摘要
目的应用RNA干扰技术沉默RAW264.7小鼠巨噬细胞中电压门控性钠通道(VGSCs/NaVs)α亚单位NaV1.9基因,建立稳定干扰细胞株,并观察其对细胞增殖活性、吞噬能力和迁移功能的影响。方法通过LipofectamineTM2000将短发夹RNA(shRNA)干扰质粒转染至RAW264.7细胞,经G418筛选获得稳定细胞株。用实时定量聚合酶链反应(RT-PCR)鉴定干扰效率,CCK-8法测定稳定细胞株的增殖活性,流式细胞术检测细胞的细胞周期及吞噬能力,Transwell小室法检测细胞的迁移功能。结果成功建立稳定干扰NaV1.9的细胞株,RT-PCR法鉴定干扰效率达80%。CCK-8法及流式细胞术结果显示,降低NaV1.9的表达使细胞增殖活性下降(P<0.05);流式细胞术结果显示,抑制NaV1.9的表达使细胞吞噬能力降低(P<0.05);Transwell小室法结果显示,干扰NaV1.9的表达使细胞迁移能力减弱(P<0.05)。结论建立了稳定干扰NaV1.9的RAW264.7细胞株,下调NaV1.9的表达使巨噬细胞增殖活性、吞噬能力和迁移功能显著降低。
Objective To establish the cell line with stable voltage-gated sodium channels （VGSCs/NaVs） cx subunit NaVI. 9 gene silencing through RNA interference （RNAi） in murine RAW264.7 macrophages, and to investigate prolifera- tion, phagocytosis and migration in this cell line. Methods The stable NaVI. 9-deficient cell line was generated by selection in G418 after the transfection of short hairpin （shRNA） plasmid with LipofectamineTM2000. RNAi efficiency was qualified by RT-PCR; proliferation ability was measured by CCK-8 assay; cell cycle and phagocytic ability were analyzed by flow cytome- try; and migrating ability was detected by Transwell migration assay. Results Stable NaVI. 9-deficient cell line was estab- lished and the expression of NaVI. 9 was reduced by 80%. CCK-8 assay and flow cytometry showed that the proliferation of the NaVl. 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 NaVl. 9-deficient cells we successfully constructed, proliferation, phagocytosis and migration were obviously inhibited.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第3期225-228,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81070121
81170238)
天津市自然科学基金(09ZCZDSF04200
11JCYBJC12000)
