2020, Vol. 27
在哺乳动物眼球中，晶体蛋白是组成其晶状体的重要结构并维持其透明度。晶体蛋白可划分为3个主要的家族：α- crystallins、β- crystallins、γ-crystallins。α-crystallins由αA-crystallin和αB-crystallin构成，是晶体蛋白主要类型。研究[1-2]发现，晶状体以外的组织细胞中也存在α-crystallins。αA-crystallin可表达于视网膜、胸腺、脾脏等部位；αB-crystallin可表达于视网膜、角膜、视神经、星形胶质细胞、Müller细胞，以及大脑、肾脏、肺、肝脏、脾脏、皮肤、心脏和骨骼肌等眼外组织[3-5]。α-crystallins是小分子热休克蛋白家族(small-molecule heat shock protein, sHSP)的重要组成部分，具有热休克蛋白的特性。
由于缺氧、炎症、外伤、血管病变等各种致病因素刺激，视网膜微血管内皮细胞缺血、缺氧，血管内皮生长因子(vascular endothelial growth, VEGF)上调，促进病理性血管增生，同时视网膜色素上皮(retinal pigment epithelial，RPE)细胞和Müller细胞分泌转化生长因子-β(transforming growth factor-β, TGF-β)、αB-crystallin等可诱导细胞向肌成纤维细胞转化，导致纤维瘢痕增生，形成一系列增生性视网膜疾病。增生性视网膜疾病主要包括增殖性玻璃体视网膜病变(proliferative vitreoretinopathy, PVR)[6]、新生血管性年龄相关性黄斑病变(neovascular age-related macular degeneration, nAMD)[7]和增生性糖尿病视网膜病变(proliferative diabetic retinopathy, PDR)[8]。PDR中异常新生血管易渗漏，从而导致视网膜水肿、出血，并在慢性刺激下形成纤维瘢痕，继发视网膜脱离或视网膜无灌注，是视力下降甚至致盲的主要原因。视网膜组织受到损伤引发增殖性改变的具体机制尚未完全清晰，已有众多文献[9-11]指出，α-crystallins可推进此过程且发挥不容忽视的作用。本文主要对α-crystallins促进视网膜血管和纤维增生的主要分子机制进行综述。
1 α-crystallins的主要功能α-crystallins的功能大致如下。(1)维持细胞骨架完整性：细胞骨架是一种复杂的动态结构，由相互连接的细丝网络组成，由微管(microtubules, MTs)、微丝(microfilaments, MFs)和中间丝(intermediate filaments, IFs)构成，有利于维持细胞形状，为细胞运动和分裂提供框架。例如，αB-crystallin作为微管蛋白/微管的分子伴侣，可在应激状态下调节微管动力学，维持细胞形态和细胞间黏附[12]。αA-crystallin与phakinin和filensin(2种晶体特异性IF蛋白)形成络合物，也可能发挥相似作用。大鼠晶状体纤维细胞研究[13-14]表明，αA-crystallin均匀分布于细胞骨架细肌丝，可保持晶状体透明度。(2)抗凋亡和细胞保护：αB-crystallin参与多种细胞凋亡通路，可直接或间接阻止应激诱导物和促凋亡因子发挥作用，例如作用于促凋亡因子Bax和Bcl-X(S)，从而抑制其向线粒体迁移[15]；抑制caspase-3和PARP(poly ADP-ribose polymerase)的激活[16]；抑制细胞色素C和caspase-8依赖的caspase-3活化[17]；与p53相互作用，防止其从细胞质转移到线粒体[18]；抑制RAS活化，抑制RAF/MEK/ERK通路[19]等。此外，αB-crystallin可在S59和S45而非S19(S19广泛非磷酸化)位点磷酸化活化后对抗大鼠脑星形胶质细胞凋亡减少。也有研究[20]提示，在人类晶状体中，αB-crystallin可在K92位点乙酰化，增强细胞保护和抗凋亡的功能，由此说明αB-crystallin蛋白修饰后可更好地发挥生物学功能，其具体机制还需进一步研究。