牛磺酸对苯扎氯铵损伤大鼠眼角膜内皮细胞无保护作用_葛健.pdf

DOI:10.12300/j.issn.1674-5817.2022.034实验动物与比较医学 Laboratory Animal and Comparative MedicineFeb.2023,43(1)牛磺酸对苯扎氯铵损伤大鼠眼角膜内皮细胞无保护作用葛 健1,孙静芬1,吴永杰2(1.上海交通大学医学院附属瑞金医院眼科,上海 200025;2.上海交通大学医学院附属瑞金医院,上海市高血压研究所,上海市高血压重点实验室,上海 200025)摘要 目的观察2%牛磺酸对苯扎氯铵损伤大鼠眼角膜内皮细胞的保护作用。方法取3只SPF级SD大鼠（共6只眼），每只眼制备6块角膜内皮层和后弹力层组织片，随机分为3组。用组织块培养法培养大鼠眼角膜内皮细胞1 d后，对照组仅加入2%牛磺酸溶液，实验组在加入2%牛磺酸溶液后再注入质量分数分别为0.01%和0.03%的苯扎氯铵溶液。继续培养1、2、4、5、6、8 d后倒置显微镜下观察各组大鼠眼角膜内皮细胞的生长情况，并用Wright染色后光学显微镜下观察内皮细胞形态。结果用0.01%苯扎氯铵和2%牛磺酸处理1 d的实验组大鼠眼角膜组织块边缘出现多角形的内皮细胞，胞体透明；2 d后多角形细胞增多，但没有出现细胞间的融合生长现象；3 d后多角形细胞减少，内皮细胞未见核分裂象；4 d后可见内皮细胞核着色加深，多角形细胞少见；5 d后内皮细胞数量减少，出现细胞体固缩死亡现象。用0.03%苯扎氯铵和2%牛磺酸处理1 d的实验组未观察到内皮细胞生长，胞体稀少散在。仅用2%牛磺酸处理的对照组1 d后组织块边缘出现多角形内皮细胞和少数类内皮多边形细胞；3 d后组织块边缘多角形细胞增多，而且有逐渐融合生长的现象；5 d后内皮细胞个数增多，而且细胞外形多呈六角形；8 d后内皮细胞形成大片状，胞体呈六角形或类圆形，出现细胞核分裂象。左右眼角膜内皮细胞生长均匀，在0.01%和0.03%苯扎氯铵处理的实验组以及对照组内左右眼内皮细胞形态均无明显差别。结论2%牛磺酸对被不同浓度的苯扎氯铵液损伤的大鼠眼角膜内皮细胞无保护作用。关键词 牛磺酸；苯扎氯铵；眼角膜内皮细胞；组织培养；SD大鼠中图分类号 R-332；R779.1 文献标志码 A 文章编号 1674-5817（2023）01-0039-05Taurine Has no Protective Effect on Rat Corneal Endothelial Cells Injured by Benzalkonium ChlorideGE Jian1,SUN Jingfen1,WU Yongjie2(1.Depavtment of Ophthalmology,RuiJin Hospital,Shanghai Jiao Tong University Medical school,Shanghai 200025,China;2.Shanghai Institute of Hypertension,RuiJin Hospital,Shanghai Jiao Tong University Medical School,Shanghai 200025,China)Correspondence to:WU Yongjie,E-mail:ABSTRACT Objective To observe the protective effect of 2%taurine on corneal endothelial cells injured by benzalkonium chloride in rats.Methods Six piece of corneal endodermis and elastic layer tissue slices were prepared from 6 eyes of 3 SPF SD rats and randomly divided into three groups.The corneal endothelial cells of rats were cultured by tissue block culture for 1 day,then the control group cells were added with 2%taurine solution,while the experimental group cells were added with 2%taurine solution and 0.01%or 0.03%benzalkonium chloride solution.After 1,2,4,5,6 and 8 days of continuous culture,the growth of corneal endothelial cells in each group was observed under an inverted microscope,and the morphology of endothelial cells was observed under an optical microscope after Wright staining.Results Treated with 0.01%benzalkonium chloride and 2%taurine for 1 day,polygonal endothelial cells appeared on the edge of corneal tissue mass,and the cells were transparent.After 2 days,the number of 人类疾病动物模型：药学专题 Animal Model of Human Disease:Pharmacology第一作者 葛 健（1964），男，副主任医师，研究方向：眼表疾病的临床和机制研究。