of hematopoietic stem cells. The SDF-1/CXCR4axis also takes important roles in tumor metastasis and (?)eovascularization, including ocular neovascularization. It was found that SDF-1can nduce endothelial progenitor cells migrate into retina ischemia region and participate in (?)eovascularization. AMD3100(commercial name Plerixafor(?)) is a specific antagonists of CXCR4. AMD3100can effectively blocking the combining of SDF-1to CXCR4and silence down cellular signal transductions of receptor CXCR4. It has been proved, by blocking SDF-1/CXCR4signal, AMD3100could inhibit tumor angiogenesis and HIV infection.
However, whether CXCR4plays role in formations of hypoxia induced retinal neovascularization is unknown yet. Therefore, in this study, mice models of oxygen-induced retinopathy and hypoxic cell model of human umbilical vein endothelial cells (HUVECs) will be established. Blocking the SDF-1/CXCR4signaling with its specific antagonist AMD3100, the formations of RNVs in OIR model, proliferations and expressions of neovascular factors in HUVECs are evaluated. Discuss the role of CXCR4in the pathogenesis and progression of hypoxia induced retinal neovascularization and the possible mechanism.
Part One
Establishment of Mice Model of Oxygen-Induced Retinopathy and Cultivation of Human Umbilical Vein Endothelial Cells
Purpose:Establish oxygen induction retinopathy (OIR) mice model; harvest human umbilical vein endothelial cells (HUVECs) by collagenase I digestion, identify and build hypoxic cell model.
Methods:Obtain20C57BL/6mice at7th postnatal days (P7), dividing into normal group (raised in normoxia until P17) and OIR model group (place a glass container with oxygen concentration at75℃up/below2℃from P7to P12, then were reared in normoxia until P17) randomly. At P17, all mice puppies were anesthetized subjected to retinal FITC-Dextran angiography or made into retinal section with HE staining. Quantify retinal NVs by counting endothelial cells (ECs) nucleus breakthrough the internal limiting membrane (ILM) of retina. Qualify retinal neovascular vessels under fluorescence microscope. Bring a segment of fresh umbilical cord (20cm-25cm) to the aseptic condition; wash the umbilical vein with D-hank's solution. Filled umbilical vein with20ml collagenase Ⅰ, digested for20min in37℃, collected and cultured cells with endothelial cell medium (ECM), observe, pass the cells and indentify them with vWF immuno-fluorescence staining. Draw cell growing curve and evaluate CoCl2(0μg/L,20μg/L,50μg/L,100μg/L,200μg/L and400μg/L) impact on HUVECs as hypoxia model by MTT assay.
Results:OIR mice possess massive ECs nucleus protruding into the ILM than normal mice number, some of them formed neovascular clusters (49sections in50sections are positive in RNV, the rate is98%). The number of neovascular ECs protruded into the ILM is (0.20±0.447) in normal group and (31.60±2.07) in OIR model, the difference is statistically significant. Retinal FITC-Dextran angiography showed irregular structures of retinal vessels expanded, with no perfusion area at the posterior pole, neovascular plexus in periphery of retina with fluorescence leakages, proving OIR modeling method to be successful. Primary harvested cells grew at the bottom in single layer. Cells were rounded, after passsges they stretched in spindle-look, rich in cytoplasm, with clear oval nucleus. Immunofluorescence stains showed numerous positive particles located in cytoplasm, while the nucleus area was negative. Cells cultivations were showed to be stable. Growing speed were exaggerated in the first2to3days after one passage; the mimic hypoxia agent CoCl2could suppress HUVEC survives as dosage increased.
Conclusion:OIR mice model can be set up as Smith described; harvest human umbilical vein endothelial cells (HUVECs) by collagenase Ⅰ digestion, and establish chemical hypoxia cell model by administrating appropriate dose of CoCl2.
Part Two
CXCR4's Role in the Pathogenesis of Oxgen-Induced Retinopathy Objective:Explore CXCR4's roles in the pathogenesis and progress of oxygen-induced retinopathy and its possible mechanism.
