人端粒酶催化亚基和SV40大T抗原永生化猪脐静脉血管内皮细胞

为建立稳定的猪血管内皮细胞系,用于猪瘟病毒(CSFV)的致病机理研究提供理想的细胞模型,本研究利用逆转录病毒分别转导人端粒酶催化亚基(hTERT)基因或SV40大T抗原(SV40 LT)基因进入猪脐静脉血管内皮细胞(SUVEC),通过G418或嘌呤霉素筛选阳性克隆,并进行细胞传代研究和细胞形态学及表型鉴定。分别获得了两种方法建立的永生化猪血管内皮细胞系SUVEC-hT和SUVEC-ST。两种细胞系均呈铺路石样单层排列生长,具有高度的增殖活性,在传代70代后不表现任何衰老细胞的特征,并保持接触生长抑制。SUVEC-hT细胞系持续表达hTERT、CD31、CD34和von Willebrand factor,并能摄取Dil标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)。SUVEC-ST细胞系持续表达SV40 LT、CD31和CD34,并能摄取Dil-Ac-LDL。两个细胞系均保持正常的核型并对CSFV敏感。结果表明通过表达外源的hTERT或SV40 LT,SUVEC已被永生化,并且保留了血管内皮细胞的主要生物学特征。

Immortalization of swine umbilical vein endothelial cells by hTERT or SV40 LT

To establish stable swine vascular endothelial cell lines as models for the study of pathogenesis of classical swine fever virus(CSFV),primary swine umbilical vein endothelial cells(SUVEC) were transduced respectively with hTERT and SV40 LT gene using retrovirus system.Positive clones were picked following the selection using G418 and puromycin respectively from hTERT and SV40 LT transduced SUVEC monolayers,followed by continuous passage and morphological and phenotypical characterization.Two immortalized swine endothelial cell lines,SUVEC-hT(carrying hTERT gene) and SUVEC-ST(carrying SV40 LT gene),were generated.They grew as a monolayer with cobblestone morphology,had high proliferation capacity with no signs of senescence even after 70 passages,and maintained contact inhibition.SUVEC-hT cell line expressed constantly hTERT,CD31,CD34 and von Willebrand factor,and displayed the uptake of dil-acetylated low density lipoprotein(Dil-Ac-LDL).SUVEC-ST cell line expressed constantly SV40 LT,CD31 and CD34,and displayed the uptake of Dil-Ac-LDL.They retained normal karyotype and were susceptive to the infection with CSFV.The results indicated immortalized SUVEC lines have been successfully established by ectopic expression of hTERT and SV40 LT,which retained endothelial cell characteristics.