【作者】 赵炜；

【导师】 惠延年； 王雨生；

【作者基本信息】 第四军医大学 ， 眼科学， 2008， 博士

【摘要】 研究背景年龄相关性黄斑变性(age-related macular degeneration, AMD)是50岁以上人群引起视力损失的首要原因。渗出型AMD是以脉络膜新生血管形成(choroidal neovascularization, CNV)为特点,由脉络膜新生的血管在视网膜色素上皮层(retinal pigment epithelium, RPE)和神经视网膜下生长的一种严重影响视力的疾病。由于出血或者瘢痕形成,CNV会引起急性或亚急性的视力丧失。尽管在形态学上已经对AMD继发的CNV进行了广泛地研究,但其具体病理机制仍然不甚明朗。研究表明,黄斑部玻璃膜疣的堆积以及变性的色素上皮改变,会导致AMD黄斑部有限的血液供给和光感受器细胞之间高氧需求之间平衡的破坏,导致RPE层相对的缺氧,上调表达一些生长因子,如血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)等,从而促进了CNV的形成。因此认为,缺氧在CNV的发生发展中具有重要的作用,而VEGF则是一个主要病理因子。已有证据提示AMD患者其RPE细胞VEGF水平升高。而缺氧条件下培养的RPE细胞,其VEGF的mRNA和蛋白表达都显著升高。近来,Ephrin/Eph受体家族——和VEGF/VEGFR同为受体酪氨酸激酶(receptor tyrosine kinase, RTK)家族,近来在血管发生中的作用也逐渐受到人们的重视。研究发现,EphrinB2/EphB4也参与了CNV的发生和发展。目前对于AMD的药物治疗,大多数是基于VEGF或者VEGF受体,如玻璃体内注射pegaptanib(Macugen)、ranibizumab (Lucentis),bevacizumab (avastin)以及全身应用的VEGF-trap。但这些抗VEGF的治疗方法都存在一个潜在的弊端,即它们的目标仅仅是多个重要血管发生因子中的一个。而其他的治疗,例如经瞳孔温热疗法(transpupillary thermotherapy, TTT)、光动力疗法(photodynamic therapy, PDT)或者玻璃体内注射曲安耐德(triamcinolone acetonide, TA),虽然可以通过部分地下调多种血管发生因子的表达而发挥抑制CNV的作用,但又存在较显著的副作用。近来,缺氧诱导因子1(hypoxia-inducible factor 1, HIF-1)逐渐被大家关注,被认为是治疗CNV的一个新靶点。HIF-1是一个异源二聚体,由组成表达的HIF-1β亚体以及氧调节的HIF-1α亚体构成。在缺氧条件下,稳定表达的HIF-1α能够诱导和血管发生、红细胞生成、糖代谢有关的多种基因表达,如VEGF、促红细胞生成素(erythropoietin,EPO)以及糖酵解酶。同时也有研究证明,在小鼠表皮皮瓣的缺氧模型上,缺氧不但可以上调HIF-1α和VEGF表达,而且也能够上调表皮上A和B类的Eph受体和Ephrin配体。在Hep3B细胞和PC-3细胞,通过针对HIF-1α的siRNA可以抑制缺氧诱导的Eph受体和Ephrin配体表达,证明HIF-1α也是缺氧条件下Ephrin/Eph受体表达的关键性因子。因此我们设想,基因缄默RPE细胞的HIF-1α表达有可能抑制血管发生。本研究中,我们利用RNA干扰(RNAi)技术,在RPE细胞上基因缄默HIF-1α,并和牛脉络膜微血管内皮细胞(choroidal microvascular endothelial cells, CEC)细胞共培养,观察其对CEC增生以及管腔形成的影响,探讨HIF-1α对血管发生的抑制作用。目的1.利用免疫磁珠法原代分离培养牛CEC,并进行鉴定;2.观察CEC上VEGFR2和EphrinB2/EphB4的表达以及缺氧对RPE细胞VEGF/VEGFR2以及EphrinB2/EphB4表达的影响;3.构建和筛选针对HIF-1α基因的shRNA载体,转染RPE细胞后检测缺氧条件下VEGF和EphrinB2/EphB4的表达;4.建立RPE/CEC共培养模型,并观察缺氧条件下RPE细胞对CEC增生、移行以及管腔形成的影响。方法1.显微分离牛脉络膜微血管,采用1 g·L-1胶原酶一步法消化,并采用免疫磁珠分选脉络膜微血管内皮细胞,使用内皮细胞培养基进行原代培养。利用光学和电子显微镜进行形态学观察,利用Von Willebrand因子免疫荧光染色及Dil-Ac-LDL吞噬实验进行细胞学鉴定,利用管腔形成实验观察内皮细胞形成管腔能力。2.在细胞培养液中加入200μM CoCl2建立人RPE细胞缺氧模型,培养0、1、3、6、12和24 h,利用免疫荧光染色观察RPE细胞及CEC上VEGFR2及EphrinB2/EphB4的表达,实时定量PCR以及Western blot观察缺氧条件下RPE细胞VEGF/VEGFR2以及EphrinB2/EphB4的表达,利用Western blot观察CEC上VEGFR2及EphrinB2/EphB4的表达,ELISA法观察RPE细胞上清中VEGF的含量;3.构建3条针对人HIF-1α基因的shRNA,利用实时定量PCR筛选抑制效率最高的shRNA。基因敲除RPE细胞HIF-1α后,利用实时定量PCR和Western blot观察缺氧条件下其对RPE细胞HIF-1α和VEGF以及EphrinB2/EphB4表达的影响,ELISA观察上清中VEGF的表达;4.利用Transwell共培养小室建立RPE细胞和CEC的增生、移行以及管腔形成模型,观察基因敲除HIF-1α后RPE细胞对CEC增生、移行以及管腔形成的影响。结果1.采用本研究改良的方法可以获得纯度高达95%的脉络膜微血管内皮细胞,外观呈细长、纺锤样,融合后呈典型铺路石样外观。电子显微镜观察可见胞浆内靠近核膜外的Weibel-Palade小体(棒状小体),免疫荧光显示Von Willebrand因子表达阳性,Dil-Ac-LDL吞噬试验阳性;在凝胶中可以形成明显管腔结构;2. (1)RPE细胞上除了存在VEGFR2表达,发现RPE细胞上存在EphB4和EphrinB2的表达(;2)化学缺氧能够以时间依赖性诱导HIF-1α在mRNA及蛋白水平的表达,在mRNA水平,HIF-1α在0 h时也有表达,3 h表达最高,随后逐渐降低,12 h时恢复到基础水平;其蛋白0 h时是没有表达的,3 h时表达到达峰值;其相应的转录产物VEGF同样也显示出呈时间依赖性表达;其mRNA水平也是在3 h时表达到达最大值,而蛋白水平则在12 h表达最高;缺氧对VEGFR2的表达也有诱导作用,其mRNA表达在3 h时达到峰值,而蛋白表达在6 h时表达最高,随后逐渐降低;(3)化学缺氧可以刺激RPE细胞表达能够上调EphB4受体转录和翻译水平的表达,在转录水平,缺氧3h时,EphB4表达增加了1.3倍;其蛋白表达也相应在6h时表达达到峰值,而随后降低。