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白内障的分子病理改变

Molecular pathological changes of cataract

来源期刊: 眼科学报 | 2021年8月 第36卷 第8期 663-668 发布时间: 收稿时间:2023/6/8 17:27:42 阅读量:4184
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白内障分子病理晶状体囊膜晶状体上皮细胞晶状体纤维
cataract molecular pathology lens capsule lens epithelial cells lens fiber
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10.3978/j.issn.1000-4432.2021.07.22
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白内障是世界范围内失明的主要原因。正常晶状体是富有弹性的形似双凸透镜的透明体,是机体内蛋白质含量最高的组织,由晶状体囊膜、晶状体上皮细胞、晶状体纤维和悬韧带构成。白内障为晶状体透明度下降,表现为晶状体混浊。近年来随着分子生物学、表观遗传学、免疫学、有机化学等学科快速发展,国内外学者对白内障也进行了大量分子水平的研究,探讨了白内障发生发展相关分子机制,为未来基因治疗和靶向药物等治疗白内障提供了理论基础。对白内障分子病理改变的了解,是白内障精准诊治的基础。
Cataract is the main cause of blindness worldwide. The normal crystalline lens is a transparent biconvex disc,with highest protein content in all human tissues. The lens is composed of capsule, lens epithelial cells, lens fiber and zonular ligment. Cataract is a decrease in the transparency of the lens, which is characterized by opacity. In recent years, with the rapid development of molecular biology, epigenetics, immunology and organic chemistry,researchers have conducted a large number of studies on the molecular basis of genetic or targeted therapy of cataract. It is important to know the molecular pathology of cataract, which is the basis of precise diagnosis and treatment of cataract.
白内障是全球首位致盲性眼病,但是目前临床眼科医生对白内障的诊断与治疗多还在宏观水平。眼科医生临床中常常是通过裂隙灯检查发现晶状体混浊以及手术摘除混浊的晶状体治疗白内障,对其分子病理机制的认识不够。近年来随着各种技术的发展,国内外研究者对白内障分子病理机制做了很多研究,有许多新的进展和发现。
正常的晶状体是双凸圆盘状透明体,由晶状体囊膜、囊膜下晶状体上皮细胞(lens epithelial cells,LECs)、晶状体纤维和悬韧带组成。白内障是指晶状体透明度下降,由透明变混浊。病理切片表现有3种[1],一是晶状体囊膜下上皮增生,二是晶状体囊膜厚度改变,三是晶状体纤维变性,纤维变短,有的呈梭形,有的呈棒状,有的变为圆形,即马氏小球(Morgagnian globules,指白内障晶状体蛋白凝固、坏死,病理切片上表现为无结构的均匀一致的小圆球[1]),而马氏小球是诊断白内障的重要病理依据。而在分子水平,晶状体囊膜、上皮细胞和晶状体纤维也都有各自的改变,不同类型的白内障分子病理机制也不尽相同,这些改变可通过免疫组织化学、荧光原位杂交(fluorescence in situ hybridization,FISH)以及电子显微镜等技术检测,辅助临床诊断。

1 晶状体囊膜的分子病理改变

晶状体囊膜,也称为晶状体基底膜,是透明而富有弹性的薄膜,是一种特殊的细胞外基质,由晶状体上皮细胞分泌产生,后由新分化的纤维细胞产生[2]。用阿尼蓝染色后可见囊膜分为2层,内层较厚,外表层较薄,悬韧带附着于外层,病理情况出现表层分离、剥落,即囊膜剥脱。后囊膜的厚度(2~3 μm)比前囊膜薄3~5倍。赤道部周围的囊膜表面有悬韧带连接点[3]。晶状体囊膜的分子构成与其他基底膜类似,主要成分是I V型胶原蛋白(最多占晶状体囊膜干重的40%),其交联集成在层粘连蛋白支架上,保持囊膜的强度和弹性。层粘连蛋白在晶状体囊膜呈极性分布,在晶状体上皮细胞侧分布较多。其他还包括巢蛋白、凝集素和一些硫酸肝素蛋白聚糖等[2]。白内障晶状体囊膜分子构成发生改变:利用免疫组织化学的方法可见I V型胶原蛋白表达减少、I型胶原蛋白表达增加,层粘连蛋白表达极性消失、在基底膜异常沉积、层粘连蛋白α4表达异常增加,衰老相关蛋白、P53、转化生长因子β1(transforming growth factor-β,TGFβ1)表达增加,并表达特异性波形蛋白、纤维连接蛋白[4]。白内障晶状体囊膜屏障作用降低,晶状体内钠、钙、钾、氯、铜、锌等离子浓度增加[5]。衰老的晶状体囊膜中晚期糖基化终末产物表达增加,可能与年龄相关性白内障以及后发性白内障的发生有关[6]

