【作者】 窦国睿；

【导师】 王雨生； 韩骅；

【作者基本信息】 第四军医大学 ， 眼科学， 2011， 博士

【摘要】 【研究背景】新生血管生成是眼内新生血管疾病,如年龄相关性黄斑变性(age related macular degeneration,AMD)、增生性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)、视网膜静脉阻塞(retinal vein occlusion,RVO)及早产儿视网膜病变(retinopathy of prematurity,ROP)等眼病共有的病理改变和重要临床表现,其相伴随的渗出、出血和纤维化等一系列病理改变可严重破坏眼的结构和功能,最终导致患者不同程度的视力障碍。在其发生机制中,多种诱发因素参与对眼底视网膜和脉络膜的破坏,组织发生过多的反应性修复,在组织学中呈现视网膜前、视网膜内或视网膜下新生血管生长的病理改变。在多种类型的眼内新生血管中,脉络膜新生血管( choroidal neovascularization, CNV)是来自脉络膜毛细血管网血管内皮细胞,通过Bruch膜的裂口迁移和增生,生长于Bruch膜与视网膜色素上皮(retinalpigment epithelium,RPE)之间、或神经视网膜与RPE之间、或位于RPE与脉络膜之间的新生血管丛。包括变性、遗传、炎症、肿瘤和外伤等多种因素均可造成RPE-Bruch膜-脉络膜毛细血管复合体的损害,因而常伴发有CNV的生成。其中,继发于AMD的CNV较多见于黄斑部,严重地损害中心视力,已成为老年人致盲的主要原因之一。CNV的发生机制尚不十分明确,但众多研究已证实多种细胞、生长因子和细胞外基质(extracellular matrix,ECM)共同构成CNV局部的微环境,并通过细胞、因子及信号通路之间的交互作用对CNV的生成发挥重要调控作用。因而,深入的机制研究为CNV的临床治疗开拓了新的方向。目前靶向血管内皮生长因子(vascular endothelial growth factor, VEGF)的制剂已经通过美国FDA认证,而临床研究发现其治疗可有效地抑制新生血管生成,且有利于改善视力。然而,VEGF同时具有维持血管正常通透和机能及神经保护的功能,长期反复使用抗VEGF药物,其安全性和疗效的稳定性有待进一步观察研究。更重要的是,在CNV的微环境中并不仅仅只有VEGF参与CNV的生成,单一抗VEGF治疗可能并不足以达到理想治疗效果,因而,深入研究CNV微环境中的其他重要因子,并探讨其治疗潜能,可为靶向治疗CNV提供新的突破口。Notch信号通路在无脊椎动物和脊椎动物进化过程中高度保守而广泛存在于各组织。最近研究表明,Notch信号通路中多种配体和受体在血管系统中表达,对胚胎及成年个体的生理性和病理性血管新生发挥关键的调控作用。值得一提的是,在胚胎期因基因单倍剂量缺失造成血管异构而最终致死的研究中,目前发现并报道的只有两个基因,即Notch信号通路中的配体分子Delta like 4 (Dll4)和VEGF,这提示Notch信号在对血管的调控中可能和VEGF具有着同等重要的地位。但不同于VEGF对血管生长进行的远位调节,目前多认为Notch信号通过膜配体和膜受体的接触发生细胞间的信号传递,从而调控血管内皮细胞、周细胞及其他相关细胞的活化和功能。最近两年来,Notch信号在眼底视网膜血管发育及某些眼内新生血管疾病中的重要作用被发现并重视,但Notch信号通路是否参与CNV的生成,且其是否具有治疗CNV的潜能等诸多问题有待研究。【目的】观察Notch信号通路对眼部及全身血管稳态的维持作用,研究Notch信号通路对CNV生长及相关重要细胞的调控作用,从新的角度探讨Notch信号通路通过骨髓来源细胞(bone marrow derived cells,BMC)参与CNV生长的调控作用,进一步探寻Notch信号通路对CNV的潜在治疗可能,为CNV治疗策略的拓展提供新的理论依据。【方法】1、建立诱导性基因敲除小鼠。交配繁育RBP-Jflox/WT、Mx-Cre和ROSA小鼠,得到Mx-Cre-RBP-Jflox/WT小鼠、Mx-Cre-RBP-Jflox/flox小鼠和Mx-Cre-RBP-Jflox-ROSA小鼠,小鼠于出生后4周开始以500μg总剂量的poly:I-C诱导(以腹腔注射的方式分5次间隔进行);2、采用组织病理学等技术,观察RBP-J/Notch信号敲除后,小鼠眼部及全身其他脏器的血管变化以及皮下接种Matrigel胶内的血管生长情况;3、利用532nm激光建立小鼠CNV模型,通过荧光素眼底血管造影(fundus fluorescein angiography,FFA)和组织病理学技术,观察RBP-J/Notch信号敲除小鼠上CNV及接受RBP-J/Notch信号敲除小鼠骨髓移植的嵌合体小鼠上CNV的严重程度;4、利用体外原代培养RBP-J/Notch信号敲除小鼠主动脉血管内皮细胞、肝血窦内皮细胞和骨髓来源内皮祖细胞(endothelial progenitor cell,EPC),研究Notch信号缺失对相关细胞生物学行为的影响。5、分别利用?-secretase抑制剂GSI体外阻断和外源性给予Dll1融合蛋白体外激活培养人RPE细胞和胚胎来源弥猴视网膜/脉络膜内皮细胞系RF/6A中的Notch信号通路,观察Notch信号对CNV中重要细胞的生物学行为的调控。6、对出生后乳鼠于天龄4d(P4)皮下注射血管靶向型Dll1-RGD融合蛋白,并对激光诱导CNV的野生型小鼠于激光诱导后1天行尾静脉注射Dll1-RGD融合蛋白,观察外源性血管靶向增强Notch信号的激活对视网膜血管发育和CNV生长的作用。【结果】1、通过繁育RBP-Jflox/WT和Mx-Cre小鼠,获得了Mx-Cre-RBP-Jflox/WT和Mx-Cre-RBP-Jflox/flox小鼠;通过繁育ROSA和Mx-Cre-RBP-Jflox小鼠交配,获得了Mx-Cre-RBP-Jflox-ROSA小鼠。Mx-Cre-RBP-Jflox/WT小鼠、Mx-Cre-RBP-Jflox/flox小鼠和Mx-Cre-RBP-Jflox-ROSA小鼠经poly:I-C诱导后,对Mx-Cre-RBP-Jflox-ROSA小鼠耳部皮肤及视网膜血管行X-gal染色可见明显蓝染,表明小鼠血管开始高效表达Cre重组酶。Cre重组酶表达的同时可通过DNA重组敲除RBP-J/Notch信号,最终得到RBP-J (-/-)小鼠,即基因敲除小鼠(KO)和RBP-J (+/-)小鼠,即为基因部分存在的对照小鼠(CON)。通过对KO小鼠和CON小鼠眼部及全身血管的观察,本研究结果显示RBP-J/Notch信号缺失时,小鼠眼部(角膜、虹膜和视网膜)及全身其他器官(肝脏和肺脏)的血管稳态的破坏,出现大量自发性新生血管。在小鼠皮下接种Matrigel胶5天后,KO小鼠皮下接种的胶内长入大量新生血管并诱发出血。体外培养的KO小鼠的主动脉血管内皮细胞增殖能力增强,体外KO小鼠的肝血窦内皮细胞管腔形成中血管萌枝增多。分子水平检测结果显示KO小鼠内皮细胞上的VEGFR2表达上调,VEGFR1表达下调,且细胞周期调控蛋白p21WAF1/CIP1转录水平表达下调。2、对RBP-J基因敲除小鼠(KO)及对照小鼠(CON)上建立激光诱导的CNV,结果显示Notch信号缺失时, FFA显示新生血管的渗漏程度加重,且生成率增加,而组织病理学统计CNV厚度和面积均大于对照组小鼠。在研究Notch信号对于CNV相关细胞的作用中,使用?