Methods:1hundred P7C57BL/6mice were divided into5groups randomly (eight mice each group):normal group, OIR models group, AMD3100large-dosage and small-dosage group, model control group. Except for normal group mice, the rests were put in the hyperoxic incubator for5days (P7to P12) and returned to normal environment till P17to establish OIR models. OIR model group received no other treatment, AMD3100large-dosage group received1μL of100μg/μL AMD3100injected into vitreous cavity once at P12when taken out from oxygen incubator; AMD3100small dosage mice received1μl of50μg/μL AMD3100injected into vitreous cavity once at P12, and model control mice received1μL of BSS at the same time. Observe HE stained-paraffin section of retina (five mice each group) and retinal FITC-Dextran angiography (five mice each group), quantify the number of endothelial cells nucleus protruding into ILM. Four mice and six mice each group are used for detection of retinal HIF-la and VEGF mRNA and protein by RT-PCR and western bolt asssy, respectively.
Results:AMD3100(large doses and small doses) reduced neovascular ECs breakthrough ILM by retinal sections. Normal group average number of ECs nucleus is for each section (0.01+/-0.12), to oxygen model group average for each section (30.33±1.51), there is a significant difference between statistical significance (t=-49.35, P＜0.01). Theaverage number of endothelial nuclei breakthrough ILM in large dose AMD3100treated group was is (13.50±1.87), compared to oxygen model there is a significant difference between statistical significance (t=17.17, P＜0.01). FITC-Dextran angiography showed retinal structures are normal in large doses of AMD3100treatment (did not see the significant new blood vessels, restrain effect of new blood vessels is dose dependent); expression level of HIF-la protein and VEGF protein were significantly lower than that in OIR model mice in a dose-dependent manner.
Conclusion:Vitreous cavity injection of CXCR4antagonist AMD3100reduced RNVs in OIR mice, and suppressed the upregulated mRNA and protein levels of HIF-1alpha and VEGF, suggesting the SDF-1/CXCR4signaling's role in the pathogenosis of hypoxia induced retinal neovascularization, this may largely related to the expression of HIF-la and VEGF.
Part Three
CXCR4's Role in Hypoxic Change of Human Umbilical Vein Endothelial Cells and its Mechanism
Objective:Discuss the effect of CXCR4signaling on hypoxic changes of cultured HUVECs and the possible mechanism.
Methods:Detect proliferations rate of HUVEC under different concentrations of AMD3100(50,100,200,400,800μmol/L), and AMD3100(50or100μmol/L) combined CoCl2(50or100μ g/L) by MTT assay. Expressions of HIF-1α and VEGF mRNA were quantified at0h,1h,2h,4h,6h,12h,24h after100μg/L CoCl2treatment in HUVECs by RT-PCR; protein levels of HIF-1alpha and VEGF in HUVECs at0h,6h,12h and24h after100μg/L CoCl2addition were detected by western bolting. AMD3100(50nM and100nM) were added to cell medium1hour ahead of100μg/L CoCl2treatment,24h later HIF-1alpha and VEGF mRNA and protein levels were evaluated.
Results:AMD3100inhibited HUVECs proliferation as its doses increased; the proliferation rate of HUVECs under800μmol/L AMD3100is80%of that in normal group. After100μg/L CoCl2were added, mRNA expression of HIF-1α and VEGF reached to the peak in6h-12h, and then dropped at24h; protein expression of HIF-1α and VEGF increased continuously from0h to24h;1h of AMD3100pretreatment suppressed100μg/L CoCl2-induced increasing of HIF-1α and VEGF both in mRNA and protein level.
Conclusion:Blocking the CXCR4signaling in hypoxic HUVECs possess an obvious anti-neovascular effect.
Summary
Inhibition of SDF-1/CXCR4signal pathway reduced hypoxia-induced retinal neovascularization in mice model; downregulated cell proliferation rates, expressions of HIF-1α and VEGF in hypoxic HUVECs. Hypoxia irritates sensitities of CXCR4receptors, the downstream signals are activated, and cytokines related to NVs are synthesized and released into retina microenviroment, inducing RNVs in the end.
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,P<0.01)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,P<0.01)；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,P<0.01)；与OIR模型组有显著差异(t=17.17,P<0.01)；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,P<0.01),且与OIR模型组有显著差异,(t=-10.17,P<0.01);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,P<0.01),与OIR模型组无显著差异(t=2.10,P>0.05)。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(P<0.01)。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。
The retinal neovascularization is a main cause of blindnesses worldwide. Retinal neovascular diseases include proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO) and age related macular degeneration (AMD). They feature in pathological retinal neovascularization induced by relative hypoxia.
