摘要
目的:构建Vasohibin-2(VASH2)慢病毒过表达载体,获得稳定表达的肝癌细胞株HepG2,并观察其对HepG2增殖的影响。方法:通过逆转录、PCR技术从细胞总RNA中获得VASH2目的基因,引入NheⅠ和PstⅠ酶切位点,连接到pTA2 vector中,经NheⅠ和PstⅠ双酶切后再连接到Lv-CMV-EGFP vector中,并对重组后的质粒进行双酶切和测序分析;VASH2过表达载体、pVSV-G及delta8.91 3个质粒共转染293T细胞,获得慢病毒感染肝癌细胞株HepG2,经流式细胞仪分选阳性细胞;提取细胞总RNA和总蛋白,利用real-time PCR和Western blot鉴定稳转细胞株中VASH2的表达,用MTT法和细胞周期法检测各组HepG2的增殖能力。结果:成功构建VASH2慢病毒过表达载体,感染肝癌细胞株HepG2后能够稳定高表达VASH2;稳定高表达VASH2的HepG2增殖能力明显增强(P<0.05)。结论:成功构建了VASH2慢病毒过表达载体,并发现VASH2可以促进HepG2的增殖能力,这为进一步研究VASH2在肝癌中的功能及作用机制奠定了基础。
Objective：To construct lentivirus-mediated expression vector of Vasohibin-2（VASH2） and infect hepatocellular carcinoma cell line HepG2,and study its effect on proliferation.Methods：The full-length VASH2 was obtained from total cellular RNA by reverse transcription and PCR.Two restriction enzyme cutting sites NheⅠand PstⅠwere introduced and linked to pTA2 vector.After restriction digestion of pTA2 vector,the target gene was inserted into the digested expression vector Lv-CMV-EGFP.Then the recombinant was evaluated by restriction digestion and sequencing.The recombinant vector,pVSV-G and delta8.91 were co-transfected into 293T cells to produce packed lentivirus.HepG2 was infected with the recombinant lentivirus and screened by flow cytometry.The expression level of VASH2 mRNA and protein in HepG2 was measured by real-time PCR and Western blot.The proliferation was detected by MTT and cell cycle analysis.Results：The lentivirus stably expressing VASH2 were successfully constructed.And the HepG2 proliferation were increased remarkably after infected with the recombinant lentiviral vector.Conclusion：The recombinant lentiviral vector expression VASH2 was successfully constructed and VASH2 can promote the proliferation of HepG2,which will provide a foundation for further study on functions and mechanisms of VASH2 in hepatocellular carcinoma.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第6期732-737,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
]国家自然科学基金(81172267
81170336)
卫生公益性行业科研专项经费(201202007)资助