-secretase抑制剂GSI阻断细胞内的Notch信号,细胞免疫组化和划痕实验中可见到体外培养RPE细胞的增殖和迁移减弱;而RF/6A增殖能力增强,但管腔形成能力减弱。3、通过骨髓移植成功建立接受KO及CON小鼠骨髓的嵌合体小鼠,并建立激光诱导的CNV,可见到接受KO小鼠骨髓移植的嵌合体小鼠CNV生成加重。KO及CON小鼠骨髓经Dio染色后移植到野生型小鼠体内,对获得的嵌合体小鼠建立的激光诱导CNV,可见CNV区域有绿色荧光细胞的浸润,且细胞上表达基质衍生因子受体(C-X-C chemokine receptor type 4,CXCR4)。接受KO小鼠骨髓移植的小鼠CNV区域的绿色荧光细胞数量减少。流式细胞技术检测提示外周血EPC数量没有明显变化,而循环血中成熟的内皮细胞数量明显增加。通过原代培养小鼠的EPC,Transwell小室实验可见KO小鼠的EPC对基质衍生因子(stroma derived factor-1,SDF-1)的趋化能力减弱, EPC上CXCR4的表达下调。4、P5及P15天新生小鼠的视网膜血管上,Notch信号的激活分别集中在顶端细胞和血管分叉处,而在野生型小鼠的激光诱导CNV区域,可见活化的Notch信号胞内段(Notch intracellular domain,NICD)的表达上调。接受Dll1-RGD融合蛋白注射的乳鼠视网膜血管发育明显较对照鼠迟缓,表现为血管分枝减少,血管内顶端细胞数量减少。在接受Dll1-RGD注射的激光诱导CNV小鼠模型中,脉络膜铺片显示其CNV的最大面积明显小于对照鼠。体外实验中,可见到RF/6A侵袭能力减弱,而管腔形成能力增强,而RPE细胞的迁移能力减弱。【结论】本研究证实,Notch信号在CNV的生成中发挥重要作用,Notch信号的缺失可加重CNV的生成,而外源性激活Notch信号对CNV的生成具有抑制作用。在成年个体中,Notch信号参与全身及眼部血管稳态的维持,信号的缺失导致血管内皮细胞的增殖能力加强及顶端细胞数量增加,这可能与Notch信号在转录水平调控VEGFR2、VEGFR1以及细胞周期调控蛋白p21 WAF1/CIP1有关。BMC同样参与了CNV的生成,其中EPC被认为可在CNV生长中发挥重要作用。KO小鼠外周血循环中的EPC数量未发生明显变化,而循环中内皮细胞的数量显著增加;Notch信号缺失时,EPC对SDF-1的趋化作用反应性降低,其原因可能是由于Notch信号缺失导致EPC上SDF-1受体CXCR4的表达下调。另外,Notch信号同时参与对RPE细胞的生物学行为的调控,可能通过影响RPE的增殖和迁移参与到CNV的消退期中。但是需要注意的是,由于我们使用的是Notch信号全身性基因敲除小鼠,信号缺失后是否除内皮细胞、BMCs和RPE细胞外其他细胞也因信号缺失参与CNV,这需要进一步研究。基于RBP-J/Notch信号的缺失导致CNV的加重,我们利用一种新型的融合蛋白Dll1-RGD以血管靶向性激活血管内皮细胞中的Notch信号通路,可见到Dll1-RGD全身性给药抑制小鼠视网膜血管发育及CNV的生成,这提示增强Notch通路的激活可能起到治疗作用,但相关分子机制和长期注射是否具有毒性作用仍需在今后工作中加以研究。更多还原

【Abstract】 BACKGROUNDOcular angiogenesis is the common pathological change and clinical feature in several ocular diseases such as age related macular degeneration (AMD), proliferateive diabetic retinopathy (PDR), retinal vein occlusion (RVO) and retinopathy of premature (ROP), with the accompany by a series of incidents including exudates, bleeding and fibrosis, which cause serious disruption in the structure and function of the eye and lead to the various extents of vision loss. It is a group of factors that commit to the disruption of retina and choroid, and correspondently, excessive reactivity of the tissue repair represents the histopathological changes of epiretinal and subretinal neovascularization.Among various ocular angiogenesis, choroidal neovascularization (CNV) is the growth of new blood vessels between Bruch’s membrane and retinal epithelium (RPE), or between neural retina and RPE, or bwtween RPE and choroidal capillary plexus, by the migration and proliferation of choroidal capillary endothelial cells from the rupture of Bruch’s membrane. Multifactors including degeneration, heredity, inflammation, tumor and trauma could cause the damage in RPE-Bruch’s membrane-choriodal capillary complex, which consequently induce the complication of CNV. AMD associated CNV is the major reason of blindness among the elderly. It is often observed in the macular area, thus causing the major loss of the central vision. To date, the specific mechanism of CNV is still unclear. However, several types of cells, growth factors and extracellular matrix (ECM) and signaling pathways constitute a microenvironment for CNV development, where the interaction of cells, growth factors and signaling pathways plays a significant role in the regulation the growth of CNV. Therefore, in-depth studies on the mechanisms of CNV may offer new candidates for its therapeutic application. The