Formation of retinal vessels involves in vasculogenesis and angiogenesis/neovascularization. Vasculogenesis refers to primary vascular network formed by endothelial progenitor cells, which generated from bone marrow hematopoietic stem cells, and differentiated into mature endothelial cells to form vessels. This is how blood vessels are developed originally. Angiogenesis are always considered as pathological processes. As in retina, when it subjected to hypoxia insults, vascular basement membrane degradated, endothelial cells sprout out from previous existed blood vessels, proliferate and form vascular lumens. In vivo and in vitro studies showed, retinas suffered from hypoxia or ischemia insults can release numerous cellular factors such as HIF-1alpha, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nsulin-like growth factor (IGF-1), transformation growth factor books (TGF-beta), etc. These factors effect on vascular endothelial cells, accelerate mitosis of endothelial cell, which lead to angiogenesis.
SDF-1belongs to the CXC chemokine sub-family, possessing cellular chemotactic unctions. SDF-1and receptor CXCR4constitute the SDF-1/CXCR4axis. When SDF-1and CXCR4combine together the downstream signals were activated, it could mediate nflammation, HIV infection and homing of hematopoietic stem cells. The SDF-1/CXCR4axis also takes important roles in tumor metastasis and (?)eovascularization, including ocular neovascularization. It was found that SDF-1can nduce endothelial progenitor cells migrate into retina ischemia region and participate in (?)eovascularization. AMD3100(commercial name Plerixafor(?)) is a specific antagonists of CXCR4. AMD3100can effectively blocking the combining of SDF-1to CXCR4and silence down cellular signal transductions of receptor CXCR4. It has been proved, by blocking SDF-1/CXCR4signal, AMD3100could inhibit tumor angiogenesis and HIV infection.
However, whether CXCR4plays role in formations of hypoxia induced retinal neovascularization is unknown yet. Therefore, in this study, mice models of oxygen-induced retinopathy and hypoxic cell model of human umbilical vein endothelial cells (HUVECs) will be established. Blocking the SDF-1/CXCR4signaling with its specific antagonist AMD3100, the formations of RNVs in OIR model, proliferations and expressions of neovascular factors in HUVECs are evaluated. Discuss the role of CXCR4in the pathogenesis and progression of hypoxia induced retinal neovascularization and the possible mechanism.
Part One
Establishment of Mice Model of Oxygen-Induced Retinopathy and Cultivation of Human Umbilical Vein Endothelial Cells
Purpose:Establish oxygen induction retinopathy (OIR) mice model; harvest human umbilical vein endothelial cells (HUVECs) by collagenase I digestion, identify and build hypoxic cell model.
Methods:Obtain20C57BL/6mice at7th postnatal days (P7), dividing into normal group (raised in normoxia until P17) and OIR model group (place a glass container with oxygen concentration at75℃up/below2℃from P7to P12, then were reared in normoxia until P17) randomly. At P17, all mice puppies were anesthetized subjected to retinal FITC-Dextran angiography or made into retinal section with HE staining. Quantify retinal NVs by counting endothelial cells (ECs) nucleus breakthrough the internal limiting membrane (ILM) of retina. Qualify retinal neovascular vessels under fluorescence microscope. Bring a segment of fresh umbilical cord (20cm-25cm) to the aseptic condition; wash the umbilical vein with D-hank's solution. Filled umbilical vein with20ml collagenase Ⅰ, digested for20min in37℃, collected and cultured cells with endothelial cell medium (ECM), observe, pass the cells and indentify them with vWF immuno-fluorescence staining. Draw cell growing curve and evaluate CoCl2(0μg/L,20μg/L,50μg/L,100μg/L,200μg/L and400μg/L) impact on HUVECs as hypoxia model by MTT assay.
Results:OIR mice possess massive ECs nucleus protruding into the ILM than normal mice number, some of them formed neovascular clusters (49sections in50sections are positive in RNV, the rate is98%). The number of neovascular ECs protruded into the ILM is (0.20±0.447) in normal group and (31.60±2.07) in OIR model, the difference is statistically significant. Retinal FITC-Dextran angiography showed irregular structures of retinal vessels expanded, with no perfusion area at the posterior pole, neovascular plexus in periphery of retina with fluorescence leakages, proving OIR modeling method to be successful. Primary harvested cells grew at the bottom in single layer. Cells were rounded, after passsges they stretched in spindle-look, rich in cytoplasm, with clear oval nucleus. Immunofluorescence stains showed numerous positive particles located in cytoplasm, while the nucleus area was negative. Cells cultivations were showed to be stable. Growing speed were exaggerated in the first2to3days after one passage; the mimic hypoxia agent CoCl2could suppress HUVEC survives as dosage increased.
