李永平

中山大学中山眼科中心510060

ABSTRACT Objective To establish immortalized human Optic Nerve Glioma cell lines in vitro for the further related research on the differentiation mechanism and regulation of tumorigenesis in human central nervous system. Methods By using LipofectamineTm2000， a gene transfection reagent， plasmid tgLS(+)LoxPHyTKcmvLT containing the simian virus 40 large T-antigene gene ( SV40Tag) was transfected into the primary cultured Optic Nerve Glioma. Colonies were isolated by Hygromycin selection and expanded by more than 50 passages. Investigate the capability of differentiation of the transfected cells with growth curve by methyl thiagolyl totrayolum experiment. The expression of SV40Tag in expanded cell lines was identified by immunocytochemistry stain，transmission electron microscope and RT-PCR. Results Particular anti- Hygromycin cell clones were acquired which can be repassaged for more than 50 passages. The total RNA were isolated from the positive cell clones， and a 372 bp fragment， which was specific for the SV40T antigene gene， was amplified. The transfected cells were expanded to immortalized cell strain. Conclusion Human optic nerve glioma cells could be cultured in vitro， and immortalized through the transfection of retroviral vector containing SV40 LT antigen.

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