文章摘要
杨东昌,母小新,刘巧玉,邓 蕾,陈 云,孙倍成.人膜连蛋白A2基因重组慢病毒载体的构建及其在肝癌细胞株中的表达[J].南京医科大学学报,2011,(2):161~165
人膜连蛋白A2基因重组慢病毒载体的构建及其在肝癌细胞株中的表达
Construction of the recombinant lentiviral vector containing human ANXA2 gene and its expression in hepatocellular carcinoma cells
投稿时间:2010-07-05  
DOI:10.7655
中文关键词: 膜连蛋白A2  慢病毒载体  肝癌
英文关键词: annexin A2  lentiviral vector  hepatocellular carcinoma
基金项目:国家自然科学基金青年基金(30901750);南京医科大学科技发展基金重点项目(NMUZ009);教育部“新世纪优秀人才支持计划”(NCET-09-0160);江苏省卫生厅“科教兴卫工程”医学重点学科(RC2007057)
作者单位
杨东昌 南京医科大学第一附属医院肝脏外科,江苏 南京 210029 
母小新 南京医科大学第一附属医院肝脏外科,江苏 南京 210029 
刘巧玉 南京医科大学第一附属医院肝脏外科,江苏 南京 210029 
邓 蕾 南京医科大学第一附属医院肝脏外科,江苏 南京 210029 
陈 云 南京医科大学微生物与免疫教研室,江苏 南京 210029 
孙倍成 南京医科大学第一附属医院肝脏外科,江苏 南京 210029 
摘要点击次数: 1392
全文下载次数: 151
中文摘要:
      目的: 构建重组慢病毒载体pWPT-ANXA2-Flag,并检测其在小鼠肝癌细胞系Hepa 1-6的表达情况?方法:利用DNA重组技术将人膜连蛋白A2(annexin A2,ANXA2)基因克隆入慢病毒表达载体pWPT中,通过酶切?测序验证后,将重组慢病毒载体pWPT-ANXA2-Flag与包装质粒pCMV-dR8.91?pCMV-VSV-G共转染人胚肾上皮细胞株293T,包装重组慢病毒LV-ANXA2-Flag,并感染小鼠肝癌细胞株Hepa 1-6,用RT-PCR检测ANXA2 mRNA转录水平, Western blot法检测ANXA2- Flag蛋白的表达情况,应用流式细胞仪检测细胞周期的变化?结果:经酶切及测序结果证实,构建了重组慢病毒载体pWPT-ANXA2-Flag;RT-PCR及Western blot结果显示慢病毒感染Hepa 1-6细胞后,ANXA2基因能够在细胞内正确的转录?翻译并稳定表达ANXA2蛋白;经过LV-ANXA2-Flag感染后,Hepa 1-6 细胞S期细胞比例明显高于对照细胞?结论:成功建立ANXA2基因慢病毒表达载体pWPT-ANXA2-Flag,包装的慢病毒能够成功感染小鼠肝癌细胞系Hepa 1-6,并使ANXA2基因得以稳定表达,并通过ANXA2促进肿瘤细胞增殖?
英文摘要:
      Objective: To construct the recombinant lentiviral vector containing human annexin A2 (ANXA2) gene and measure the expression level of ANXA2 in hepatocellular cell line of Hepa 1-6. Methods:The ANXA2 fragment was amplified by PCR and subcloned into the lentiviral vector pWPT. The resultant lentivirus were confirmed by PCR, restriction enzyme digestion and DNA sequencing. Recombinant lentivirus (LV-ANXA2-Flag) were produced from 293T by transient calcium-phosphate co-transfection with pWPT-ANXA2-Flag, CMVR8.91and pCMV-VSV-G. Hepa 1-6 cells were infected by LV-ANXA2-Flag lentivirus, and the expression of ANXA2 was confirmed by RT-PCR and Western blotting. Cell cycles were measured via flow cytometry. Results:The recombinant lentiviral vector carried the ANXA2 gene was successfully constructed. RT-PCR and Western blot analysis revealed that the ANXA2 gene can be correct transcripted and translated in Hepa 1-6 cell which infected by LV-ANXA2-Flag. After infected,the Hepa 1-6 cells were higer than control cells in percentage of S phase cells. Conclusion: The recombinant lentiviral vector pWPT-ANXA2-Flag was constructed successfully, it could be used to transfect hepatocellular carcinoma cells. ANXA2 could be stablely expressed and promote the proliferation of Hepa 1-6 cell.
查看全文   查看/发表评论  下载PDF阅读器
关闭