目的研究蒸馏水处理对体外培养的人晶状体上皮细胞(LECs)活性的影响。
方法收集2020年5—12月在南京大学医学院附属鼓楼医院确诊为年龄相关性白内障并行超声乳化白内障吸除联合人工晶状体植入术的患者156例156眼，将手术过程中收集的晶状体前囊膜标本156片等分为312小片。采用计算机取随机数法将其中157小片样本分为正常对照组23小片、阳性对照组10小片、平衡盐溶液(BSS)浸泡组61小片和蒸馏水浸泡组63小片，其中正常对照组无任何处理；阳性对照组直接用质量分数4%组织细胞固定液固定；BSS浸泡组中20小片浸泡1 min、21小片浸泡2 min、20小片浸泡3 min；蒸馏水浸泡组中20小片浸泡1 min、23小片浸泡2 min、20小片浸泡3 min。另选取125小片模拟白内障手术过程中的注吸过程，按照上述BSS浸泡组和蒸馏水浸泡组处理相应时间后，用装有BSS的瓶子在70 cm高度冲洗1 min。采用锥虫蓝－伊红染色法检测细胞活力，计算LECs密度、死亡数、死亡率及脱落百分率。将剩余小片分为正常对照组、BSS浸泡组和蒸馏水浸泡1、2、3 min组，其中BSS浸泡组浸泡3 min，其余分组处理同前，光学显微镜和透射电子显微镜下观察各组LECs结构。
结果BSS浸泡各组LECs边界清晰，形态与正常对照组相比无明显差异。蒸馏水浸泡各组随时间延长，LECs逐渐胀大，死亡细胞边界不清。各组LECs密度、死亡数和死亡率总体比较差异均有统计学意义( F＝13.459、98.918、130.600，均 P<0.001)，其中蒸馏水浸泡2 min和3 min LECs密度均低于正常对照组，蒸馏水浸泡1、2、3 min组LECs死亡数和死亡率均高于正常对照组，蒸馏水浸泡1、2、3 min组LECs死亡数和死亡率均高于BSS浸泡相同时长组，差异均有统计学意义(均 P<0.05)。正常对照组，BSS浸泡1、2、3 min组和BSS浸泡联合冲洗组仅检测到少量LECs脱落。浸泡不同时间点，蒸馏水浸泡联合冲洗组LECs脱落明显，且随着蒸馏水浸泡时间的增加，LECs脱落范围增大。各组LECs脱落百分率总体比较差异有统计学意义( F＝123.670， P<0.001)，其中蒸馏水浸泡组、蒸馏水浸泡联合冲洗组处理不同时间LECs脱落百分率均高于正常对照组，蒸馏水浸泡组随处理时间延长LECs脱落百分率明显升高，差异均有统计学意义(均 P<0.05)。光学显微镜下显示蒸馏水浸泡1、2、3 min组细胞破坏，其中浸泡2 min和3 min组LECs部分脱落。透射电子显微镜下可见蒸馏水浸泡1、2、3 min组细胞溶解破坏，亚细胞器肿胀，细胞间连接破坏；其中浸泡1 min和2 min组细胞与囊膜连接疏松，浸泡3 min组LECs与囊膜部分分离。
结论蒸馏水浸泡可导致LECs死亡，其作用强度呈时间依赖性，蒸馏水浸泡联合冲洗可去除晶状体囊膜LECs。
ObjectiveTo investigate the effect of distilled water on the viability of human lens epithelial cells (LECs) cultured in vitro.
MethodsA total of 156 anterior capsule specimens were collected from 156 patients (156 eyes) who were diagnosed with age-related cataract during phacoemulsification combined with intraocular lens implantation from May to December 2020 in Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School.The 156 specimens were divided into 312 small pieces.Of the 312 pieces, 157 pieces were divided into normal control group (23 pieces), positive control group (10 pieces), balanced salt solution (BSS) immersion group (61 pieces) and distilled water immersion group (63 pieces) using computer-generated random numbers.Normal control group received no treatment.Positive control group was directly fixed with a mass fraction of 4% histiocytes fixative solution.For the 61 pieces in BSS immersion group, 20 pieces were soaked for 1 minute, 21 pieces for 2 minutes, and 20 pieces for 3 minutes.For the 63 pieces in distilled water immersion group, 20 pieces were soaked for 1 minute, 23 pieces for 2 minutes, and 20 pieces for 3 minutes.Another 125 pieces were selected to simulate the cataract aspiration-irrigation according to the treatment in BSS immersion group and distilled water immersion group respectively, plus rinse in a bottle containing BSS at a height of 70 cm for 1 minute.Cell viability was detected by trypan blue-eosin staining.LECs density, dead cell count, cell death rate and percentage of shedding (%) were calculated.Of the remaining 30 pieces, every 15 pieces were divided into normal control group, BSS immersion group, and distilled water immersion for 1, 2 and 3 minutes groups, with 3 pieces in each group.BSS immersion group was immersed for 3 minutes, and the other four groups were treated as mentioned above, and the LECs structure of the four groups was observed by light microscopy and transmission electron microscopy.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (No.2019-248-01). Written informed consent was obtained from each subject.
ResultsThe boundaries of LECs in BSS treatment groups were clear, and there was no significant difference in morphology compared with normal control group.With time increasing, LECs in distilled water treatment groups gradually swelled, and the boundaries of dead cells were not clear.There were significant differences in LECs density, dead LECs count and LECs mortality ( F=13.459, 98.918, 130.600; all at P<0.001). The LECs density was lower in 2-minute and 3-minute distilled water treatment groups than in normal control group, showing statistically significant differences (both at P<0.05). The dead LECs count and LECs mortality were higher in 1-minute, 2-minute and 3-minute distilled water treatment groups than in normal control group and BSS treatment groups for the same time, and the differences were statistically significant (all at P<0.05). Only a few shed LECs were seen in normal control group, 1-minute, 2-minute and 3-minute BSS treatment groups, and BSS immersion combined rinse group.After different time of soaking, there were more shed LECs in distilled water immersion combined rinse group, and the range of LECs shedding increased with the extension of distilled water immersion.There was a significant difference in the shedding percentage of LECs among different groups ( F=123.670, P<0.001). The shedding percentages of LECs at different time points were higher in distilled water immersion groups and distilled water immersion combined rinse groups than in normal control group, and the difference was statistically significant (all at P<0.05). The shedding percentage of LECs increased significantly in distilled water immersion groups with the extension of immersion.Light microscopy showed that the cells were destroyed in 1-minute, 2-minute and 3-minute distilled water treatment groups, and some LECs shed in the 2-minute and 3-minute treatment groups.Transmission electron microscopy showed cell lysis and destruction, suborganelles swelling, disruption of intercellular junctions in 1-minute, 2-minute and 3-minute distilled water treatment groups, loose attachment between cells and capsule in the 2-minute and 3-minute treatment groups, and cell detachment from capsule in the 3-minute treatment group.
ConclusionsDistilled water immersion leads to LECs death in a time-dependent manner, and distilled water immersion combined with rinse can remove LECs on the lens capsule.
张文文,张荣沛,刘亚军,等. 蒸馏水处理对体外人晶状体上皮细胞活性的抑制作用[J]. 中华实验眼科杂志,2023,41(06)：527-535.
DOI:10.3760/cma.j.cn115989-20220731-00355版权归中华医学会所有。
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张文文：设计实验、实施研究、采集数据、统计分析、文章撰写；张荣沛：设计实验、实施研究、采集数据、统计分析；刘亚军、何自芳、张司：实施研究、采集分析数据；解正高：设计实验、指导研究、对文章的知识性内容作批评性审阅
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