摘要
目的 用端粒酶催化亚单位(hTERT)和猿猴病毒 40大T抗原(SV40大T)异位表达建立永生化人脐静脉内皮细胞系。方法 构建含有hTERT和SV40大TcDNA片段的逆转录病毒载体,转染人原代脐静脉内皮细胞,连续传代培养。通过细胞免疫组织化学观察、Westernblot分析hTERT和SV40大T抗原表达、PCR ELISA检测端粒酶活性、ELISA分析内皮特异性E 选择素和RT PCR测定内皮细胞脂肪酶进行细胞形态学和功能学鉴定。结果 转染的人脐静脉内皮细胞为扁平多角形,呈单层铺路石状镶嵌排列。有Ⅷ因子、SV40大T、hTERT表达、E 选择素和内皮细胞脂肪酶表达阳性。转染细胞中端粒酶活性经测定在第 12代时为 0 36, 在第 50代时为 0 38, 而对照的原代细胞在第 1代时为 1 12,第 3代时仅为 0 06。结论 导入外源性端粒酶催化亚单位和SV40大T抗原能使人脐静脉内皮细胞永生化,保持内皮细胞性状不改变。
Objective To immortalize human umbilical vein endothelial cells(HUVECs) by ectopic expression of the telomerase reverse transcriptase enzyme (hTERT), and by Simian Virus 40 Large T (SV40LT) antigen without malignant transformation Methods Two different retroviruses that contained hTERT/SV40LT cDNA fragment and drug resistance gene were constructed, and were used to transfect normal primary HUVECs The transfected cells were screened with 500 μg/ml G418 and 4 μg/ml puromycin Drug resistance cell clones were selected 3 days after transfection and cultured for further studies An under inverted microscope and a scanning electron microscope were used to observe the morphology and growth of the cells The expression of Ⅷ factor and transfected DNA fragments were detected for identification of the endothelial origin and successful transfection And the expression of E selectin and endothelial lipase with or without the stimulus of TNF α were also assayed to analyze the biological activity of the transfected cells Results The cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping Immunocytochemical staining showed the existence of Ⅷ factor SV40LT/hTERT antigen expressed by the transfected cells was detected,while the contrasts had non expression Telomerase activity of the cell was detected in the transfected cells, which was 0 36 at 12 th passage and 0 38 at 50 th passage However, the activity in the normal HUVECs was 1 12 at the first passage and 0 06 at the third passage assayed by PCR ELISA Both E selectin and endothelial lipase were all specific in endothelial cells The expressions of these two were also detected And the expression of E selectin can be up regulated with the stimulus of TNF α, while the expression of endothelial lipase was not unregulated significantly Conclusion Ectopic expression of hTERT and SV40LT can effectively immortalize HUVECs without tumorigenesis
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2005年第2期166-169,共4页
Chinese Journal of Cardiology
基金
浙江省科技厅资助项目(021110240)
