维生素A修饰的纳米载体靶向肝星状细胞沉默TLR4基因并抑制其激活的体外实验研究  

Vitamin A-modified nanocarrier for targeted TLR4 siRNA delivery to hepatic stellate cells for anti-fibrotic treatment: an experimental study in vitro

作　　者：陈志伟[1] 韩世松 安泳橙 周智美[1] 陈烨[1] 林立腾 朱康顺 CHEN Zhiwei;HAN Shisong;AN Yongcheng;ZHOU Zhimei;CHEN Ye;LIN Liteng;ZHU Kangshun(Department of Minimally Invasive Intervention,Laboratory of Interventional Radiology,Second Affiliated Hospital of Guangzhou Medical University,Guangzhou,Guangdong Province 510220,China)

机构地区：[1]广州医科大学附属第二医院微创介入科/介入放射学实验室,510220

出　　处：《介入放射学杂志》2020年第10期1008-1015,共8页Journal of Interventional Radiology

基　　金：国家自然科学基金(81873920)。

摘　　要：目的制备由维生素A(VA)修饰的纳米载体聚乙二醇(PEG)-聚乙烯亚胺(PEI),用于靶向肝星状细胞(HSC)(LX-2细胞株)输送Toll样受体(TLR)4小干扰RNA(siRNA)。观察和评估VA-PEGPEI对LX-2细胞转染效率、TLR4/核因子(NF)-κB信号通路和α-平滑肌肌动蛋白(SMA)表达的影响。方法采用细胞计数试剂盒(CCK)-8检测纳米基因药物细胞毒性。通过流式细胞术和荧光显微镜观察纳米药物转染效率。脂多糖(LPS)刺激LX-2细胞,并用不同多聚体[VA-PEG-PEI/TLR4 siRNA(siTLR4)或PEG-PEI/siTLR4]进行转染。蛋白质印迹法(WB)和免疫荧光法分别检测LX-2细胞TLR4/NF-κB信号通路蛋白水平和α-SMA表达。结果高浓度(40μg/mL)VA-PEG-PEI/siTLR4孵育下LX-2细胞仍具有80%以上存活率,表明纳米药物细胞毒性较低。LPS刺激的LX-2细胞TLR4/NF-κB信号通路水平升高,这种作用经VA-PEG-PEI/siTLR4处理后显著减弱。同时,VA-PEG-PEI/siTLR4能有效地使LX-2细胞失活,这可通过降低纤维化标志物α-SMA表达证明。结论VA-PEG-PEI/siTLR4能高效转染siRNA,有效下调LX-2细胞中TLR4/NF-κB信号通路和α-SMA表达。本体外实验研究表明,VA-PEG-PEI/siTLR4具有治疗肝纤维化的巨大潜力。Objective To prepare the polyethylene glycol and polyethyleneimine(PEG-PEI)modified by vitamin A(VA)for targeted TLR4 siRNA delivery to hepatic stellate cells(LX-2 cells),to evaluate the transfection efficiency,the level of TLR4/NF-kappa B signaling pathway and theα-SMA expression in LX-2 cells treated with PEG-PEI-VA.Methods The cytotoxicity of the nanodrug was evaluated by CCK-8 assay.The transfection efficiency of the nanodrug was measured by flow cytometry and fluorescence microscopy.LX2 cells were stimulated by lipopolysaccharide(LPS)and transfected with different polyplexes(VA-PEG-PEI/siTLR4 or PEG-PEI/siTLR4).The protein levels of TLR4/NF-kappa B signaling pathway andα-SMA expression in LX2 cells were respectively assessed by Western blot assay and immunofluorescence.Results LX2 cells were incubated with VA-PEG-PEI/siTLR4 at a high concentration of 40 ug/mL,and at least 80%of them remained viable,indicating that the cytotoxicity of the nanodrug was very low.The level of TLR4/NFkappa B signaling pathway of LX2 cells stimulated by LPS was obviously increased,such an effect was significantly detracted by VA-PEG-PEI/siTLR4 treatment.Moreover,VA-PEG-PEI/siTLR4 could effectively deactivate LX-2 cells as evidenced by the reduced level of fibrotic markerα-SMA.Conclusion VA-PEGPEI/siTLR4 can achieve a highly efficient siRNA transfection,and effectively downregulate the TLR4/NF-kappa B signaling pathway andα-SMA expression in LX-2 cells.This in vitro investigation indicates that VAPEG-PEI/siTLR4 has great potential for the treatment of liver fibrosis.(J Intervent Radiol,2020,29:1008-1015)

关 键 词：肝纤维化 纳米载体 肝星状细胞 TOLL样受体4 小干扰RNA 

分 类 号：R735[医药卫生—肿瘤]