携带miRNA质粒的新型聚乙烯亚胺纳米凝胶抑制大鼠Kupffer细胞核因子κB的表达

发布时间：2018-12-15 19:29

【摘要】：目的用新型聚乙烯亚胺纳米凝胶(PEI-NP)携带小RNA(miRNA)质粒沉默大鼠Kupffer细胞(KC)核因子κB(NF-κB)的表达。方法凝胶电泳检测PEI-NP装载miRNA质粒的容量;PEI-NP/miRNA质粒转染RAW264.7细胞后分别采用CCK-8法检测细胞毒性,annexinⅤ-FITC/PI双染色结合流式细胞术检测RAW264.7细胞凋亡和转染率,实时定量PCR(qRT-PCR)检测RAW264.7细胞NF-κB mRNA水平,Western blot法检测RAW264.7细胞NF-κB蛋白水平,透射电镜观察阳离子聚合物在RAW264.7细胞和肝组织KC内的分布;免疫组织化学技术检测KC内NF-κB的表达。结果 PEI-NP/miRNA质粒质量比为20∶1时载荷率接近100%,体外转染RAW264.7细胞48 h,转染率为(33.63±1.94)%。与对照组相比,PEI-NP/miRNA转染的RAW264.7细胞NF-κB蛋白含量显著降低,透射电镜观察到RAW264.7细胞内有大量PEI-NP。肝脏组织透射电镜只在KC内观察到PEI-NP分布;与对照组相比,免疫组织化学染色结果显示,KC内NF-κB蛋白水平明显降低。结论 PEI-NP携带miRNA质粒能成功转染体外培养的巨噬细胞和体内肝脏KC并沉默NF-κB的表达。
[Abstract]:Aim to silence the expression of (KC) nuclear factor 魏 B (NF- 魏 B) in rat Kupffer cells using a novel polyvinyleneimine nanogel (PEI-NP) carrying small RNA (miRNA) plasmid. Methods the capacity of PEI-NP loaded with miRNA plasmid was detected by gel electrophoresis. After transfection of PEI-NP/miRNA plasmid into RAW264.7 cells, cytotoxicity was detected by CCK-8 assay, apoptosis and transfection rate of RAW264.7 cells were detected by annexin 鈪，

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