摘要
目的 探讨Wistar大鼠晶体上皮细胞原代培养方法 ,在形态学方面及晶体蛋白的特异性表达方面 ,对原代和第二代细胞进行鉴定。方法 在无菌条件下 ,取新生Wistar大鼠乳鼠晶体 ,解剖镜下剔除粘附的虹膜及悬韧带 ;室温条件下 ,于终浓度 0 0 8%的胰蛋白酶液中消化 5~ 10min后离心 ,用 199培养液调细胞至适当浓度 ,37℃ 5 %CO2 条件下培养 ,3~ 4d换液 1次 ;待细胞长成连续单层后 ,以 1∶2或 1∶3传代。结果 原代及第二代细胞形态呈典型的上皮细胞表型 ,从第三代起细胞形态发生了较大变化 ,提示细胞已开始分化。以Northern印渍杂交对培养细胞晶体蛋白的特异性表达进行了鉴定 ,发现原代及第二代细胞内仅存在α晶体蛋白mRNA。结论 原代和第二代细胞尚未进入分化状态 ,可用作进一步实验的材料。
Objective To probe the primary culture method of Wistar rats lens epithelial cells,and identify the morph and crystallins expression of primary and secondary generation cells.Methods Wistar rat suckling lenses were taken out under the condition without bacterium,and the attaching iris and ligament were rejected under the anatomical glasses.At room temperature,after the lenses having been digested with 0.08% trypsin for 5~10min,the aqueous phase was centrifugalized at 800~1000rpm.The lens epithelial cells were adjusted to a suitable concentration with medium 199 and were cultured at 37℃ with 5% CO 2,the cell medium was replaced per 3~4 days.After having grown into a continuous monolayer,the cultured cells were passed on from generation to generation by the rate of 1∶2 or 1∶3.Results The morph of the primary and the secondary generation cells showed a typical epithelial cell phenotype,but the cell phenotype changed greatly after the third generation and this suggested that the cells had differentiated.The special expression of crystallins from the cultured cells was identified by Northern blot,α crystallin mRNA was only found in the primary and the secondary generation cells.Conclusion The primary and secondary generation cells had not differentiated yet,and can be used as experiment matcrial.
基金
黑龙江省教委资助项目