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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 a Vasohibin-2 (VASH2) lentiviral vector and obtain a stable HepG2 cell line, and observe its effect on the proliferation of HepG2 cells. Methods: The target gene of VASH2 was obtained from total cellular RNA by reverse transcription and polymerase chain reaction (PCR). NheⅠ and PstⅠ restriction sites were introduced into pTA2 vector and ligated into Lv-CMV-EGFP vector after digestion with NheⅠ and PstⅠ The recombinant plasmids were double-digested and sequenced. VASH2 overexpression vector, pVSV-G and delta8.91 plasmids were co-transfected into 293T cells to obtain lentivirus-infected hepatoma cell line HepG2. Flow cytometry The total RNA and total protein of cells were extracted. The expression of VASH2 was detected by real-time PCR and Western blot. The proliferation of HepG2 cells was detected by MTT assay and cell cycle assay. Results: VASH2 lentivirus overexpression vector was successfully constructed and infected with HepG2 cells stably high expression of VASH2. The proliferation of HepG2 cells stably expressing VASH2 was significantly enhanced (P <0.05). CONCLUSION: VASH2 lentivirus overexpression vector was successfully constructed and found that VASH2 can promote the proliferation of HepG2, which lays a foundation for further study on the function and mechanism of VASH2 in hepatocellular carcinoma.