Lipidoid作为新型脂质载体对肝脏非实质细胞的靶向作用实验研究

Study on the targeting effect of a new type of lipid carrier Lipidoid on liver non- parenchymal cells

目的探讨新型脂质载体Lipidoid对肝脏的细胞靶向效率,为抗肝纤维化治疗提供一定实验室依据。方法C57BL/6小鼠分别给予CCl_4灌胃建立肝纤维化模型,同时给予Lipidoid、Lipidoid/siRNA-TIMP-1尾静脉注射,通过Masson染色观察各组小鼠肝脏纤维化情况,并通过实时荧光定量PCR和蛋白免疫印迹检测Lipidoid/siRNA-TIMP-1对TIMP-1表达水平的影响;通过对小鼠尾静脉注射绿色荧光(GFP)标记的Lipidoid-GFP,取其肝组织进行冰冻切片分别进行Desmin及F4/80染色,评价Lipidoid-GFP对肝脏非实质细胞的靶向作用;通过对小鼠分离的原代肝星状细胞SW-HSC、小鼠巨噬细胞系(RAW264.7)转染Lipidoid-GFP,流式细胞仪分别检测上述两种细胞中GFP荧光强度。结果通过对肝组织进行Masson染色发现,在CCl_4诱导的肝纤维化小鼠模型中,给予Lipidoid/siRNA-TIMP-1治疗可显著降低胶原纤维的沉积;实时荧光定量PCR和蛋白免疫印迹结果显示Lipidoid/siRNA-TIMP-1可以显著抑制TIMP-1基因的表达;肝组织冰冻切片Desmin及F4/80染色结果显示,Lipidoid-GFP主要被肝脏肝星状细胞和Kuffer所捕获,肝星状细胞、Kuffer细胞捕获Lipidoid-GFP的阳性率分别为13.4%、43.8%;原代肝星状细胞SW-HSC、小鼠巨噬细胞系(RAW264.7)转染Lipidoid-GFP后经流式细胞仪检测,结果显示转染Lipidoid-GFP的SW-HSC及RAW264.7GFP阳性率分别为66.9%、75.8%,均显著高于对照组。结论小鼠实验结果表明,Lipidoid/siRNA-TIMP-1可有效降低肝脏TIMP-1水平,发挥抗肝纤维化作用。Lipidoid作为一种新型脂质载体可有效感染肝脏非实质细胞(肝星状细胞和Kuffer细胞)。

Objective To explore the efficiency of a new type of lipid carrier targeting on liver cells. Methods C57BL/6 mice were given with carbon tetrachloride（ CCl_4） by gastric lavage in order to establish liver fibrosis models. Lipidoid control or Lipidoid /siRNA- TIMP- 1was administered by tail intravenous injection. The degree of liver fibrosis was evaluated by Masson staining. The levels of mRNA and protein of tissue inhibitor of metalloproteinase 1（ TIMP- 1） were measured by quantitative real- time PCR（ qRT- PCR） and Western blot respectively.The targeting effect of Lipidoid- GFP to non- parenchymal cells of liver was evaluated by Desmin and F4 /80 immunofluorescent staining after administration of Lipidoid- GFP. Primary hepatic stellate cells SW- HSC were isolated from C57 BL /6 mice. Lipidoid- GFP was transfected in primary SW- HSC and murine macrophage cell line- RAW264. 7. The intensity of fluorescence of GFP was evaluated by flow cytometry. Results The Masson staining of liver tissue showed that treatment with Lipidoid / siRNA- TIMP- 1 could significantly alleviate the degree of collagen deposition in CCl_4- induced liver fibrosis. The mRNA and protein levels of TIMP- 1 measured by qRT- PCR and Western blot were markedly decreased. The Desmin and F4 /80 staining of liver tissue showed that Lipidoid- GFP was mainly captured by HSCs and Kupffer＇s cells,and their positive rates were 13. 4% and 43. 8% respectively. Flow cytometric analysis showed that positive rates of GFP in SW- HSC and RAW264. 7 cells transfected with Lipidoid- GFP were 66. 9% and 75. 8% respectively. Conclusion The treatment with Lipidoid / siRNA- TIMP- 1 can dramatically suppress the expression of TIMP- 1 and attenuate the fibrosis of liver in mice. Lipidoid,as a new type of lipid carrier,can effectively transfect the non- parenchymal cells（ HSCs and Kupffer＇s cells） of liver.