current concept of anti-VEGF has been approved by FDA, and verified its effectiveness in the inhibition of new vessels growth and the improvement of vision. However, due to the physiological function of VEGF including maintenance of vascular permeability and neural protection, extensive observation are required on the safety and curative stability with long term and repeated usage of anti-VEGF drugs. More notably, in the microenvironment of CNV, VEGF is absolutely not the single growth factor which is involved in the complicated angiogenic process, thus studies on other key factors in this condition and their therapeutic potentials could be the new target in the CNV clinical treatment.Notch signaling pathway is highly conserved pathway that widely expresses in all tissues in the development of vertebrate and invertebrate. Recent studies have identified the expressions of several ligands and receptors of Notch signaling pathway in vascular system, which play a critical role in the regulation of physiological and pathological angiogenesis during the embryonic and postnatal stage. It is notable that in the studies that loss of even single allele of gene (haploinsufficiency) can cause embryonic lethality due to the vascular abnormity, only two genes have been confirmed with this effect: VEGF and Notch signaling ligand Delta like 4 (Dll4), indicating the equivalent importance of Notch signaling with VEGF in the development of the vascular system. But, the regulatory modulation of Notch signaling is distinct from VEGF. Notch signaling pathway functions in neighboring cells via the binding of Notch ligands and receptors, whereas VEGF is secreted and exert a remote effect to the distant cells, in the control of the activation and function of EC, mural cells and other types of cell. In recent years, it has been unveiled and emphasized that Notch signaling pathway is vital in the development of retinal vasculature and some ocular angiogenesis, but involvement of Notch signaling in the development of CNV and its correspondent therapeutic potential remain in investigation.AIMSTo investigate the contribution of Notch signaling in the maintenance of ocular and systemic vascular homeostasis and to elucidate the regulatory role of Notch signaling in the development of CNV as well as the effects of Notch signaling on the key cells in ocular angiogenesis and its underlying mechanisms, with a further study is to investigate the possibility of its therapeutic potential in the treatment of CNVMETHODS1. RBP-Jflox/WT, Mx-Cre and ROSA mice were mated to get Mx-Cre-RBP-Jflox/WT, RBP-Jflox/flox and Mx-Cre-RBP-Jflox-ROSA mice. Four weeks after birth, the above mice were underwent injection with 500μg polyI-C (five times in total) to get the final RBP-J knockout (KO) mice and control (CON) mice. The activity of Cre in endothelium was observed by X-gal staining;2. By using the histopathological techniques, the vascular changes in the eyes and other organs after the knockout of RBP-J/Notch signaling were observed, as well as the vessel growth in the subcutaneous Matrigel gel;3. RBP-J KO chimeric mice and CON chimeric mice were developed by transplanting bone marrow cells from RBP-J KO and CON mice to