防腐剂对兔角结膜上皮细胞毒性及玻璃酸钠保护作用的实验研究  

Cellular toxicity of preservatives in corneal and conjunctival epithelial cells and cytoprotective action of sodium hyaluronate

作　　者：张琛[1] 王志昕[1] 邓世靖[1] 孙旭光[1] 

机构地区：[1]首都医科大学附属北京同仁医院同仁眼科中心

出　　处：《眼科新进展》2008年第2期85-89,共5页Recent Advances in Ophthalmology

摘　　要：目的观察玻璃酸钠(sodium hyaluronate,SH)对3种眼科常用防腐剂致兔角膜和结膜上皮细胞毒性的保护作用。方法体外常规方法培养兔角膜和结膜上皮细胞,将不同浓度防腐剂羟苯乙酯(0.3g·L-1、0.6g·L-1和1.2g·L-1)、山梨酸(1g·L-1和2g·L-1)以及苯扎溴铵(0.025g·L-1、0.05g·L-1和0.1g·L-1)分别单独和联合4g·L-1SH与培养细胞作用,并于作用后1h、3h、5h,利用LIVE/DEAD细胞活性/毒性检测方法,观察细胞酯酶及细胞膜透通性变化,计算细胞存活率。结果3种防腐剂与细胞作用不同时间后,角膜和结膜上皮细胞存活率均低于对照组,并且与浓度和作用时间呈依赖关系(P<0.05)。其中0.1g·L-1苯扎溴铵对细胞存活率影响最大,作用1h后,角膜和结膜上皮细胞存活率均为0;0.3g·L-1羟苯乙酯影响最小,作用5h后角膜和结膜上皮细胞存活率分别为(80.89±7.81)%和(55.00±7.81)%;二者相比差异均有统计学意义(P<0.05)。各时间点结膜上皮细胞的存活率均低于角膜上皮细胞(P<0.05)。在各时间点羟苯乙酯-SH组及山梨酸-SH组,角膜和结膜上皮细胞存活率均高于同浓度防腐剂组(P<0.05)。作用3h后,0.05g·L-1浓度苯扎溴铵-SH组与同浓度防腐剂组相比,细胞存活率明显增高(P<0.05),其余浓度的苯扎溴铵-SH组与同浓度防腐剂组相比,细胞存活率差异无统计学意义(P>0.05)。结论3种防腐剂中苯扎溴铵对角结膜的毒性最大,羟苯乙酯毒性最小。3种防腐剂对结膜上皮细胞的毒性作用大于角膜上皮。SH对羟苯乙酯和山梨酸所致细胞毒性均有较好的保护作用,但其保护作用随防腐剂作用时间的增加而减弱。Objective To investigate the cytoprotective action of sodium hyaluronate （SH） on cellular toxicity of corneal and conjunctival epithelial cells induced by 3 commonly used preservatives. Methods Rabbit corneal epithelial cells and conjunctival epithelial cells were cultured in vitro, and then treated with 3 preservatives in different concentrations and preservative-SH,respectively, ethyl hydroxybenzoate （0.3 g · L^- 1 ,0.6 g · L^- 1, 1.2 g · L^- 1 ）, sorbic acid （ 1 g· L^- 1, 2 g· L^- 1）,benzalkonium bromide （0.025 g· L^- 1,0.05 g· L^- 1,0.1 g· L^- 1） and 4 g· L^- 1 SH. After 1 hour, 3 hours and 5 hours, intracellular esterase activity and cell membrane permeability were analyzed with calcein-AM/ethidium-homodimer fluorescent viability stain. Cell survival rates in three groups were evaluated. Results Compared with the control group, the corneal and conjunctival epithelial cell survival rates in preservative groups were lower after treated with 1 hour,3 hours and 5 hours,and there was concentration-time dependency （P〈0.05）. The cell survival rates was lowest in 0.1 g · L^- 1 benzalkonium bromide and the survival rates of corneal and conjunctival epithelial cells were 0 after treated with 1 hour;It was highest in 0.3 g · L^- 1 ethyl hydroxybenzoate and the survival rates of corneal epithelial cells and conjunctival epithelial cells were （80.89 ± 7.81 ）% and（55.00 ± 7.81 ）% ,respectively, after treated with 5 hours, and there was significant difference （ P 〈 0. 05 ）. In all preservative groups,the corneal epithelial cell survival rates were higher than the conjunctival epithelial cell （ P 〈 0.05 ）. The cell survival rates in ethyl hydroxybenzoate- SH and sorbic acid-SH groups were higher than that in preservative group with the same concentration （P〈0.05）.After 3 hours treatment,cell survival rate of 0.05 g · L^- 1 benzalkonium bromide-SH group was higher than that in preservative group with the same concentration （ P 〈 0.05 ） ; there were no differ

关 键 词：防腐剂 羟苯乙酯 山梨酸 苯扎溴铵 玻璃酸钠 角膜上皮细胞 结膜上皮细胞 

分 类 号：R772.2[医药卫生—眼科]