白内障 1922 PO0032
氯苯甲烷铵和噻吗心胺对人晶状体上皮细胞损伤作用的比较研究（Comparing the effection of Benzalkonium Chloride and Timolol in Suffering Human Lens Epithelial Cell）
项鹂 刘谊
四川省成都市416医院眼科 610051
目的：通过体外培养人晶状体上皮细胞株(SRA01/04)，加入不同浓度的氯苯甲烷铵和噻吗心胺，探讨它们对人晶状体上皮细胞的损伤及刺激细胞分泌炎性介质的作用。
材料和方法：体外培养人晶状体上皮细胞株(SRA01/04)于含10％小牛血清的DMEM培养基中至细胞长满融合，胰酶消化后均匀种在六孔板中。将噻吗心胺和氯苯甲烷铵分别配制为0.5％和0.01％（均为噻吗心胺眼液中所使用浓度），然后将其稀释为不同浓度：噻吗心胺稀释10倍、20倍、30倍、60倍和100倍；氯苯甲烷铵稀释100倍、200倍、300倍、600倍和1000倍。将上述不同稀释倍数的噻吗心胺和氯苯甲烷铵逐一加入六孔板中作用于人晶状体上皮细胞，每种浓度作用3孔细胞，余3孔细胞不加任何药物设为正常对照组。孵育48小时后显微镜下观察细胞形态的改变并离心提取细胞上清液，浓缩冻成干粉状，加少量DMEM培养基将其溶解，再做酶联免疫吸附实验（enzyme-linkedimmunosorbentassay，ELISA）检测上清液中所含白介素-1（interleukin 1，IL-1）和前列腺素E2（prostaglandin E2，PGE2）的浓度并与正常对照组比较，以了解药物对细胞毒性作用的差异；同样，将SRA01/04均匀种在96孔板中，滴加不同稀释倍数的噻吗心胺和氯苯甲烷铵，孵育48小时后用MTT还原测定法（Mono-nuclear cell direc cytotoxicity assay，MTT）检测细胞活性。
结果：实验发现，0.01％的氯苯甲烷铵和0.5％的噻吗心胺作用人晶状体上皮细胞24小时后，前者导致细胞全部死亡，后者尚有部分细胞存活；48小时后两者作用的细胞大部分脱壁；在同样稀释100倍的情况下，氯苯甲烷铵作用的细胞72小时后部分脱壁死亡，而噻吗心胺作用的细胞形态大小正常，且可见细胞分裂增殖。药物作用细胞48小时后提取上清液行ELISA检测发现，与正常对照组相比，细胞分泌PGE2和IL-1的量取决于药物作用的浓度：噻吗心胺在高浓度时对细胞有直接杀伤作用，稀释20倍时对细胞的炎性刺激作用最强（P<0.05），当浓度逐渐降低时，对细胞的毒性作用随之减弱；而氯苯甲烷铵在高浓度时直接导致细胞死亡，随着稀释倍数增加，细胞存活率升高，但对细胞的作用转变为慢性炎性刺激，即使在稀释1000倍的情况下刺激细胞分泌炎性介质的量也明显高于正常对照组（P<0.05）。
结 论：氯苯甲烷铵作为噻吗心胺眼液的防腐剂，是对人晶状体上皮细胞造成损伤并刺激其强烈分泌IL-1和PGE2等炎症介质的主要原因。可推测噻吗心胺眼液的使用与白内障发生之间存在一定联系。
ABSTRACT
Objective To investigate the effects of benzalkonium chloride and timolol maleate with different concentraion on cell’s damage and the induction of the inflammatory mediators by culturing human lens epithelial cells (SRA01/04).
Method Cells from a human lens epithelial cell line(SRA01/04) were cultured in vitro till confluenced. Then, dispersed the cells with trypsogen and transferred the cells into 6-well-plates. The amounts of benzalkonium chloride (0.01%) and timolol(0.5%) used in eyedrops were diluted into different concentrations: benzalkonium chloride were diluted into ×100 to×1000 dilutions while timolol into×10 to×100 dilutions. Then, durgs with different concentrations were added into medium. After 48 hours culturing, cell’s morphological changes were observed and cell-free culture supernatants were collected for detecting prostaglandin E2(PEG2) and interleukin 1 (IL-1) by enzyme-linked immunosorbent assay(ELISA). Thus, we are able to investigate which durg is likely more harmful to human lens epithelial cells. We also cultured the cells in an 96-well-plate, added the drugs with different concentrations into it, and then measured the cell’s activity by MTT after 48 hours.
Results Both the concentrations of benzalkonium chloride and timolol used in eye drops showed an extremely toxicity to the human lens epithelial cells: the former one let the cells completely died after 24 hours culturing, and the latter only part of the cells remained alive. Then, 48 hours later, almost all the cells in those two mediums were detached from the culture and died; Being cultured for 72 hours at the same dilution of×100, we saw part of the cells in medium with benzalkonium chloride died, while with timolol still alive--the cell shape is normal and even proliferation and elongation could be obsevered; Comparing with the control gourp, we detected the secretions of PEG2 and IL-1 by ELISA after 48 hours culturing, and the data shows that: the amounts of those two inflammatory mediators depended on the dose of the drugs: timolol with high concentration would kill the cells directely, and turned into harmless when it was diluted. At the dilution of ×20, it showed the strongest stimulation to the cells(P<0.05); Benzalkonium chloride, on the other hand, could not only kill the cells at the high concentration, but also make chronic inflammatory stimulate at low concentration, especially when it was diluted at ×1000 (P<0.05).
Conclusions As a result, benzalkonium chloride, a preservative in eyedrops containing in timolol, should be the agent most damaging to the human lens epithelial cells, and strongly stimulates the cells to secrete the inflammatory mediators. This may indicates the relationship between timolol and cataract, especially to patients who have to chose timolol in controlling intraocular pressure for a long term.
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