【作者】 朱洁；

【导师】 王雨生；

【作者基本信息】 第四军医大学 ， 眼科学， 2008， 博士

【摘要】 研究背景脉络膜新生血管(choroidal neovascularization,CNV)是年龄相关性黄斑变性(age related macular degeneration,AMD)导致视力丧失的最主要的原因之一,其发病机制至今尚不十分清楚。视网膜色素上皮(retinal pigment epithelium,RPE)细胞可以通过调节自身多种基因的表达,对缺氧和代谢变化等周围环境的刺激产生快速的应答。在CNV发生的启始阶段,RPE细胞分泌细胞因子和生长因子,促进CNV的生成。随后,增生的脉络膜血管穿破Bruch膜生长至RPE和/或视网膜下,形成CNV。CNV的发生机制十分复杂,受到生长因子和细胞黏附信号等多种因素的影响。参与调控CNV生成的RPE细胞接受多种来源的信号,如可溶性细胞因子信号和细胞外基质(extracellular matrix,ECM)信号等。黏着斑激酶(focal adhesion kinase,FAK),一种非受体型酪氨酸激酶,在细胞骨架和整合素/细胞因子受体信号的链接中发挥重要的作用,并参与调控细胞增生、存活、移行和分化等细胞进程。ECM和可溶性细胞因子均可活化FAK。进来的研究还表明,FAK参与了新生血管的应答,并且在病理性视网膜新生血管发生中发挥重要的调控作用。然而,FAK在CNV发生中的作用尚未见相关报道。目的和内容探讨FAK在CNV发生中的作用,进一步阐明CNV的发生机制。在建立激光诱导的大鼠CNV模型基础上,观察FAK的表达与CNV生成的相关性;体外观察多种已知与CNV生成相关的危险因素的作用下,培养的人RPE细胞中FAK的表达;运用小干扰RNA(small interfering RNA,siRNA)特异性抑制RPE细胞FAK的表达,观察FAK信号通路在CNV发生中的作用。方法⑴激光击穿Bruch膜,建立挪威(Brown Norway,BN)大鼠CNV模型。分别于激光光凝后1、3、7和14天,免疫荧光和Western blot方法观察大鼠CNV中FAK的表达;⑵体外原代培养人RPE细胞,分别暴露于缺氧、H2O2氧化衰老、ECM成分和吞噬光感受器外节膜盘(out segment of photoreceptors,POS)等刺激因素。Western blot方法观察RPE细胞中FAK的表达;⑶运用siRNA技术特异性抑制RPE细胞FAK的表达,RT-PCR、Western blot和ELISA等方法观察缺氧条件下RPE细胞缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达;⑷原代培养牛脉络膜微血管内皮细胞(choroidal endothelial cells,CEC),建立RPE-CEC共培养体系。采用MTT和结晶紫染色等方法观察特异性抑制FAK表达的RPE细胞对CEC增生和移行的影响。结果⑴在激光诱导的BN大鼠CNV生成早期(光凝后1d、3d),RPE-脉络膜复合体中FAK表达显著升高,随后表达逐渐下降。免疫荧光检测显示,FAK的表达上调主要定位于参与CNV生成的RPE细胞中;⑵体外实验表明,目前已知的多种与CNV生成相关的影响因素,如缺氧、氧化衰老、POS代谢产物在胞内堆积以及Bruch膜破裂导致的RPE与ECM成分的接触等,均可不同程度上调RPE细胞内FAK的表达;⑶成功构建了pSilencer/FAK表达载体,通过脂质体介导转染RPE细胞可有效下调FAK的表达。运用siRNA特异性抑制RPE细胞FAK的表达后,可以显著抑制缺氧诱导的RPE细胞HIF-1α和VEGF的表达上调;⑷成功原代培养牛CEC细胞,利用细胞共培养体系观察到RPE细胞与CEC共培养可以刺激CEC的增生和移行,缺氧条件下刺激作用增强。而运用siRNA特异性抑制RPE细胞FAK的表达后,可以显著抑制缺氧条件下RPE细胞对CEC增生和移行的诱导作用。结论本研究首次证实FAK信号参与了激光诱导的大鼠CNV的生成,提示CNV发生早期,活化的RPE细胞内FAK表达的上调参与CNV生成的调控。体外实验表明,多种已知与CNV生成相关的刺激因素均可不同程度上调RPE细胞FAK的表达,进一步提示FAK在CNV发生过程中可能发挥重要调控作用。特异性抑制RPE细胞FAK的表达,可以显著抑制缺氧诱导的RPE细胞HIF-1α和VEGF的表达上调,并进而抑制CEC增生和移行,进一步明确了FAK对CNV生成的调控作用。综上,FAK信号参与了CNV生成过程的调控,此类研究在国内外尚未见报道。针对FAK的siRNA可以抑制CNV生成,这将为临床防治CNV性疾病提供新思路。更多还原

