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目的:通过构建人血管内皮生长因子165(humanVEGF_(165),hVEGF_(165)的慢病毒载体,感染小鼠单核巨噬细胞RAW264.7,建立稳定高表达人VEGF165的小鼠巨噬细胞系。方法:将聚合酶链反应(PCR)扩增得到的hVEGF_(165)和慢病毒载体pLVX-IRES-ZsGreen1双酶切后连接,构建慢病毒表达载体pLVX-hVEGF_(165)-IRES-ZsGreen1。再经双酶切和测序鉴定后,进行病毒包装及浓缩。将该慢病毒载体感染RAW264.7细胞,利用绿色荧光蛋白ZsGreen1进行2次流式分选。用Realtime-PCR、WesternBlot分别检测各组细胞中hVEGF165的mRNA和蛋白表达;ELISA分别检测细胞上清中人VEGF和小鼠VEGF的含量。结果:酶切及测序结果示慢病毒表达载体pLVX-hVEGF165-IRES-ZsGreen1构建正确;流式分选后得到高纯度的ZsGreen1-hVEGF_(165)-RAW264.7细胞。Realtime-PCR、WesternBlot显示该细胞特异高表达hVEGF_(165)基因和蛋白(P均<0.01)。ELISA显示该细胞分泌人和小鼠VEGF均显著增加(P均<0.01)。结论:成功构建hVEGF_(165)慢病毒表达载体,并建立稳定高表达hVEGF_(165)的小鼠巨噬细胞系。为深入研究该细胞的功能、机制及应用提供充足稳定的细胞来源。
OBJECTIVE: To establish a murine macrophage cell line stably expressing human VEGF165 by infecting mouse monocyte-macrophage RAW264.7 cells with lentiviral vector containing human VEGF165 and hVEGF165. Methods: The lentiviral expression vector pLVX-hVEGF_ (165) -IRES-ZsGreen1 was constructed by digesting hVEGF_ (165) amplified by polymerase chain reaction and lentiviral vector pLVX-IRES-ZsGreen1. After double enzyme digestion and sequencing, the virus was packaged and concentrated.The RAW264.7 cells were infected with lentiviral vector and then were screened by green fluorescent protein ZsGreen1 for 2 times.Real time PCR and Western Blot were used to detect each group The expression of hVEGF165 mRNA and protein were detected by ELISA, and the levels of VEGF and VEGF were detected by ELISA respectively.Results: The recombinant plasmid pLVX-hVEGF165-IRES-ZsGreen1 was constructed correctly by flow cytometry The highly purified ZsGreen1-hVEGF_ (165) -RAW264.7 cells were obtained after selection.Realtime-PCR and Western Blot showed that the cells were highly expressed hVEGF_ (165) gene and protein (all P <0.01) Mouse VEGF were significantly increased (P <0.01) .Conclusion: The hVEGF_ (165) lentiviral vector was successfully constructed and a murine macrophage cell line stably expressing hVEGF_ (165) was established.To study the function, mechanism and application of hVEGF_ (165) source.