在缺氧条件下,相应EphrinB2受体在转录以及翻译水平下调表达;(2)CEC上存在VEGFR2、EphrinB2配体和EphB4受体的表达;3. (1)shRNA1、2、3的抑制效率分别为77%,62%和54%,选择shRNA1(pshHIF-1α)对RPE细胞进行基因干扰实验;(2)和pDNA转染对照组相比,3 h时相应HIF-1α及VEGF mRNA及蛋白水平表达均下降,其中HIF-1αmRNA和转染对照组相比下降了72.6%,而VEGF mRNA下降了75.6%;(3)pshHIF-1α转染组细胞上清中VEGF表达量最低,和pDNA对照转染组相比降低了58%(P<0.01);(4)3 h时,RPE细胞上EphB4受体的表达在经过RNAi处理后,和转染对照组相比其mRNA表达降低了73.2%,而对EphrinB2配体的mRNA表达影响不大。相应在蛋白水平上,可见RNAi处理后,在缺氧3h时EphB4受体的表达上调受到了抑制,表达降低;4.在共培养系统内,RPE细胞HIF-1α被基因敲除后, CEC的增生、移行以及管腔形成显著受到了抑制,和对照组转染组相比,第3、4及5天CEC的增生率分别下降了40.2%, 36.6%和36.8%;在5 h时移行下降了49.6%,48 h时管腔形成降低了40.4%。结论1.成功分离并纯化了牛CEC,可在短期内快速简便地获得足够数量的内皮细胞;2. RPE细胞存在VEGF/VEGFR2及EphrinB2/EphrB4系统的表达,缺氧条件下和HIF-1α的表达相关,提示EphirnB2/EphB4在CNV的发生中具有一定的作用;同时验证了CEC上VEGFR2的表达,是RPE细胞分泌VEGF的作用点;3.筛选出效率最高的pshHIF-1α,证实其可有效地抑制HIF-1α表达,从而降低VEGF表达,并提示EphB4的表达和HIF-1α相关;4.在体外对RPE细胞HIF-1α进行基因干扰可抑制血管发生,为治疗CNV性疾病提供了可能的治疗方向,其结果国内外未见报道。更多还原

【Abstract】 BackgroundAge-related macular degeneration (AMD) is the leading cause of visual loss in persons more than 50 years of age. The exudative form of the disease is, characterized by choroidal neovascularization (CNV), in which newly formed vessels from the underlying choroid grow beneath the retinal pigment epithelium (RPE) and the neuroretina. CNV may cause acute or subacute blindness because of bleeding or scar formation. Although the morphology of angiogenesis in CNV secondary to AMD has been described in detail, the pathogenesis is still poorly understood.The deposition of drusen, the basal liner deposit, and the degenerative pigmentary changes have all been shown to be closely associated with the increased risk of CNV. The relative hypoxia caused by the disturbed balance between the limited blood supply in the macula and the high oxygen demand by the photoreceptors may also contribute to the formation of CNV by up-regulating the expression of growth factors, such as VEGF. Therefore, hypoxia was considered to play an important role and VEGF is a major pathogenic factor in the development of CNV. Several lines of evidence implicate increased levels of VEGF in retinal pigment epithelium (RPE) from patients with AMD. In cultured RPE cells, the VEGF expression was significantly up-regulated at both the mRNA and protein levels after exposure to hypoxia. As one of RTKs families, Ephrin/Eph receptor family was paid more attentions in angiogenesis by researchers. It was said that this family took part in the development of CNV, too.At present, several novel therapies for CNV have emerged based on antagonism of VEGF or the VEGF receptor, such as intravitreal administration of pegaptanib (Macugen) or ranibizumab (Lucentis), bevacizumab (avastin) and a systemically delivered, modified VEGF receptor (VEGF-Trap). However, a potential drawback of these therapies is that only one of multiple potentially important angiogenic factors is targeted. Other therapies, such as thermal laser, photodynamic therapy (PDT) or intravitreal triamcinolone, are believed to exert their effects partly through the down-regulation of multiple angiogenic factors. These therapeutic methods, however, all have significant side effects.Recently, hypoxia-inducible factor 1 (HIF-1), which is a transcription factor that regulates genes such as VEGF and erythropoietin (EPO) involved in the response to hypoxia, has been proposed as a novel therapeutic target. HIF-1 is a heterodimer composed of HIF-1αand HIF-1βsubunits. HIF-1βis constitutively expressed, while HIF-1αis induced by hypoxia. HIF-1 transactivates a repertoire of genes, including VEGF, which mediate angiogenesis, cell proliferation/survival, and glucose/iron metabolism to hypoxia. At the same time, a study indicated that hypoxia up-regulates not only HIF-1αand VEGF expression, but also Ephs and ephrins of both A and B subclasses in the mouse skin. In addition, in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins was abrogated by siRNA-mediated down-regulation of HIF-1α. These novel findings show that HIF-1αis also a key factor for expression of Ephrin/Eph.Therefore, we hypothesized that silencing the HIF-1αin RPE cells could inhibited angiogenesis. In the present study, we used coculture systems to investigate the effect of RPE cells knocked down HIF-1αon the proliferation, migration and tube formation of CECs.Purpose1. To establish a rapid and convenient method for purification and primary culture of bovine CEC in vitro and provide an in vitro model for CNV diseases.2. To observe expressions of VEGFR2, EphrinB2/EphB4 in CECs and observe VEGF/VEGFR2, EphrinB2/EphB4 expressions in RPE cells under hypoxia.3. To construct and select the most efficient short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1α) shRNA vector and observe the expressions of corresponding ligands and receptors after RPE cells were transfected by pshHIF-1α.4. To observe the effects of RPE cells transfected by pshHIF-1αon the proliferation, migration and tube formation of CECs, respectively.Methods1. Bovine choroidal microvascular vessels were isolated from choroidal tissues by microdissection and digested with 1 g?L-1 collagenase. CECs were purified selectively by immunomagnetic beads and were cultured with endothelial culture medium. The characteristics of CECs were observed by light and electron microscopy and were identified by immunofluorescence of Von Willebrand factor and Dil-Ac-LDL phagocytosis. CECs grew as tubes in a collagen gel.2. RPE cells were cultured under chemical