Human lens epithelial cell line
Human lens epithelial cell line
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DOI：
10.1006/exer.1998.0551
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发表时间：
1998-11-01
影响因子：
3.4
通讯作者：
Reddy, VN
中科院分区：
文献类型：
--
作者：
Ibaraki, N;Chen, SC;Reddy, VN
虽然原代培养的人晶状体上皮细胞（HLE）提供了重要的ir；关于上皮在正常晶状体和白内障形成中的作用，缺乏细胞系阻碍了对这些细胞的代谢和分子生物学的广泛研究。因此，我们开发了一种HLE细胞系。用含有SV40大T抗原的质粒载体DNA转染HLE细胞原代培养物。对永生化细胞的形态、生长速度、核型以及结晶蛋白、醛糖还原酶和其他酶的表达进行了表征。永生细胞的一个克隆SRA 01/04形成了一个单层细胞，持续生长超过130代。同工酶表型显示SRA 01/04为人类来源，染色体数量在次四倍体范围内。Western blot分析显示，细胞表达极低水平的晶体蛋白(α （a)和β (B2)）和醛糖还原酶。通过逆转录聚合酶链反应（RT-PCR）检测α和β结晶蛋白在传代早期和晚期的信使RNA （mRNA）。对HLE细胞系和原代培养中α (A)和β (B2)结晶蛋白的PCR产物进行序列分析，发现与已发表的人α (A)和β (B2)序列100%匹配。这些特征在细胞系的早期和晚期传代中都没有改变。这是在已建立的人类细胞系中首次报道α (A)和α (A)和β (B2) mRNA转录本的存在。这一新的HLE细胞系使得对上皮在晶状体和白内障形成中的作用进行许多未来的研究成为可能。(C) 1998学术出版社。
Although primary cultures of human lens epithelial (HLE) cells provide important ir;formation concerning the role of epithelium in normal lens and cataract formation, the lack of a cell line precludes a broad range of studies on the metabolism and molecular biology of these cells. We have, therefore : developed an HLE cell line. Primary cultures of HLE cells were transfected with plasmid vector DNA containing a large T antigen of SV40. The immortalized cells were characterized with regard to morphology, growth rate, karyotype, and expression of crystallins, aldose reductase, and other enzymes. A single clone of the immortalized cells, SRA 01/04, formed a monolayer and grew constantly over 130 passages. Isozyme phenotype showed that SRA 01/04 was of human origin, and the chromosome counts were in the hypotetraploid range. Western blot analysis showed that the cells expressed a very low level of crystallins (alpha(A) and beta(B2)) and aldose reductase. Messenger RNA (mRNA) for both alpha and beta crystallins was detected by reverse transcription polymerase chain reaction (RT-PCR) in both early and late passages. Sequence analysis of the PCR products, corresponding to alpha(A) and beta(B2) crystallins in the cell line and in primary cultures of HLE, revealed a 100% match with published human alpha(A) and beta(B2) sequences. These characteristics were unchanged in the cell line in early and late passages. This is the first report of the presence of alpha(A) and transcripts of mRNA for both alpha(A) and beta(B2) in an established human cell line. This new HLE cell line makes it possible to undertake many future studies on the role of epithelium in lens and cataract formation. (C) 1998 Academic Press.