adult C57BL/6J mice and comfirmed by flow cytometry one month later. CNV were induced in experimental mice (RBP-J KO and CON mice, RBP-J KO chimeric and CON chimeric mice) by laser injury of Bruch’s membrane and the severity of CNV was observed by and fundus fluorescence angiogram (FFA) and histopathological techniques.4. The expressions and regulatory functions of Notch signaling molecules were studied by using mice retinal flatmount, in vitro cultures of the aortic endothelial cells, liver sinusoidal endothelial cells and bone marrow derived endothelial progenitor cells from KO mice, primary culture of human RPE cells and rhesus monkey choroid–retinal endothelial cell line RF/6A.5. The effect of activation of Notch signaling was observed by ?-secretase inhibitor GSI and exogenous recombinant Dll1 treatment on the RF/6A and RPE cells.6. To observe the effect of activation of Notch signaling in vivo, neonatal C57BL/6J mice and C57BL/6J mice with laser induced CNV were respectively treated with subcutaneous and intravenous injection of murine Dll1-RGD. Retnial flatmount and RPE-choroidal-scleral complex flatmount were used in this part.RESULT1. Mx-Cre-RBP-Jflox/WT and Mx-Cre-RBP-Jflox/flox mice were obtained by mating RBP-Jflox/WT and Mx-Cre;Mx-Cre-RBP-Jflox-ROSA was obtained by mating Mx-Cre-RBP-Jflox and Mx-Cre-ROSA. Mx-Cre-RBP-Jflox/WT, Mx-Cre-RBP-Jflox/flox and Mx-Cre-RBP-Jflox-ROSA mice were undergone 500μg poly:I-C injection (5 times in total). The skin and retinal vasculatures of Mx-Cre-RBP-Jflox-ROSA exhibited blue staining by X-gal, indicating that the Cre expression in mice vescular system. The expression of Cre simutanously knocked out RBP-J gene by DNA recombination, which finaly resulted in production of RBP-J (-/-) mice named Knockout mice (KO) and RBP-J (+/-) named control mice (CON). Through the observation on the ocular and systemic vascular system of KO mice and CON mice, we found the lack of RBP-J/Notch signaling caused spontaneously angiogenesis in the iris, cronea, retina, liver and lung. Five days after Matrigel subcutanous implantation in KO and CON mice, a large mount of neovessels grew into the gel of KO mice with extensive bleeding. In addition, in the vitro studies, we found the proliferation of arotic endothelial cells was greatly enhanced and the sprouting of liver sinus endothelial cells was increased. The expression of VEGFR2 was upregulated, while the expression of VEGFR1 was downregulated. And also, cell cycle regulatory protein p21WAF1/CIP1 was downregulated in the transcriptional level.2. When Notch signaling was inhibited, laser induced CNV on KO mice was more severe than that in CON mice. FFA exhibited that the leakage and the incidence of CNV were enchanced in KO mice. The area and thickness of CNV in KO mice were increased statistically than those in KO mice. In vitro studies, inhibition of Notch signaling ?