【摘要】 目的：建立鼠晶状体上皮细胞体外培养的模型。方法：应用组织块贴片法和酶逐步消化法对10～14d的SD大鼠晶状体上皮细胞进行体外培养,在相差显微镜下观察其生长规律。结果：组织块贴片法在加入培养基4～5d后见细胞生长，2wk细胞融合。而酶逐步消化法在加入培养基后7d左右见细胞贴壁，2wk左右见细胞融合。结论：鼠晶状体上皮细胞体外培养较困难，本试验采用酶逐步消化方法和组织块贴片法。成功地建立了鼠晶状体上皮细胞体外培养的模型,为研究后发性白内障发病机制提供了基础。
【关键词】 晶状体上皮细胞；细胞培养；鼠
Culture systemin vitro of rat cataract lens epithelial cells
QuanChen Xiong, LuYan Zhang, YuPing Zheng, XiaoHua Wang, ZhaoHui Feng
Department of Ophthalmology, the Second Affiliated Hospital of Xian Jiaotong University, Xian 710004, Shaanxi Province, China
Correspondence to: QuanChen Xiong. Department of Ophthalmology, the Second Affiliated Hospital of Xian Jiaotong University, Xian 710004, Shaanxi Province, China. [email protected]
AbstractAIM: To establish a culture system in vitro of rat cataract lens epithelial cells.METHODS: Cataract lens epithelial cells of postnatal 1014 days SD rat were cultured by tissue block adherence and gradual enzymatic digestion. Cultured cells growth was observed by phasecontrast microscopy.RESULTS: In the culture of rat lens epithelial cells by tissue block explant, cells attached and started growing from the 4th5th day, and cell fusion occurred during 2 weeks. In the culture by enzymatic digestion, cells didnt attach until the 7thday, while cell fusion was found in 2 weeks.CONCLUSION: Rat cataract lens epithelial cells are delicate and difficult to be cultured. We have successfully set up the culture system of rat lens epithelial cells, which might provide a methodological foundation for research on pathogenesis of posterior capsule opacification.
KEYWORDS: cataract lens epithelial cells; cell culture; rat
0引言
白内障是常见致盲眼病。 后发性白内障是白内障囊外摘除术后的主要远期并发症,是白内障手术后视力下降的主要原因,术后2mo～5a的该病发生率成人约为50%，儿童约为100%[1]。研究表明晶状体上皮细胞的异常增殖和分化与后发性白内障的发生密切相关[2]。晶状体上皮细胞的体外培养是观察晶状体上皮细胞生长的形态、功能变化的基础,也是为进一步从分子水平研究晶状体上皮细胞的生物学特性提供基础,从而为进一步研究后发性白内障的预防和治疗提供细胞学基础。
1材料和方法
1.1材料
DMEM/F12培养基（Gibco）, 胰蛋白酶（Gibco）, 胎牛血清（Hyclon）,CO2恒温培养箱,倒置相差显微镜和照相系统(Nikon )晶状体上皮细胞取自2wk大小的清洁级SD大鼠，动物由西安交通大学医学院第二医院动物场提供。
1.2方法
1.2.1组织块贴片法
将晶状体囊膜片用手术剪刀剪成约1×1mm2小块,然后将组织小块分散贴到经多聚赖氨酸处理过的培养瓶中,再将培养瓶倒置使有组织块一面朝上,加入DMEM/F12培养基(内含有200mL/L胎牛血清,100kU/L青霉素,100mg/L链霉素)，放入50mL/L CO2培养箱内37℃培养2～4h后将培养瓶翻过来,使组织块浸入培养基内,继续放入50mL/L CO2培养箱内37℃培养。定时观察细胞的生长情况。
1.2.2酶逐步消化法
将晶状体囊膜片用1.25g/L胰蛋白酶消化3min,,在倒置显微镜下观察,确定有细胞游离出来，收集上清，沉淀再次消化,收集上清,共消化3次,收集各次消化的上清,离心800r/min 8min,弃上清,沉淀中加入DMEM/F12培养基(内含有200mL/L胎牛血清,100kU/L青霉素,100mg/L链霉素)混匀,接种于经多聚赖氨酸处理过25mL培养瓶中,37℃CO2培养箱中培养。定时观察细胞的生长情况。
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