Conclusion:OIR mice model can be set up as Smith described; harvest human umbilical vein endothelial cells (HUVECs) by collagenase Ⅰ digestion, and establish chemical hypoxia cell model by administrating appropriate dose of CoCl2.
Part Two
CXCR4's Role in the Pathogenesis of Oxgen-Induced Retinopathy Objective:Explore CXCR4's roles in the pathogenesis and progress of oxygen-induced retinopathy and its possible mechanism.
Methods:1hundred P7C57BL/6mice were divided into5groups randomly (eight mice each group):normal group, OIR models group, AMD3100large-dosage and small-dosage group, model control group. Except for normal group mice, the rests were put in the hyperoxic incubator for5days (P7to P12) and returned to normal environment till P17to establish OIR models. OIR model group received no other treatment, AMD3100large-dosage group received1μL of100μg/μL AMD3100injected into vitreous cavity once at P12when taken out from oxygen incubator; AMD3100small dosage mice received1μl of50μg/μL AMD3100injected into vitreous cavity once at P12, and model control mice received1μL of BSS at the same time. Observe HE stained-paraffin section of retina (five mice each group) and retinal FITC-Dextran angiography (five mice each group), quantify the number of endothelial cells nucleus protruding into ILM. Four mice and six mice each group are used for detection of retinal HIF-la and VEGF mRNA and protein by RT-PCR and western bolt asssy, respectively.
Results:AMD3100(large doses and small doses) reduced neovascular ECs breakthrough ILM by retinal sections. Normal group average number of ECs nucleus is for each section (0.01+/-0.12), to oxygen model group average for each section (30.33±1.51), there is a significant difference between statistical significance (t=-49.35, P＜0.01). Theaverage number of endothelial nuclei breakthrough ILM in large dose AMD3100treated group was is (13.50±1.87), compared to oxygen model there is a significant difference between statistical significance (t=17.17, P＜0.01). FITC-Dextran angiography showed retinal structures are normal in large doses of AMD3100treatment (did not see the significant new blood vessels, restrain effect of new blood vessels is dose dependent); expression level of HIF-la protein and VEGF protein were significantly lower than that in OIR model mice in a dose-dependent manner.
Conclusion:Vitreous cavity injection of CXCR4antagonist AMD3100reduced RNVs in OIR mice, and suppressed the upregulated mRNA and protein levels of HIF-1alpha and VEGF, suggesting the SDF-1/CXCR4signaling's role in the pathogenosis of hypoxia induced retinal neovascularization, this may largely related to the expression of HIF-la and VEGF.
Part Three
CXCR4's Role in Hypoxic Change of Human Umbilical Vein Endothelial Cells and its Mechanism
Objective:Discuss the effect of CXCR4signaling on hypoxic changes of cultured HUVECs and the possible mechanism.
Methods:Detect proliferations rate of HUVEC under different concentrations of AMD3100(50,100,200,400,800μmol/L), and AMD3100(50or100μmol/L) combined CoCl2(50or100μ g/L) by MTT assay. Expressions of HIF-1α and VEGF mRNA were quantified at0h,1h,2h,4h,6h,12h,24h after100μg/L CoCl2treatment in HUVECs by RT-PCR; protein levels of HIF-1alpha and VEGF in HUVECs at0h,6h,12h and24h after100μg/L CoCl2addition were detected by western bolting. AMD3100(50nM and100nM) were added to cell medium1hour ahead of100μg/L CoCl2treatment,24h later HIF-1alpha and VEGF mRNA and protein levels were evaluated.
Results:AMD3100inhibited HUVECs proliferation as its doses increased; the proliferation rate of HUVECs under800μmol/L AMD3100is80%of that in normal group. After100μg/L CoCl2were added, mRNA expression of HIF-1α and VEGF reached to the peak in6h-12h, and then dropped at24h; protein expression of HIF-1α and VEGF increased continuously from0h to24h;1h of AMD3100pretreatment suppressed100μg/L CoCl2-induced increasing of HIF-1α and VEGF both in mRNA and protein level.
Conclusion:Blocking the CXCR4signaling in hypoxic HUVECs possess an obvious anti-neovascular effect.
Summary
Inhibition of SDF-1/CXCR4signal pathway reduced hypoxia-induced retinal neovascularization in mice model; downregulated cell proliferation rates, expressions of HIF-1α and VEGF in hypoxic HUVECs. Hypoxia irritates sensitities of CXCR4receptors, the downstream signals are activated, and cytokines related to NVs are synthesized and released into retina microenviroment, inducing RNVs in the end.
引文
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