眼科研究 2000年第4期第18卷 实验研究
作者:陈凤华 陈翠真 王津津
单位:陈翠真(100005北京市眼科研究所);陈凤华(现在首都医科大学99博士班);王津津(首都医科大学附属北京同仁医院)
关键词:晶状体上皮细胞;MTT;增殖
摘要 目的通过建立小牛晶状体上皮细胞的传代培养,观察晶状体上皮细胞的离体生长特性。方法组织块接种培养;用计数法及MTT法测细胞生长曲线;绘制细胞分裂指数曲线。结果采用组织块培养法晶状体上皮细胞原代及传代培养生长良好;MTT及细胞计数法所测细胞生长过程基本一致;细胞分裂指数曲线显示细胞在培养第5天达分裂高峰。结论组织块法是晶状体上皮细胞离体培养的较好方法;晶状体上皮细胞具有较好的离体生长能力;MTT比色法是检测晶状体上皮细胞的有效方法。
分类号 R776
Investigation of cultured lens epithelial cells and its growth characteristics
Chen Fenghua Chen Cuizhen Wang Jinjin
(Beijing Institute of Ophthalmology,Beijing 100005)
Abstract ObjectiveTo investigate the growth characteristics of bovine lens epithelial cells by in vitro lens epithelial cells culture.MethodsBovine lens epithelial cells were planted and cultured in condition of 37℃,5%CO2.The cell growth curves were examined by cell counting and MTT colorimetry,exponential curve of cell fission was also measured.ResultsLens epithelial cells grow well during planting culture.Most of the pieces attached to the glass or plastic surface during the first 24-hour’s culture and the signs of cell growth were seen at the edges of the fragments afte 2~3 days culture.These cells lay together with features of epithelial cells,forming a monolayer.Mostly the cells formed a confluent layer in about 2~3 weeks.The cells reached confluence at 7~10 days in the first passage culture.The cell growth Curve measured by counting was similar to what was measured by MTT colorimetry;the fission of lens epithelial cells reached climax after 5 days’ subculture.ConclusionThe planting culture is a good method for lens epithelial cells,which grow and proliferate well during in vitro primary culture and subculture.MTT colorimetry is an effective way to measure cell growth and proliferation.
Key words lens epithelial cells MTT proliferation
晶状体上皮细胞对维持晶状体的正常代谢和功能起着重要作用。晶状体上皮细胞的功能损害、形态改变或异常增殖、移行,与白内障及后囊混浊的发生密切相关[1]。因此,成功地建立晶状体上皮细胞的原代和传代培养方法,对研究白内障和后囊混浊的发生机理及药物防治很有价值。本文探讨了晶状体上皮细胞的原代及传代培养方法,并观察了细胞离体生长的部分生长特性。
1 材料与方法
1.1 主要试剂
DMEM培养基、Trypsin为美国Gibco公司产品,胎牛血清为美国Hyclone公司产品,MTT为美国Sigma公司产品。
1.2 主要仪器
日本Olympus倒置相差显微镜,德国Heraeus CO2细胞培养箱,美国Biorad MR-4500酶标仪。
1.3 牛晶状体上皮细胞的原代培养
新生牛眼购自北京南郊牛奶公司,死后立即无菌取材,放入4℃冰盒中带回实验室。在无菌条件下,剔除肌肉组织,暴露干净的巩膜,浸泡在含320U/ml庆大霉素的生理盐水中1h。用无菌生理盐水冲洗3次,在超净台中切开眼球,经后部取出晶状体,仔细截取晶状体前囊,放入含青霉素200U/ml,链霉素250μg/ml及20%胎牛血清的DMEM培养基中。将前囊皮剪成1mm2大小的碎片,均匀分种于50ml培养瓶中,使培养基刚好盖过瓶底,置37℃,5%CO2,95%空气的培养箱中培养,2天后补加培养基,以后约每3天更换培养基。
1.4 晶状体上皮细胞的传代培养
原代培养的细胞在培养瓶底长满融合后,用0.02%EDTA,0.25%胰酶消化后传代,以1∶ 2或1∶ 3的分种率分种传代。
1.5 用细胞计数法测定细胞生长曲线[2]
收集生长良好的细胞,经胰酶消化,台盼蓝计数,以1×104/ml细胞密度接种于24孔板中,每孔加入含20%胎牛血清的DMEM培养基1ml,于37℃,5%CO2的培养箱中培养,每日消化4孔,用血细胞计数板计数,将4孔细胞计数的平均值绘制成细胞生长曲线。
1.6 用MTT法测定细胞生长曲线[3]
将细胞消化后以1×104/ml细胞密度接种于96孔板,每孔加入含20%胎牛血清的DMEM培养基100μl,于37℃,5%CO2的培养箱中培养,按下述方法每日测4孔:向每孔中加入20μlMTT溶液(5mg/ml),于37℃,5%CO2的培养箱中培养16h,每孔再加100μl20%SDS(十二烷基磺酸钠),50%DMF(二甲基甲酰胺),37℃孵育6~7h,用酶标仪测定其OD值,测定波长为600nm,将每日均值绘制成细胞生长曲线。
1.7 细胞分裂指数计数法[2]
按计数法检测细胞生长曲线的细胞接种方法,将细胞悬液接种于事先放置有小盖玻片的培养瓶内,每日取4个小玻片,用95%酒精固定,吉姆萨或HE染色。对每一时间组的玻片各取细胞多、中、少3个区域各1区,共数1000个细胞,计算出每1000个细胞中的分裂相平均数,绘制成细胞分裂指数曲线图。
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