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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 investigate the cell-targeting efficiency of Lipidoid, a novel lipid carrier, and to provide a laboratory basis for the treatment of hepatic fibrosis. Methods C57BL / 6 mice were given intragastric administration of CCl4 to establish model of hepatic fibrosis. At the same time, mice were injected with Lipidoid, Lipidoid / siRNA-TIMP-1 through the tail vein. Masson staining was used to observe liver fibrosis of mice in each group. PCR and Western blotting were used to detect the effect of Lipidoid / siRNA-TIMP-1 on TIMP-1 expression. Lipidoid-GFP labeled with green fluorescent protein (GFP) was injected into the tail vein of mice and the liver tissues were harvested for frozen section. Desmin And F4 / 80 staining to evaluate the targeting effect of Lipidoid-GFP on hepatic non-parenchymal cells. The primary hepatic stellate cells SW-HSC isolated from mice and the macrophage cell line RAW264.7 were transfected with Lipidoid -GFP, flow cytometry were detected in the above two cells GFP fluorescence intensity. Results Masson staining of liver tissue showed that the treatment of Lipidoid / siRNA-TIMP-1 in CCl 4 -induced hepatic fibrosis mice model could significantly reduce the deposition of collagen fibers. Real-time quantitative PCR and Western blotting results showed that Lipidoid / siRNA-TIMP-1 can significantly inhibit the expression of TIMP-1 gene; the frozen sections of Desmin and F4 / 80 of liver tissue showed that Lipidoid-GFP was mainly captured by hepatic stellate cells and Kuffer, hepatic stellate cells, The positive rate of Lipidoid-GFP was 13.4% and 43.8%, respectively. The primary hepatic stellate cells SW-HSC and the mouse macrophage cell line RAW264.7 were transfected with Lipidoid-GFP and then detected by flow cytometry. The results showed that the positive rates of SW-HSC and RAW264.7GFP transfected with Lipidoid-GFP were 66.9% and 75.8%, respectively, which were significantly higher than that of the control group. Conclusion The results of mouse experiments show that Lipidoid / siRNA-TIMP-1 can effectively reduce the level of liver TIMP-1 and exert anti-hepatic fibrosis. Lipidoid, a novel lipid carrier, is effective in infecting hepatic nonparenchymal cells (hepatic stellate cells and Kuffer cells).