【Abstract】 Background Choroidal neovascularization (CNV) is now known the leading cause of vision loss in age related macular degeneration (AMD), while its pathogenesis is still poorly understood. Retinal pigment epithelium (RPE) cells are known to quickly respond and adapt to environmental stresses such as ischemia and metabolic changes by expressing a number of various genes. In the early stages of CNV development, RPE produce cytokines and growth factors promoting CNV development. Subsequently, proliferating choroidal vessels surmount Bruch’s membrane and spread in the subpigment epithelial and subretinal space to form a CNV membrane.The underlying mechanism of CNV is multifactorial and complex. Growth factors and cell adhesion molecules have been implicated in CNV. RPE cells participating in angiogenesis get signals from several sources, such as soluble stimulators and extracellular matrix (ECM). Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by the integrins or growth factor receptors to intracellular cytoskeletal and signaling proteins, thus controlling essential cellular processes such as growth, survival, migration and differentiation. Extensive evidence has shown that FAK is activated in response to both the ECM and soluble signaling factors. Recent work from several laboratories points out the importance of FAK in influencing distinct steps of the angiogenic response and suggests a critical role of FAK in pathological retinal angiogenesis. But to our knowledge, no study investigating the effect of FAK on CNV formation has been reported previously.Purpose It would be helpful for our understanding of CNV to study the effect of FAK on CNV formation. In this study, we investigate the expression of FAK in the experimental CNV models and evaluate the effect of several CNV risk factors on the expression of FAK in cultured RPE cells. Then we used siRNA specific blockade of FAK in RPE cells, seek to evaluate the role of FAK in key steps involved in CNV.Methods⑴The BN rats underwent laser rupture of Bruch’s membrane to induce CNV and were killed at 1, 3, 7, and 14 days after laser injury. Immunofluorescenc and Western blot were processed to detect FAK protein.⑵Cultured human RPE cells were exposed to hypoxia, H2O2, ECM and out segment of photoreceptors (POS), differently. The expression of FAK and pFAK in cultured RPE cells were examined by Western blot.⑶RPE cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot.⑷Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal endothelial cells (CEC), respectively.Results⑴FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. Immunocytochemistry evaluations suggest that the strong up-regulation of FAK expression was located in RPE cells participating in choroidal neovascularization.⑵In vitro experiment shows hypoxia, H2O2, ECM and POS, which were known initiate CNV response, can induce FAK expression in cultured RPE cells.⑶FAK siRNA-treated RPE cells exhibited an inhibition in the expression of HIF-1 and VEGF in response to hypoxia, which indicated that FAK involved in hypoxia signaling in RPE cells.⑷The absence of FAK in RPE cells show inability in reducing the proliferation and migration of CEC under hypoxia condition.Conclusions In summary, we indicate the first time that FAK pathway activation plays a role in the development of laser-induced CNV in rat. In vitro experiment shows FAK pathway involves in several CNV initiated signaling in cultured RPE cells. The absence of FAK effectively reduces gene expression of hypoxia-induced HIF-1αand VEGF in RPE cells in vitro. Furthermore, our data provide strong evidence that specific blockade of FAK in RPE cells resultes in the inhibition of proliferation and migration of CEC. Combining the observation above, this is the first demonstration that FAK modulates the formation of CNV. It is reasonable to propose that FAK siRNA potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorder, which could represent a therapeutically relevant strategy for the inhibition of CNV.更多还原