2 晶状体上皮细胞分子病理改变

晶状体前囊膜下有一层上皮细胞,分布在从中央区到赤道部的整个范围,而后囊膜下没有上皮细胞。根据形态不同,LECs分为3个区,中央区细胞为立方形,排列整齐,体积较大;中周部细胞欠规则,细胞核靠近基底部;赤道部细胞变长,核呈圆形,可有分裂象。晶状体上皮细胞微环境改变,如角膜溃疡穿孔后晶状体囊膜与溃疡接触、外伤性后晶状体囊膜破裂、青光眼急性发作等,LECs可发生增生、化生,甚至萎缩变性,形成白内障[7]

2.1 LECs细胞膜蛋白表达改变

正常LECs膜表面表达多种分子通道和转运体,包括缝隙连接蛋白43(connexin 43,Cx43)和Cx50[8]。Cx43和Cx50形成具有不同性质的缝隙连接和半通道,在细胞发育中起重要作用。年龄相关性白内障LECs细胞膜Cx50表达降低,而Cx43表达改变不明显[9]
水通道蛋白(aquaporins,AQPs)是一种重要的膜蛋白,根据渗透梯度转运细胞内外的水分子。LECs细胞膜上主要表达AQP1和AQP5两种水通道蛋白。免疫组织化学结果显示白内障LECs的2种水通道蛋白表达均显著增加,提示水通透性增加。
晶状体组织中钙离子浓度增加激活蛋白水解酶,介导晶状体蛋白的水解,是白内障发生重要的分子机制[10]。研究表明白内障LECs细胞膜上钙通道蛋白表达增加,钙离子通透性增强。细胞膜ATP依赖的钙泵(plasma membrane Ca2+-ATPase,PMCA)是晶状体钙离子去除的主要机制,在遗传性白内障模型中荧光免疫组织化学发现LECs细胞膜PMCA代偿性表达增加[11]
综上,白内障LECs细胞间连接蛋白、水通道蛋白和钙通道蛋白等表达改变,细胞间连接稳定性下降,细胞膜水、钙离子等通透性增加。
2.2 LECs细胞膜脂质改变随着质谱等技术的日益发展,内障患者晶状体脂质组成被发现发生了明显变化。而膜脂质发生细微的、甚至目前手段无法检测到的变化都可能导致晶状体透明性下降。几乎所有类型的白内障在晶状体的各个区域均发生了膜结构的重组[12]。白内障LECs细胞膜发生曲折、液泡以及层状小体(lamellar bodies)。晶状体上皮细胞特有的层状小体是附着在细胞膜上的晶状体蛋白外面包绕一层脂质结构。白内障LECs细胞膜层状小体明显增加。早在1881年,学者[13]发现白内障患者晶状体的胆固醇水平升高。随后,有研究[14]发现人晶状体中的脂质氧化的主要产物丙二醛浓度随着年龄的增加和白内障的发生而增加。人类晶状体中的主要磷脂为髓磷脂,但是甘油磷脂的氧化、含量降低在白内障中更为显著。与年龄相近的正常晶状体相比,白内障晶状体中甘油磷脂的总量要少得多。

