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ENGLISH ABSTRACT
吲哚菁绿对人晶状体上皮细胞生物学行为和转分化的抑制作用及其机制
刘亚军
赵英迪
张文文
张司
何自芳
陈菲菲
解正高
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20220817-00380
Inhibitory effect of indocyanine green on biological behavior and transdifferentiation of human lens epithelial cells and its mechanism
Liu Yajun
Zhao Yingdi
Zhang Wenwen
Zhang Si
He Zifang
Chen Feifei
Xie Zhenggao
Authors Info & Affiliations
Liu Yajun
Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210009, China
Zhao Yingdi
Department of Anatomy, Wannan Medical College, Wuhu 241002, China
Zhang Wenwen
Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210009, China
Zhang Si
Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210009, China
He Zifang
Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210009, China
Chen Feifei
Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210009, China
Xie Zhenggao
Department of Ophthalmology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210009, China
·
DOI： 10.3760/cma.j.cn115989-20220817-00380
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摘要

目的研究吲哚菁绿(ICG)对人晶状体上皮细胞(HLECs)生物学行为和转分化的抑制作用及其机制。

方法将HLECs细胞系分为空白对照组、5%葡萄糖溶液(GS)组和0.5%、1.5%、2.5% ICG组，分别用平衡盐溶液、5% GS以及0.5%、1.5%和2.5%的ICG溶液处理3 min，然后在新鲜培养基中孵育24 h。采用流式细胞术检测HLECs凋亡水平；采用Western blot法检测HLECs凋亡相关蛋白Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤因子2(Bcl-2)、半胱氨酸蛋白酶3(caspase-3)和caspase-9表达水平；采用细胞计数试剂盒8(CCK-8)和5-溴-2-脱氧尿嘧啶(EdU)法检测HLECs增生过程；采用细胞划痕实验检测HLECs迁移能力；采用Transwell法检测HLECs迁移和侵袭过程。采用Western blot法检测HLECs转分化相关蛋白α-平滑肌肌动蛋白(α-SMA)、N-钙黏蛋白(N-cadherin)、纤维连接蛋白(FN)和波形纤维蛋白(vimentin)表达水平。

结果空白对照组、5% GS组、0.5% ICG组、1.5% ICG组和2.5% ICG组细胞凋亡率分别为(4.35±0.60)%、(4.63±0.19)%、(8.17±0.69)%、(13.90±0.33)%和(23.08±1.12)%，总体比较差异有统计学意义( F＝412.74， P<0.05)，其中0.5% ICG组、1.5% ICG组和2.5% ICG组细胞凋亡率明显高于空白对照组和5% GS组，差异均有统计学意义(均 P<0.05)。0.5% ICG组、1.5% ICG组、2.5% ICG组caspase-3、caspase-9和Bax蛋白相对表达量明显高于空白对照组和5% GS组，1.5% ICG组、2.5% ICG组Bcl-2蛋白相对表达量较空白对照组和5% GS组降低，差异均有统计学意义(均 P<0.05)。0.5% ICG组、1.5% ICG组和2.5% ICG组EdU阳性细胞率明显低于空白对照组和5% GS组，差异均有统计学意义(均 P<0.05)。0.5% ICG组、1.5% ICG组、2.5% ICG组细胞生存率明显低于空白对照组和5% GS组，差异均有统计学意义(均 P<0.05)。细胞划痕实验结果显示，0.5% ICG组、1.5% ICG组、2.5% ICG组细胞迁移率明显低于空白对照组和5% GS组，差异均有统计学意义(均 P<0.05)。Transwell实验结果显示，0.5% ICG组、1.5% ICG组、2.5% ICG组细胞迁移数和侵袭细胞数明显少于空白对照组和5% GS组，差异均有统计学意义(均 P<0.05)。0.5% ICG组、1.5% ICG组和2.5% ICG组α-SMA、N-cadherin和FN蛋白相对表达量明显低于空白对照组和5% GS组，1.5% ICG组和2.5% ICG组vimentin蛋白相对表达量较空白对照组和5% GS组降低，差异均有统计学意义(均 P<0.05)。

结论ICG可促进HLECs凋亡，抑制其增生、迁移、侵袭和转分化，并呈浓度依赖性。

白内障;囊膜混浊;吲哚菁绿;晶状体上皮细胞;生物学行为;转分化
ABSTRACT

ObjectiveTo investigate the inhibitory effect of indocyanine green (ICG) on biological behavior and transdifferentiation of human lens epithelial cells (HLECs) and its mechanism.

MethodsHLECs were divided into blank control group, 5% glucose solution (GS) group and 0.5% ICG group, 1.5% ICG group and 2.5% ICG group, which were treated with balanced salt solution, 5% GS and 0.5%, 1.5% and 2.5% ICG solutions for 3 minutes, respectively, and then were incubated in fresh medium for 24 hours.The apoptosis level of HLECs was detected by flow cytometry.The expression levels of apoptosis-related proteins, Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), caspase-3 and caspase-9 were detected by Western blot.Cell proliferation was detected via the cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay.The migration ability of HLECs was detected by cell scratch assay.Cell migration and invasion were determined by Transwell assays.The expression levels transdifferentiation-related proteins, α-smooth muscle actin (α-SMA), nerve calcium adhesion protein (N-cadherin), fibronectin (FN) and vimentin were assessed by Western blot.

