[NaV1.9基因沉默对RAW264.7细胞增殖、吞噬作用及迁移的抑制作用]
[Inhibitory effect of NaV1.9 gene silencing on proliferation, phagocytosis and migration in RAW264.7 cells].
作者信息
Luo Yuechen, Zhou Xin, Ji Wenjie, Sun Haiying, Lu Ruiyi, Jiang Tiemin, Li Yuming
机构信息
Tianjin Medical University, Tianjin, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Mar;29(3):225-8.
OBJECTIVE
To establish the cell line with stable voltage-gated sodium channels (VGSCs/NaVs) α subunit NaV1.9 gene silencing through RNA interference (RNAi) in murine RAW264.7 macrophages, and to investigate proliferation, phagocytosis and migration in this cell line.
METHODS
The stable NaV1.9-deficient cell line was generated by selection in G418 after the transfection of short hairpin (shRNA) plasmid with Lipofectamine TM2000. RNAi efficiency was qualified by RT-PCR; proliferation ability was measured by CCK-8 assay; cell cycle and phagocytic ability were analyzed by flow cytometry; and migrating ability was detected by Transwell migration assay.
RESULTS
Stable NaV1.9-deficient cell line was established and the expression of NaV1.9 was reduced by 80%. CCK-8 assay and flow cytometry showed that the proliferation of the NaV1.9-deficient cell line was inhibited (P<0.05). Flow cytometry revealed that phagocytic ability was reduced in the cell line (P<0.05). Transwell migration assay demonstrated that migrating ability was depressed in the cell line (P<0.05).
CONCLUSION
In the stable NaV1.9-deficient cells we successfully constructed, proliferation, phagocytosis and migration were obviously inhibited.
目的
通过RNA干扰（RNAi）在小鼠RAW264.7巨噬细胞中建立稳定沉默电压门控钠通道（VGSCs/NaVs）α亚基NaV1.9基因的细胞系，并研究该细胞系的增殖、吞噬和迁移情况。
方法
用Lipofectamine TM2000转染短发夹（shRNA）质粒后，通过G418筛选产生稳定的NaV1.9缺陷细胞系。通过RT-PCR鉴定RNAi效率；用CCK-8法检测增殖能力；用流式细胞术分析细胞周期和吞噬能力；用Transwell迁移试验检测迁移能力。
结果
成功建立了稳定的NaV1.9缺陷细胞系，NaV1.9的表达降低了80%。CCK-8法和流式细胞术显示，NaV1.9缺陷细胞系的增殖受到抑制（P<0.05）。流式细胞术显示，该细胞系的吞噬能力降低（P<0.05）。Transwell迁移试验表明，该细胞系的迁移能力下降（P<0.05）。
结论
在我们成功构建的稳定的NaV1.9缺陷细胞中，增殖、吞噬和迁移明显受到抑制。
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