猿肾病毒40大T抗原基因永生化大鼠神经前体细胞株的构建  

Construction of immortalized rat neural progenitor cell strain by transfection with simian virus 40 large T antigene gene

作　　者：高峰[1] 田玉科[1] 杨辉[1] 安珂[1] 张传汉[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉学教研室,武汉市430030

出　　处：《中华麻醉学杂志》2005年第8期597-600,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助项目(30170905)

摘　　要：目的建立猿肾病毒40大T抗原基因(SV40Tag)永生化大鼠神经前体细胞株,为细胞移植治疗和转基因治疗提供稳定的细胞来源。方法利用脂质体介导的基因转染技术将含有SV40Tag的质粒pCMVSV40T/PUR转染原代培养的新生大鼠神经前体细胞,经嘌呤霉素筛选,阳性克隆扩大培养并连续传代。应用巢蛋白抗体进行细胞鉴定,5%胎牛血清诱导细胞分化后,应用免疫细胞化学法检测其分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern印迹杂交和免疫细胞化学法检测SV40Tag在转染细胞中的表达。结果转染细胞经筛选培养后获得1 个阳性细胞克隆,免疫细胞化学结果显示细胞的巢蛋白和微管相关蛋白2染色为阳性,增殖能力较强。5%胎牛血清可诱导转染细胞分化为微管相关蛋白2阳性和胶质纤维酸性蛋白阳性细胞。Southern印迹杂交结果显示转染细胞基因组中存在SV40Tag cDNA,并可检测到SV40Tag mRNA及其蛋白的表达。转染细胞经扩大培养,命名为永生化神经前体细胞。贴壁培养的神经前体细胞,群体倍增时间为(22.9±2.7)h,传代、冻存和复苏对细胞形态及生长无明显影响。结论成功地构建了SV40Tag永生化的大鼠神经前体细胞株。Objective To establish immortalized neural progenitor cell strain and provide stable cell resource for cell-transplantation and gene therapies. Methods Plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigen gene （SV40Tag） were transfected into the primary cultured neural progenitor cells （NPCs） of newborn rat using lipofectin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Anti-nestin antibodies were used to identify the cultured cells. The specific molecular marker of neurons and astrocytes were detected using immunocytochemistry method to investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by RT-PCR, Southern blot and immunocytochemistry method. Results One anti-puromycin cell clone was obtained, which was microtubule-associated protein-2 （MAP-2） positive cells With the capability of proliferation and could differentiate into MAP-2 or glial fibrillary acidic protein positive cells. The existence of SV40Tag cDNA and the expression of mRNA and protein of SV40Tag were confirmed in transfected cells. The transfected cells were expanded to immortalized cell strain maintained for more than 50 passages, named as immortalized neural progenitor cell （INPC）. INPCs were elliptical or triangular cells with two or three short axons. The population doubling time of INPC was （22.9 ± 2.7）h, subculture, freezing and recovering had no effect on cellular shape and proliferation of INPC. Conclusion Immortalized neural progenitor cell strain was established successfully. It may provide stable cell resource for the basic researches and cell-transplantation therapies with NPC.

关 键 词：猴病毒40 抗原 多瘤病毒转化 干细胞 永生化神经前体细胞 基因永生化 新生大鼠 细胞株 大T抗原 SV40Tag 病毒 

分 类 号：Q813[生物学—生物工程]