利用慢病毒载体构建稳定过表达人VEGF_(165)的小鼠巨噬细胞系

Establishment of Mouse Macrophage Cell Line Stably Overexpressing Human VEGF_(165) by Lentiviral Vector

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 construct human VEGFI65 lentiviral vector and establish the high and stable expression in mouse macrophage cell line RAW264.7. Methods： Human VEGF165 was amplified by using PCR, and it was connected to the lentiviral vector pLVX-IRES-ZsGreenl after double enzymatic digestion to generate pLVX-hVEGF^65-IRES-ZsGreenl vector. The recombination vector was confirmed by PCR and DNA sequencing after double enzymatic digestion, then packaged and concentrated. RAW264.7 cells were infected with hVEGF165 lentiviral vector and sorted by flow cytometry twice according to green fluorescence protein ZsGreenl. Human VEGF165 expression on these sorted cells was confirmed by Real time PCR and Western Blot; Human and mouse VEGF165 expression in supernatant was detected separately by ELISA. Results： Enzymatic digestion and sequencing analysis indicated that pLVX-hVEGF165-IRES-ZsGreenl vector was successfully constructed. High purified ZsGreenl-hVEGF165-RAW264.7 cell line was established by flow cytometry. Human VEGF165 mRNA and protein expressed specifically and highly in the lentiviral hVEGF165 infected cell line （P〈0.01）. Human and mouse VEGF165 expression in supematant both was increased significantly in lentiviral hVEGF165 infected cells compared with that in control cells （P〈0.01）. Conclusions： Human VEGF165 lentiviral expression vector was successfully constructed and a stable expression macrophage cell line was established, providing the cell source for the further study of the function, mechanism and application.