TGF-β ⅡR单链抗体／轻链恒定区／鱼精蛋白截短体融合蛋白携带miR-29b抗肝纤维化的研究

Effect of miR-29b transfected by anti-TGF-β ⅡR ScFv/Ck/tP fusion protein on liver fibrosis

目的观察TGF-β ⅡR单链抗体／轻链恒定区／鱼精蛋白截短体融合蛋白（简称新型载体）携带miR-29b对体外培养肝星状细胞（HSC）的转染效率和治疗效果，为今后肝纤维化基因治疗提供新裁体。方法脂质体、新型载体和慢病毒携带miR-29b治疗体外培养的HSC，荧光显微镜和流式细胞术观察转染效率，实时荧光定量RT-PCR和WesternBlot技术分析collagen αl（I）mRNA和蛋白表达水平。结果与对照组比较，慢病毒组的转染效率最高，为70％；其次为新型载体组，为58％；最后为脂质体组，为29％。各载体组collagen αl（I）mRNA表现出不同的下降，与对照组比较，慢病毒组下降70％（t=6．316，P〈0．01），新型载体组为50％（t=4．082，P〈0．01），脂质体组为38％（t=3．014，P〈0．05）。各载体组collagen αl（I）蛋白也表现出不同的下降，慢病毒组下降为59％（t=4．209，P〈0．01）；新型载体组为41％（t=4．033，P〈0．01；）；脂质体组为27％（t=2．842，P〈0．05）。结论笔者构建的新型载体具有较高的转染效率，并且携带miR-29b具有较好的抗肝纤维化效果。

Objective To observe the transfection efficiency and anti-fibrotic effect of miR-29b transfected by anti-TGF-β ⅡR R ScFv/Ck/tP fusion protein （new vector） in hepatic stellate cell （HSC）, and to provide a new vector in gene therapy for liver fibrosis. Methods The liposome vector, new vector, and lentiviral vector were used as transfection reagents to transfect miR-29b into HSC. Transfection efficiency was observed under fluorescence microscope and flow cytometry. Collagen cd （I） mRNA and protein expres- sion in different groups were analyzed by real-time RT-PCR and Western Blot, respectively. Results Com- pared to the control, transfection efficiencies in lentiviral vector, new vector, and liposome vector groups were about 70%, 58% , and 29% , respectively. Collagen α1 （I） mRNA expression in lentiviral vector, new vector, and hposome vector groups was decreased by about 70%, 50%, and 38%, respectively （ （ t =6.316, P 〈0. 01;t =4.082, P 〈0.01;t =3.014, P 〈0.05）. Collagen αl（I） protein expression in lentiviral vector, new vector, and liposome vector groups was decreased by about 59%, 41% , and 27% , respectively （t =4.209, P 〈0.01; t =4.033, P 〈0.01; t =2.842, P 〈0.05）. Conclusions The new vector constructed by us has a high transfection efficiency. MiR-29b transfected by the new vector has a good anti-liver fibrosis effect.