E-mail：；孙静芬（1967），女，博士，副主任医师，研究方向：青光眼的临床和视神经保护机制研究。E-mail：通信作者 吴永杰（1967），男，副研究员，研究方向：动物实验和实验动物研究。E-mail：39实验动物与比较医学 Laboratory Animal and Comparative MedicineFeb.2023,43(1)polygonal cells increased,and there was no fusion growth between cells.After 3 days,the number of polygonal cells decreased and no mitotic signs were observed in endothelial cells.After 4 days,the endothelial nuclei were deeply stained and polygonal cells were rare.After 5 days,the number of endothelial cells decreased,and cell body shrinkage and death occurred.In the experimental group treated with 0.03%benzalammonium chloride and 2%taurine for 1 day,no endothelial cell growth was observed and the cells were sparsely-scattered.In control group,polygonal endothelial cells and a few endothelium-like polygon cells appeared at the edge of tissue blocks after 1 day.After 3 days,the number of polygonal cells at the edge of tissue blocks increased,and there was a phenomenon of gradual fusion growth.After 5 days,the number of endothelial cells increased,and the cells were mostly hexagonal.After 8 days,the endothelial cells formed large sheets,the cell bodies were hexagonal or round,and the nuclei were divided.The growth of corneal endothelial cells in the left and right eyes was uniform,and there was no significant difference in the morphology of the left and right eye endothelial cells in the 0.01%and 0.03%benzalammonium chloride treatment groups and the control group.Conclusion 2%taurine had no protective effect on corneal endothelial cells injured by benzalammonium chloride.Key words Taurine;Benzalkonium chloride;Corneal endothelial cells;Tissue culture;SD rats对于眼结膜和角膜疾病及部分内眼疾病，目前最主要的用药治疗方式是眼药水滴用。很大部分眼药水都添加防腐剂，其中苯扎氯铵是被广泛使用的防腐剂之一。苯扎氯铵是一种具有阳离子表面活性的广谱杀菌剂，能改变菌体细胞膜的通透性，使菌体细胞质内的物质外渗，阻碍菌体细胞代谢，从而起到灭菌作用。因为在低浓度时有较好的安全性和有效性，苯扎氯铵一直以低浓度形式被用作滴眼液的防腐剂，至今已有近70年历史1。近年来，临床上广泛使用含有苯扎氯铵的眼科药物，其对角膜和结膜的潜在损害也逐渐引起人们的重视。临床上时常可以观察到眼科药物造成的细点状的角膜上皮炎，或者因长期用药造成的角膜组织糜烂和眼内外眦皮肤溃烂，以及使用眼药水造成的眼结膜长期充血和眼干涩、刺痛等临床表现。研究发现，低浓度的苯扎氯铵对角膜细胞、角膜神经有损伤，对眼表组织有毒性反应2；在结膜囊内滴用含苯扎氯铵的眼药水后，药物和苯扎氯铵都能以渗透压梯度和主动运输方式进入眼球内，从而对球内组织产生作用，其中苯扎氯铵可能会损伤眼组织。因此，如何减少和抑制眼药水中防腐剂对眼组织的损害就显得尤为重要。牛磺酸又称氨基乙磺酸，最早从牛黄中分离得到3。牛磺酸广泛存在于动物的心肌、骨骼肌、神经组织和眼组织中，尤以大脑和心肌中的浓度最高。牛磺酸能抑制神经递质的传导，维持神经细胞膜的稳定性4-5，也能帮助钠、钾、钙在细胞内外运动，并且在眼、血管和胆囊中具有解毒和抗氧化作用6-8。本研究拟通过使用质量分数为2%的牛磺酸，观察其对苯扎氯铵损害的大鼠眼角膜内皮细胞生长有无保护作用。由于临床上常使用的眼药水一般含质量分数为0.003%0.03%的苯扎氯铵，因此本研究设计用0.01%和0.03%的苯扎氯铵溶液损伤大鼠眼角膜，同时加入含2%牛磺酸溶液的大鼠眼角膜内皮细胞培养液，以此观察角膜内皮细胞的生长情况，从而判断牛磺酸是否对日常使用浓度的苯扎氯铵损伤的眼角膜内皮细胞有保护作用。1材料与方法1.1实验动物SPF级SD大鼠3只，10周龄，1雌2雄，体质量250280 g，购自浙江维通利华实验动物技术有限公司SCXY（浙）2019-000