-secretase inhibitor GSI reduced the proliferation and migration of RPE; While GSI promoted the proliferation but weakened the tube formation of RF/6A.3. Chimeric mice were obtained by successfully transplanted bone marrow from KO and CON mice to wild type C57BL/6J mice. Laser induced CNV were set up in the chemiric mice one month after bone marrow transplantation. The severity of CNV in the KO chimeric mice was enhanced. Bone marrow derived cells from KO and CON mice were stained by Dio dye and transplanted in to the wild type C57BL/6J mice. Cells with green fluorenscein were observed in the laser induced CNV area, and expression of CXCR4 was detected on these cells. There was no significant change in the number of EPC in the peripheral blood; however, the mature endothelial cells were increased in the circulation. In the study of the vitro culture of EPC, EPC from KO mice exhibited less responsiveness to the chemotaxis to SDF-1, which was possibly caused by the downregulation of SDF-1 receptor CXCR4.4. On the retinal vasculatures of P5 and P15 neonatal C57BL/6J mice, the activation of Notch signaling was respectively expressed in the tip cells and the vascular branches. And Notch signaling was also activated in the CNV area of C57BL/6J mice. After subcutaneously injection of Dll1-RGD to the neonatal mice, the tip cells and branches were decreased compared with the control group. After intravenously injection of Dll1-RGD in the C57BL/6J mice with laser induced CNV model, the area of CNV was markedly reduced in the comparesion with the control group. In vitro study, after the Dll1 treatment, the invasion capacity of RF/6A was weakened, but its tube formation was enhanced. For RPE cells, Dll1 treatment caused the reduction of cell migration.CONCLUSIONIn this study, we identified that Notch signaling plays an important regulatory role in CNV. Lack of Notch signaling exacerbates the CNV formation, wherease enhancement of Notch signaling alleviates the CNV formation. In adult, Notch signaling contributes to the maintainance of ocular and systemic vascular system. When Notch signaling is inhibited, the EC’s proliferation and specification to tip cells could be enhanced, which is possibly due to the transcriptional regulation of VEGFR2, VEGFR1 and p21 by Notch signaling.BMCs also participate in the CNV formation, of which EPC is considered as a dominating type in the growth of CNV. The comparision on the number of EPCs and ECs suggests that Notch may modulate the differentiation of EPC to EC. In addition, the reduced responsiveness of EPC to SDF-1 caused by lack of Notch signaling is possibly due to the downregulated expression of CXCR4. Notch signaling could also regulate the biological function of RPE cell including cell proliferation and migration, which finally commit to the progression of CNV. However, it is notated that, as the mice model we used is systemic gene knockout, whether Notch signaling regulates other types of cells in the CNV formation is still in investigation.Based on the observation that lack of RBP-J/Notch signaling exacerbates the CNV formation, we adopted a new fusion protein called Dll1-RGD to specifically activate the Notch signaling in vascular endothelial cells. Systemic treatment of Dll1-RGD could inhibite the formation of mice retinal vasculatrures and the growth of CNV, indicating that activation of Notch signaling might have a therapeutic potential for the treatment of CNV. But more work including the toxic effect and its associated molecular mechanism are needed in future studies.更多还原