| 贺玉娟 袁世锦 陈星星 李玉红 晏丹.利用慢病毒载体构建稳定过表达人VEGF165的小鼠巨噬细胞系[J].,2015,15(33):6401-6405 |
| 利用慢病毒载体构建稳定过表达人VEGF165的小鼠巨噬细胞系 |
| Establishment of Mouse Macrophage Cell Line Stably OverexpressingHuman VEGF165 by Lentiviral Vector |
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| DOI： |
| 中文关键词: 慢病毒载体 单核巨噬细胞 血管内皮生长因子 稳定过表达 |
| 英文关键词: Lentiviral vector Macrophage Vascular Endothelial Growth Factor Stable overexpression |
| 基金项目:国家自然科学基金项目（81200103）；湖北省卫生和计划生育委员会基金项目（JX6B101） |
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| 中文摘要: |
| 目的：通过构建人血管内皮生长因子165 (human VEGF165, hVEGF165)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hVEGF165 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-hVEGF165-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Real time-PCR、Western
Blot 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-hVEGF165-RAW264.7 细胞。Real time-PCR、Western Blot 显示该细胞特异高表达hVEGF165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hVEGF165慢病毒表达载体，并建立稳定高表达
hVEGF165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。 |
| 英文摘要: |
| ObjectiveTo construct human VEGF165 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-ZsGreen1 after double enzymatic digestion to generate pLVX-hVEGF165-IRES-ZsGreen1 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 ZsGreen1. 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-ZsGreen1 vector was successfully constructed. High purified ZsGreen1-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 supernatant both was increased significantly in lentiviral hVEGF165 infected
cells compared with that in control cells (P<0.01).Conclusion: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. |
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