ResultsThe apoptosis rates of blank control group, 5% GS group, 0.5% ICG group, 1.5% ICG group and 2.5% ICG group were (4.35±0.60)%, (4.63±0.19)%, (8.17±0.69)%, (13.90±0.33)% and (23.08±1.12)%, with a statistically significant difference in the overall comparison ( F=412.74, P<0.05). The apoptosis rate was significantly higher in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The relative expressions of caspase-3, caspase-9 and Bax proteins were significantly higher in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the relative expression of Bcl-2 protein was lower in 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all at P<0.05). The rate of EdU-positive cells was significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG groups than in blank control group and 5% GS group (all at P<0.05). The survival rate of cells was significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The migration rates of scratch cells were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all P<0.05). The number of migrating cells and the number of invading cells were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group (all at P<0.05). The relative expressions of α-SMA, N-cadherin and FN were significantly lower in 0.5% ICG group, 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the relative expression of vimentin was lower in 1.5% ICG group and 2.5% ICG group than in blank control group and 5% GS group, and the differences were statistically significant (all at P<0.05).

ConclusionsICG can promote HLECs apoptosis and inhibit HLECs proliferation, migration, invasion and transdifferentiation in a concentration-dependent manner.

Cataract;Capsule opacification;Indocyanine green;Lens epithelial cells;Biological behavior;Transdifferentiation
Xie Zhenggao, Email: mocdef.3ab6178eixgz

Liu Yajun and Zhao Yingdi contributed equally to the article

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引用本文

刘亚军,赵英迪,张文文,等. 吲哚菁绿对人晶状体上皮细胞生物学行为和转分化的抑制作用及其机制[J]. 中华实验眼科杂志,2023,41(12)：1160-1168.

DOI:10.3760/cma.j.cn115989-20220817-00380

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据报道，在5年随访期内年龄相关性白内障术后后囊膜混浊(posterior capsule opacification，PCO)的发生率高达22.6% [ 1 ]，在儿童患者中其发生率可高达100% [ 2 ]。PCO的发生与白内障术中前囊膜及赤道部囊膜上残留的晶状体上皮细胞(lens epithelial cells，LECs)发生增生、迁移、侵袭和转分化为肌成纤维细胞、基质沉积和收缩有关 [ 3 ]。药物抑制PCO发病机制中的这些关键细胞事件似乎是预防PCO的一种有价值方法。目前临床工作中经常使用的治疗PCO的有效方法是YAG激光后囊切开术，但这种治疗方式可能会损伤人工晶状体(intraocular lens，IOL)，有发生黄斑囊样水肿、眼压升高和视网膜脱离的风险 [ 4 , 5 ]。近年来，通过抑制、清除或杀死残留LECs来减少PCO发生的各种药物已被广泛研究，其中抑制LECs增生的药物包括塞来昔布、氟尿嘧啶和西罗莫司 [ 6 , 7 , 8 ]；阻碍迁移的药物包括甲氨蝶呤、厄洛替尼和微小RNA-34a [ 9 , 10 , 11 ]；阻碍上皮－间质转分化(epithelial-mesenchymal transiton，EMT)的药物包括山梨醇、白藜芦醇和THZ1 [ 12 , 13 , 14 ]；清除LECs的药物包括乙二胺四乙酸(ethylenediamine tetraacetic acid，EDTA)、蒸馏水和高渗盐水 [ 15 , 16 , 17 ]。这些药物仍主要用于细胞和动物实验，尚未广泛应用于临床。因此，需要一种有效且安全的PCO预防药物。吲哚菁绿(indocyanine green，ICG)在眼科临床上广泛用于荧光素眼底血管造影、内界膜染色和晶状体前囊染色 [ 18 , 19 ]。研究表明，0.50 mg/ml ICG对LECs的活性影响很小；低于0.075 mg/ml时，ICG对LECs活性无影响，0.06% ICG孵育人视网膜色素上皮细胞(human retinal pigment epithelial cells，HRPEs)后72 h，细胞存活率仍能达到近90%；0.062 5 mg/ml和0.02 mg/ml的ICG均不会引起RPE细胞存活率的变化 [ 20 , 21 , 22 ]。然而，越来越多的细胞实验显示了高浓度ICG具有细胞毒性作用，0.25%的ICG可使HRPEs存活率降至约30%；ICG浓度为5 mg/ml时，孵育时间超过5 min，6 h后细胞存活率明显下降，孵育20 min后无活RPE细胞 [ 22 , 23 , 24 ]。ICG是否能够抑制人晶状体上皮细胞(human lens epithelial cells，HLECs)的生物学行为尚不清楚。本研究拟观察ICG对HLECs生物学行为的抑制作用并探讨其机制，为PCO的预防方法研究提供实验依据。
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Dong N ， Tang X ， Yuan XY ，et al. TAK1 promotes epithelial-mesenchymal transition of lens epithelial cells[J]．Chin J Ophthalmol， 2016，52(4)∶278－284. DOI： 10.3760/cma.j.issn.0412-4081.2016.04.009 .
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[32]
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