αA-crystallin也具有类似的细胞保护作用，如激活未折叠蛋白反应(unfolded protein response, UPR)[21]。(3)作为分子伴侣，促进细胞内外分子正确折叠，增强其作用，与细胞色素c、caspase-6相互作用[22]，与Bax和Bcl-X(S)高度亲和[23]，抑制促凋亡因子从细胞质向线粒体迁移从而保护线粒体完整性。αB-crystallin还与内皮生长因子(vascular endothelial growth factor, VEGF)共同定位在RPE内质网上，从而调控VEGF介导的脉络膜血管新生[24]。(4)作为外泌体蛋白发挥胞外作用：如星形胶质细胞将αB-crystallin以外泌体形式分泌出胞，调节炎症反应[25]。此外，尽管αB-crystallin在S59位点被广泛磷酸化，但在胶质瘤细胞中，模拟磷酸化可显著减少通过外泌体分泌的αB-crystallin，由此推测αB-crystallin以非磷酸化形式在胞外分泌[26]，其胞外作用有待研究。
2 α-crystallins与增生性视网膜疾病 2.1 α-crystallins与糖尿病视网膜病变糖尿病视网膜病变(diabetic retinopathy, DR)是糖尿病重要的微血管病变之一，也是糖尿病患者视力丧失的主要原因。糖尿病引起视网膜缺血缺氧，导致新生血管在视网膜表面增生，不成熟的新生血管出血渗出加重病变。αB-crystallin领域一个重要的研究方向是其与多种蛋白包括凋亡、细胞骨架、信号转导等相关蛋白的相互作用。基于伴侣蛋白的特性，αB-crystallin与这些蛋白之间可能存在天然联系，尤其在某些疾病中，αB-crystallin可能间接参与其病理过程。以下主要阐述α-crystallins促进视网膜新生血管的过程。
在氧诱导视网膜病变(oxygen-induced retinopathy, OIR)和脉络膜新生血管(choroidal neovascularization, CNV)大鼠模型中敲减αB-crystallin能使视网膜新生血管病理过程中表达增高的VEGF和缺氧诱导因子(hypoxia inducible factor-1, HIF-1α)的表达情况维持在低水平。研究[24]利用免疫沉淀证实了αB-crystallin与VEGF共同定位在RPE内质网，并发现αB-crystallin在低氧下保护VEGF而间接促进血管新生。另一方面，Dong等[27]发现，在人眼视网膜组织上，磷酸化αB-crystallin(αB-crystallin活化形式)在CD31+视网膜内皮细胞内显著表达，并与VEGF、p38-丝裂原活化蛋白激酶(p-p38MAPK)共同定位在PDR黄斑前膜。磷酸化αB-crystallin(尤其是S59位点磷酸化)可作为VEGF、p-p38 MAPK的伴侣蛋白，使其正确折叠而减少降解，参与形成PDR视网膜的新生血管[27]。此外，PDR患者玻璃体内αB-crystallin、VEGF表达量较正常人显著增加，其中，全视网膜光凝(pan-retinal photocoagulation, PRP)术后患者玻璃体内这2种蛋白表达有所下降[28]。因此，体内和体外实验均证明，αB-crystallin直接或者间接地促进视网膜血管新生。αA-crystallin似乎也有类似作用。有研究[29]显示，体外内皮细胞培养联合OIR、CNV模型，敲减αA-crystallin同样可通过VEGF和VEGFR2通路抑制眼部血管新生。
α-crystallins通过促进VEGF表达和正确折叠而增加视网膜新生血管发生。另外，αB-crystallin也能上调表达一些内皮细胞黏附蛋白而增强白细胞募集，影响内皮细胞炎症激活过程[30]。是否存在其他影响因素及如何将该特性应用到治疗上尚需进一步研究。
2.2 α-Crystallins与年龄相关性黄斑变性年龄相关性黄斑变性(age-related macular degeneration, AMD)是导致老年人发生不可逆视力丧失的主要疾病之一，临床上主要分为萎缩性(干性)AMD和渗出性(湿性)AMD。早期萎缩性AMD的特征是黄斑内的色素变化和玻璃膜疣的形成。随着疾病进展，感光细胞、RPE细胞和脉络膜血管内皮细胞进行性变性，最终可发展为地图状萎缩(geographic atrophy, GA)；渗出性AMD眼底特征为脉络膜新生血管膜(choroidal neovascularmembrane, CNVM)生成，常伴有出血和渗出。
