CAJ | 학술논문

目的观察TGF-βⅡR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载体.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Western Blot技术分析collagen α(1) (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 α1(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 α1 (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％(f=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.
목적 관찰TGF-βⅡR단련항체/경련항정구/어정단백절단체융합단백(간칭신형재체)휴대miR-29b대체외배양간성상세포(HSC)적전염효솔화치료효과,위금후간섬유화기인치료제공신재체.방법 지질체、신형재체화만병독휴대miR-29b치료체외배양적HSC,형광현미경화류식세포술관찰전염효솔,실시형광정량RT-PCR화Western Blot기술분석collagen α(1) (Ⅰ) mRNA화단백표체수평.결과 여대조조비교,만병독조적전염효솔최고,위70％;기차위신형재체조,위58％;최후위지질체조,위29％.각재체조collagen α1(Ⅰ) mRNA표현출불동적하강,여대조조비교,만병독조하강70％(t=6.316,P＜0.01),신형재체조위50％(t=4.082,P＜0.01),지질체조위38％(t =3.014,P＜0.05).각재체조collagen α1 (Ⅰ)단백야표현출불동적하강,만병독조하강위59％(t=4.209,P＜0.01);신형재체조위41％(t =4.033,P＜0.01;);지질체조위27％(f=2.842,P＜0.05).결론 필자구건적신형재체구유교고적전염효솔,병차휴대miR-29b구유교호적항간섬유화효과.
Objective To observe the transfection efficiency and anti-fibrotic effect of miR-29b transfected by anti-TGF-β Ⅱ 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 α1 (Ⅰ) mRNA and protein expression in different groups were analyzed by real-time RT-PCR and Western Blot,respectively.Results Compared 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 liposome 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 α1(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.