hypoxia. Immunofluorescence was used to test the EphrinB2/EphB4 expression on RPE cells. Real-time RT-PCR and Western blot were used to observe the effect of hypoxia on expression of EphrinB2/EphB4 on RPE cells. ELISA measured the secreted VEGF in supernatant of RPE cells.3. Three RNAi vectors targeting HIF-1αwere constructed and Real-time PCR selected the most efficient shRNA. Real-time RT-PCR and Western blot were used to observe the effect of hypoxia on expression of HIF-1α, VEGF and EphrinB2/EphB4 in RPE cells transfected by pshHIF-1α. ELISA was used to measure the secreted VEGF in supernatant of the RPE cells.4. Three kinds of coculture models were used to observe the effects of RPE cells transfected by pshHIF-1αon the proliferation, migration and tube formation of CECs, respectively.Results1. 95% of the cultured cells were CECs affirmed by immunofluorescence of Von Willebrand factor and Dil-Ac-LDL phagocytosis. The cultured CECs were slender and spindle-shaped. After confluence, the cells had cobblestone appearance. Weibel-Palade body was found near nuclear membrane by electron microscopy. The staining of Von Willebrand factor was positive and the cells can phagocytose the Dil-Ac-LDL.2. Human RPE cells could express EphrinB2/EphB4 and chemical hypoxia could up-regulate EphB4 receptor expression at mRNA and protein levels. At mRNA level, EphB4 receptor expression increased 2.3 fold and its protein expression reach peak value at 6 h. Correspondingly, the EphrinB2 ligand expression was down-regulated under hypoxia. Bovine CECs could express EphrinB2/EphB4.3. RPE cells were cultured under hypoxia and RNAi technique was used to knock down the HIF-1αgene by pshHIF-1αin RPE cells. mRNA and protein expression of HIF-1αand VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. ELISA measured the secreted VEGF in supernatant of the three experiment groups.4. Proliferation, migration and tube formation of CECs were significantly inhibited by the knocked down RPE cells compared with the control in the coculture system. Proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on day 3, 4 and 5, respectively. Migration reduced by 49.6% at 5 h and tube formation decreased by 40.4% at 48 h.Conclusions1. Bovine CECs can be successfully isolated and purified with this modified method by which enough CEC can be got easily for CNV study;2. Human RPE cells could express EphrinB2/EphB4, which were related with HIF-1αexpression. It suggested the role of EphrinB2/EphB4 in CNV.3. The most efficient pshHIF-1αwas selected and was used for transfection, which could inhibit expression of HIF-1αand VEGF. Expression of EphB4 receptor was related with HIF-1α.4. RNAi of HIF-1αin RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1α. Similar research has not been reported yet at home and abroad.更多还原