人角膜上皮细胞体外培养的生长曲线及几种抗生素的影响

The Growth Curve of Cultured Human Corneal Epithelial Cells in Vitro and the Effects of Antibiotics

目的通过人角膜上皮细胞培养研究细胞的生长曲线和抗生素的影响作用。方法组织块接种后在标准条件下培养，传代细胞进行细胞计数和３Ｈ－ＴｄＲ测定，并加入抗生素观察细胞的生长情况。结果角膜上皮细胞呈膜状扩展增殖，形成单层时间为１３～１５天，群体倍增水平５．９４，具有上皮细胞的特征。传代细胞接种后第４天进入指数生长期，３Ｈ－ＴｄＲ渗入增加。结论加入不同浓度的三种抗生素表明庆大霉素超过１．０ｍｇ／ｍｌ，妥布霉素超过２．０ｍｇ／ｍｌ浓度具有明显的细胞毒性作用，氯霉素介于二者之间。

ObjectiveTo study the growth curve of adult human corneal epithelial cells in vitro and the effects of some antibiotics on it.MethodsHuman corneal epithelial cells were planted and cultured in a standard condition.The cells’proliferation and cytotoxicity of three kinds of antibiotics were measured by the growth curve and 3H TdR incorporation.ResultsHuman corneal epithelial cells formed a monolayer in about 13～15 d in primary culture,with features of corneal epithelia in vivo including irregular polygonal shape.The cells reached confluence at 7～8 d in the first passage culture and inoculated cells entered logarithmic growth stage from the 4th d accompanying the increased 3H TdR incorporation.Three kinds of antibiotics,i.e.,gentamicin,tobramycin,chloramphenicol(concentrations of 0.2,0.5,1.0, 2.0 ,5.0mg/ml medium)were added in culture medium after corneal epithelial cells were inoculated for 72 h to investigate the changes in the growth curve, 3H TdR incorporation and the monolayer defect repair time.The proliferation of the cells was not affected by low dose gentamicin(0.2mg/ml),but was inhibited at 0.5mg/ml and even cellular toxicity was shown at 1.0mg/ml or more.The longer the exposure was,the higher the toxicity.However,tobramycin produced no effect on the cells’ growth at a concentration of 1.0mg/ml or less,but obvious cytotoxicity at 2.0mg/ml or more.The effects of chloramphenicol were ranged between the two previously designed drugs.ConclusionCultured human corneal epithelium is a practical and simple method for screening various pharmaceutical agents and corneal epithelial cytotoxicity.