Genomic aberrations and cellular heterogeneity in SV40-immortalized human corneal epithelial cells.
Genomic aberrations and cellular heterogeneity in SV40-immortalized human corneal epithelial cells.
复制标题
DOI：
10.1167/iovs.08-2239
复制
发表时间：
2009-02
影响因子：
4.4
通讯作者：
K. Yamasaki;S. Kawasaki;R. Young;H. Fukuoka;H. Tanioka;Mina Nakatsukasa;A. Quantock;S. Kinoshita-
中科院分区：
文献类型：
--
作者：
K. Yamasaki;S. Kawasaki;R. Young;H. Fukuoka;H. Tanioka;Mina Nakatsukasa;A. Quantock;S. Kinoshita-
目的 猿猴病毒 (SV)40 永生化人角膜上皮 (HCE-T) 细胞已广泛用作人角膜上皮细胞的体外模型。评估该细胞系的基因组畸变和细胞异质性。方法为了定量测量基因组畸变，进行了基于阵列的比较基因组杂交（CGH）分析。为了鉴定细胞异质性，分析了细胞形态、生长动力学、跨上皮电阻和转染/转录效率。针对一些获得或丢失的基因座进行实时 PCR 和染色体荧光原位杂交 (cFISH)，以评估基因组异质性。收集该细胞系的表达序列标签 (EST) 以评估 HCE-T 细胞和正常角膜上皮细胞之间基因表达谱的差异。 Southern印迹分析和反向PCR分析用于确定SV40大T抗原基因(LTAG)的基因组整合位点。结果阵列CGH分析表明HCE-T细胞的基因组内容与正常健康基因组不同。细胞功能测定、实时 PCR 和 cFISH 的结果强烈表明，HCE-T 细胞由大量异质细胞群组成。 SV40 大 T 抗原的基因组整合位点位于 9 号染色体的 p22.1。 结论 结果表明 HCE-T 细胞的基因组内容发生了改变，并且它们由异质细胞群组成。在进行实验或解释使用该细胞系的研究结果时应考虑这一点。
PURPOSE Simian virus (SV)40-immortalized human corneal epithelial (HCE-T) cells have been widely used as an in vitro model of human corneal epithelial cells. The nature of this cell line was assessed for genomic aberrations and cellular heterogeneity. METHODS For the quantitative measurement of genomic aberrations, array-based comparative genomic hybridization (CGH) analysis was performed. For identification of cellular heterogeneity, cell morphology, growth kinetics, transepithelial electrical resistance, and transfection/transcriptional efficiency were analyzed. Real-time PCR and chromosomal fluorescent in situ hybridization (cFISH) against some gained or lost loci were performed, to assess genomic heterogeneity. Expressed sequence tags (ESTs) for this cell line were collected to assess differences in the gene expression profiles between HCE-T cells and normal corneal epithelial cells. Southern blot analysis and inverse PCR analyses were used to determine the genomic integration site of the SV40 large T antigen gene (LTAG). RESULTS Array CGH analysis demonstrated that the genomic content of HCE-T cells is different from the normal healthy genome. The results from cellular functional assays, real-time PCR, and cFISH strongly indicated that HCE-T cells consist of a significant number of heterogeneous cell populations. The genomic integration site of the SV40 large T antigen was at p22.1 of chromosome 9. CONCLUSIONS The results indicate that HCE-T cells have an altered genomic content and that they are composed of heterogeneous cell populations. This should be considered when conducting experiments or interpreting the results of studies that use this cell line.