摘要
目的探讨0．1％奥洛他定、0．025％富马酸酮替酚及0．1％吡嘧司特钾对体外培养的人角膜上皮细胞的毒性作用。方法实验研究。使用无血清角膜细胞培养基（K-SFM）进行人角膜上皮细胞的体外培养；将传2～4代的细胞暴露于0．1％奥洛他定（A组）、0．025％富马酸酮替酚（B组）、0．1％吡嘧司特钾（C组）3种滴眼液，以无血清角膜细胞培养基将滴眼液分别稀释到50．0％、20．0％、4．0％、0．8％的浓度，与细胞共同孵育的时间分别为10、30min，2、6、12、24h。滴眼液所致细胞毒性的评估采用光学显微镜和扫描电镜对细胞进行的形态学观察、钙贡绿素和乙锭同型二聚体活性与毒性荧光双染色及四唑氮蓝还原法（MTT Assay）。统计学方法使用单因素方差分析、组间两两比较SNK-q检验。结果（1）形态学改变：于光镜下观察，随着药物浓度的增加、作用时间延长，细胞连接逐渐松解、细胞变圆、脱落。于扫描电镜下观察，3组药物均可减少细胞表面的微绒毛层，细胞膜出现裂孔。滴眼液稀释到20．0％时，A组细胞膜形态的改变较B、C组轻。（2）细胞活性与毒性荧光染色：乙锭同型二聚体阳性提示细胞膜受损、通透性升高。20．0％浓度时A组6、12、24h的胞膜受损细胞的百分比低于B、C组[A组分别为（29．7±2．6）％、（36．9±3．2）％、（51．2±4．3）％；B组分别为（36．5±3．1）％、（48．5±4．3）％、（75．5±3．8）％；C组分别为（37．1±2．2）％、（52．7±3．4）％、（71．1±5．1）％]，差异均有统计学意义（6h时，qA-B=3．27，P=0．031；qA-C=4．31，P=0．023）。钙贡绿素阴性提示细胞质内酯酶活性丧失。A、B、C组药物作用于细胞后，随着浓度和作用时间的提高，细胞内酯酶活性逐渐降低，但3组之间差异无统计学意义[作用24h时A、B、C组无酯酶活性细胞的百分比�
Objective To investigate the cytotoxic effect of three kinds of topical ocular anti-allergic agent, including olopatadine 0. 1% （A group）, ketotifen fumarate 0.025% （B group） and pemirolast postassium 0. 1% （ C group） , on cultured human corneal epithelial cells in vitro. Methods Primary human corneal epithelial cells were cultured with keratinocyte serum-free medium. The cells were exposed to three kinds of topical ocular anti-allergic agent for a period of 10 rain, 30 min, 2 h, 6 h, 12 h and 24 h. Toxicity was examined in three ways. MTI＇ assay was used to quantify cytotoxicity. Cell membrane permeability and intracellular esterase activity were analyzed with live-dead viability staining of fluorescent calmein-AM/ethidium homodimer. The morphologic analysis was performed by light and scanning electron microscopy. Statistical methods adopted one-way ANOVA （analysis of variance ） and Student-Newman-Keuls q test between each group. The P-value of 0. 05 was considered statistically significant. Results （ 1 ） Morphologic changes： The Findings under the light microscopy were demonstrated that cells became round or edematic and detached from dishes after exposure to topical ocular anti-allergic agent. The cellular damage was more severe with longer exposure time and increasing concentration. Likewise, the electron microscopy examination showed reduced microville with longer exposure time and increasing concentration. The cellular changes of 20. 0% olopatadine 0. 1% were reduced when compared to the other agents. （2） Live/dead viability/cytotoxicity assay： Ethidium homodimeris permeates damaged cell memberanes and results in red fluorescence. These results indicated that cell membrane damage caused by 20. 0% olopatadine 0. 1% at 6, 12,24 h was less than those of ketotifen fumarate 0. 025% and pemirolast postassium 0. 1%. The data of A group were （29.7 ±2. 6）%, （36. 9 ±3.2）%, （51.2 ±4. 3）%, B group were （36.5 ±3.1）%, （48. 5± 4.3）%, （75.5±3.8）% and C group were
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2010年第1期43-50,共8页
Chinese Journal of Ophthalmology
关键词
上皮
角膜
上皮细胞
细胞
培养的
毒理学
眼药水
组胺拈抗药
Epithelium,corneal
Epithelial cells
Cells, cultured
Toxicology
Ophthalmic solutions
Histamine antagonists