晚期湿性AMD黄斑区视网膜下纤维化涉及一个重要的过程：上皮间质化转变(epithelial-mesenchymal transition, EMT)。肌成纤维细胞是CNVM的主要细胞构成，表现为α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)免疫反应阳性。组织学研究[31-32]表明，肌成纤维细胞来源于骨髓源性细胞和RPE细胞等。黄斑区RPE损伤后，RPE细胞失去其顶端-基底极性，向迁移性的肌成纤维细胞转化，伴上皮细胞标志物(如CD31、VE-cadherin)表达减少和间质标志物(α-SMA、细胞外基质蛋白)表达增加[33-34]。α-crystallins与此表型转化密不可分。对24例AMD眼和25例正常眼黄斑区Bruch膜-脉络膜复合物进行定量蛋白质组分析，显示AMD眼中α-crystallins表达明显增加[35]。近年来，研究[11]发现αB-crystallin作为SMAD4分子伴侣，可增强其核内转移和聚集，激活TGF-β/Smad通路，参与EMT形成，调控湿性AMD晚期视网膜下纤维化发展。同时也有不少关于αB-crystallin在继发性白内障和肺纤维化等其他内脏器官EMT作用的研究[36-37]。
因此，α-crystallins如何作用于AMD尤其是湿性AMD继发的视网膜下纤维化，值得进一步研究。另外，除了参与EMT过程，探索α-crystallins是否也参与视网膜、脉络膜微血管内皮细胞间质化转变(endothelial-to-mesenchymal transition, endoMT)，有利于深入了解α-crystallins与AMD的关系。
2.3 α-crystallins与增生性玻璃体视网膜病变PVR是孔源性视网膜脱离(rhegmatogenous retinal detachment, RD)手术修复的严重并发症，特征是在视网膜前表面形成纤维膜，称为视网膜前膜(epiretinal membrane, ERM)，其收缩导致视网膜结构扭曲和皱褶，最终发生牵拉性视网膜脱离(retinal detachment, RD)[38]。合理的治疗手段是通过手术去除活跃的细胞和细胞膜，必要时切除整个玻璃体。研究[39]证明，ERM成分复杂，包括RPE细胞、成纤维细胞、肌成纤维细胞、胶质细胞和巨噬细胞等，RPE细胞外泌体分泌的αB-crystallin也在ERM表达。
通过对玻璃体液进行蛋白组学和基因发现，约60%玻璃体蛋白分子量和蛋白形态符合细胞外泌体特征[40]。因此外泌体在玻璃体和视网膜之间的运输作用不容忽视，同时外泌体对于ERMs的作用仍值得思考。一项研究[41]证实，阻碍RPE细胞αB-crystallin表达可改变外泌体标志物(LAMP1、LAMP3)分布。因此，αB-crystallin除参与构成ERM外，还可促进外泌体合成。结合临床32例RD患者玻璃体样本αB-crystallin表达量显著增加的结果[28]，推测αB-crystallin除调控视网膜下纤维化外也可能促进ERM形成，加重视网膜损害。
3 α-crystallins的应用前景目前增生性视网膜疾病的治疗方法比较有限，主要有激光光凝、玻璃体切除额外纤维血管膜、抗血管生成药物等。为了提高临床疗效，有必要研发抑制视网膜血管增生和纤维化的新药。近年基于α-crystallins分子伴侣特性，研发了mini-αA peptide和mini-αB peptide[42]，将其与α-crystallins重组为蛋白多聚颗粒、重组腺病毒[43-44]。此类多肽伴侣可防止蛋白质聚集和纤维形成，保护突变晶体蛋白，同时抗氧化应激、抗凋亡等。近年来发现一种可以阻断αB-crystallin与VEGF-16联系的小分子抑制剂[45]，或可将其应用于新生血管性视网膜疾病[46]。因此，研发出阻断α-crystallins与SMAD通路联系的抑制剂，或将使治疗CNV和视网膜下纤维化成为可能。
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