Growth and differentiation of human lens epithelial cells in vitro on matrix.
Growth and differentiation of human lens epithelial cells in vitro on matrix.
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发表时间：
2000-11
影响因子：
4.4
通讯作者：
Eleanor A. Blakely;K. Bjornstad;Polly Y. Chang;M. P. McNamara;Edmund Chang;G. Aragon;Serena P. Lin;GeMing Lui;Jon R. Polansky
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作者：
Eleanor A. Blakely;K. Bjornstad;Polly Y. Chang;M. P. McNamara;Edmund Chang;G. Aragon;Serena P. Lin;GeMing Lui;Jon R. Polansky
目的 表征体外生长的非永生化人晶状体上皮 (HLE) 细胞的生长和成熟。方法 从 18 周产前晶状体建立的 HLE 细胞维持在补充有碱性成纤维细胞生长因子 (FGF-2) 的培养基中的牛角膜内皮 (BCE) 细胞外基质 (ECM) 上。通过核型分析、细胞形态学和生长动力学研究、逆转录聚合酶链式反应 (RT-PCR)、免疫荧光和蛋白质印迹分析来表征培养物的身份、生长和分化。结果 HLE 细胞具有男性、人类二倍体 (2N = 46) 核型。呈指数增长的细胞的群体倍增时间为24小时。培养15天后，细胞形态发生变化，出现明显的透镜体形成。逆转录聚合酶链反应 (RT-PCR) 表明 αA- 和 βB2- 晶状体蛋白、成纤维细胞生长因子受体 1 (FGFR1) 和主要内在蛋白 (MIP26) 在指数生长中表达。蛋白质提取物的 Western 分析显示，随着培养时间的延长，三种免疫学上不同类别的晶状体蛋白（αA-、αB- 和 betaB2-晶状体蛋白）呈阳性表达。通过蛋白质印迹分析，p57（KIP2）（一种已知的终末分化纤维细胞标记物）的表达在指数培养物中可检测到，并且在汇合后水平增加。使用免疫荧光在汇合培养物中检测到 MIP26 和 γ-晶状体蛋白表达，但在指数生长的细胞中未检测到。结论 HLE 细胞在源自 BCE 细胞的 ECM 上可在含有 FGF-2 的培养基中维持长达 4 个月。随着培养时间的推移，细胞表现出晶状体纤维细胞分化的形态特征，并表达晶状体纤维细胞分化的蛋白质标记。该体外模型将有助于研究辐射诱发的白内障发生和其他晶状体毒性研究。
PURPOSE To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.