2.3 LECs胞内蛋白表达改变

晶状体蛋白通过短程相互作用保持晶状体透明。衰老或者疾病损伤导致晶体蛋白错误折叠、聚集成不溶性淀粉样物质,形成白内障。人晶状体有3个主要蛋白质家族:α、β和γ晶状体蛋白[15]。α晶状体蛋白分为α晶状体蛋白A(αA晶状体蛋白)和α晶状体蛋白B(αB晶状体蛋白),属于分子伴侣小热休克蛋白家族,是脊椎动物晶状体的主要蛋白质成分,在维持γ-晶状体蛋白等其他晶状体蛋白的水溶性中起重要作用[16]。年龄相关性和先天性白内障LECs中的可溶性αA和αB晶状体蛋白表达均较正常对照组减少。可溶性α晶状体蛋白与γ晶状体蛋白等其他蛋白质结合形成不可溶蛋白是晶状体蛋白变性的重要机制[17]
在机体衰老过程中,β-晶状体蛋白被高度修饰,白内障β-晶状体蛋白亚基数量改变并组成不同的寡聚体,推测其可能在衰老和白内障形成中起重要作用[18-19],但是目前对β-晶状体蛋白的结构和功能研究仍然较少,其在白内障发生发展中的作用仍需进一步研究。
白内障γ晶状体蛋白出现聚集、变性,是白内障重要的分子改变。未聚集的γ晶状体蛋白作为单体存在于晶状体中[20]。人类γ晶状体蛋白家族由5个成员组成:γA、γB、γC、γD和γ S晶状体蛋白。它们在序列和分子质量上高度相似,但表达有一定的时序性。γA和γD在晶状体发育早期表达,因此主要存在于晶状体核;γS在晶状体发育后期产生,在晶状体皮质更为多见[21-22]
晶状体中高浓度还原型谷胱甘肽是维持组织透明度重要的分子基础。氧化损伤是白内障形成的重要机制之一。一方面,白内障LECs细胞内活性氧ROS、H2O2、NO等增加[23],另一方面,清除氧化损伤的能力下降,如还原型谷胱甘肽(glutathione,GSH)、超氧化物歧化酶、过氧化物酶、过氧化氢酶等含量降低[24]。白内障LECs细胞质和胞核中的GSH浓度均降低,并且在细胞核中下降比例更高,提示细胞核更易受氧化应激的影响[25]
此外,LECs发生凋亡、焦亡等程序性死亡也参与白内障的发病过程,细胞呈现相关的分子病理改变。细胞凋亡是一种程序性死亡,白内障晶状体上皮细胞凋亡增加。凋亡分子表达谱具有特征性,如Bcl2等抑制凋亡基因和蛋白表达减少、Bax等促凋亡基因和蛋白表达增加[26]。细胞焦亡是继细胞凋亡和坏死之后发现的一种新的细胞程序性、伴有炎性因子参与的死亡方式。新近研究[27]表明白内障LECs焦亡分子标志物Caspase-1和Gasdermin蛋白GSDMD表达上调,提示LECs焦亡参与了白内障的发生。
近来有研究[28]发现:晶状体混浊部位的LECs自噬相关蛋白LC3-II和自噬相关基因ATG7 、ATG5、Beclin-1表达增加,提示自噬参与白内障发病过程。自噬是真核细胞清除细胞内聚集、变性蛋白及受损细胞器,进而维持细胞内稳态的一种自我保护机制。

2.4 LECs核酸改变

白内障早期LECs即出现DNA损伤。在白内障LECs中,几乎检测不到DNA链断裂,但核苷酸组成改变:氧化嘧啶和嘧啶二聚体含量降低,氧化嘌呤的含量增高。白内障LECs中,通过聚合酶链式反应(polymerase chain reaction,PCR)的方法可检测到编码α、β和γ晶状体蛋白基因CRYA、CRYB和CRYG[29]、编码连接蛋白基因GJA3和GJA8[30]、编码内源性膜蛋白基因MIP和LIM2[31-32],以及代谢相关基因包括葡萄糖代谢相关基因GALK1等多种基因突变[33]。另外,我们在年龄相关性白内障中发现氧化损伤修复基因OGG1、WRN等基因突变[34]。氧化损伤修复基因OGG1、WRN、ERCC6等也呈现表观遗传学改变(如甲基化等)。部分研究也发现一些转录因子的DNA结合区发生突变是绕核性等白内障致病因素[35]
近年来,非编码RNA如微小RNA(microRNA,miRNA)、长链非编码R N A ( long non - coding RNA,lncRNA)以及环状RNA(circular RNA,circRNA)等在白内障研究领域被广泛报道。微小RNA(miRNA)是长约22 nt的非编码RNA,广泛存在于从病毒到人类的各种生物中,通过与mRNA结合,阻断蛋白编码基因的表达,抑制蛋白翻译,与多种疾病的发病相关[36]。白内障LECs内miR-34a miR-15a5p、miR-15a-3p和miR-161-5p等miRNA表达明显增[37-38],miR-125b和miR-16-1-3p则表达降低。在糖尿病白内障LECs中miR-30a表达降低[39-40]。另外,miR-138、miRNA-4328在白内障患者前囊膜和房水中高表达[41]
LncRNA是长度大于200 nt的非编码RNA,在表观遗传调控、细胞周期调控和细胞分化调控等众多生命活动中发挥重要作用。白内障LECs中lncRNA TUG1和lncRNA KCNQ1OT1表达增加[42-43],lncRNA PLCD3-OT1表达降低[44]。LncRNA MIAT在年龄相关性白内障(age-related cataract,ARC)患者血浆和房水中显著升高,被认为是ARC特异性生物标志物[45]。CircRNA分子呈封闭环状结构,不受RNA外切酶影响,富含miRNA结合位点,在细胞中起到miRNA海绵的作用,进而解除miRNA对其靶基因的抑制作用,升高靶基因的表达水平,发挥调控作用[46]。白内障LECs中circHIPK3表达显著下调,认为与白内障的发生相关[47]

3 晶状体纤维分子病理改变

晶状体纤维主要由晶状体蛋白组成,成熟晶状体纤维中的蛋白质合成和周转停止,使晶状体纤维蛋白长期保持可溶性和透明。蛋白质翻译后修饰,包括磷酸化、脱酰胺基化、N、C截断、乙酰化和甲基化,导致晶状体蛋白质相互作用被破坏,形成高分子聚合物并与细胞膜结合,晶状体蛋白分子3级、4级结构均发生改变,发生白内障[48-52]。其中N、C端截断被认为是白内障晶状体蛋白的标志性翻译后修饰。α晶状体蛋白的N123残基在维持其伴侣功能方面起着至关重要的作用。当发生N端截断,晶状体蛋白分子伴侣功能减弱,导致白内障的发生[50]。蛋白糖基化,如γB晶状体蛋白糖基化,是糖尿病性白内障发生的重要分子改变。因此,晶状体蛋白质组学变化被认为是白内障发生主要的分子病理变化。
晶状体蛋白聚集有2种结果,形成高度有序的淀粉样物质或者形成高度无序的无定形聚集物[17]。无定形聚集物一般不会导致显著的蛋白质折叠疾病[53]。淀粉样物质是一类高度有序和稳定的蛋白质聚集体,组织中淀粉样物质沉积是多种疾病的病理改变,如阿尔茨海默氏症和帕金森病[53]。利用透射电镜和二维红外光谱法可检测到衰老和白内障晶状体组织中微量淀粉样物质存在,未成年人透明晶状体中未发现淀粉样改变[54]
随着精准医学时代的到来,白内障的诊断与治疗必然朝着早发现、早治疗、基因/药物治疗替代手术治疗等方向发展。掌握白内障的分子病理机制是早期精准诊断白内障的前提,也是未来基因/药物治疗白内障的理论基础。目前对白内障的分子病理的研究仍在不断的探索中。相信随着生物化学、遗传学、分子生物学等学科不断发展、研究方法不断改进,白内障的分子病理机制会进一步被阐明,白内障的诊断和治疗也将进入精准的分子诊断和治疗水平。
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1、国家自然科学基金 (81670852,81770906,81974129);中国博士后科学基金 (2017M610343);江苏省科技计划 项目 (BE2016699);南通市科技计划项目 (MS22019012)。
This work was supported by the National Natural Science Foundation (81670852, 81770906, 81974129), China Postdoctoral Science Foundation (2017M610343), Technology and Science Foundation of Jiangsu Province (BE2016699), and Technology and Science Foundation of Nantong (MS22019012), China.()
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