纳米壳聚糖携带miR-29b转染肝星状细胞的实验研究

Study on miR-29b expression plasmid transfection with nanochitosan into hepatic stellate cells

目的:观察纳米壳聚糖携带miR-29b表达质粒对大鼠肝星状细胞的转染效率和治疗效果。方法:制备载miR-29b表达质粒的纳米壳聚糖,并转染大鼠肝星状细胞,荧光显微镜和流式细胞仪观察转染效率,实时荧光定量RT-PCR分析Ⅰ型胶原α1链mRNA的表达水平。结果:紫外分光光度计分析纳米壳聚糖的载药率为91.56%。凝胶电泳阻滞实验显示纳米壳聚糖与质粒结合较好。纳米壳聚糖携带miR-29b表达质粒的转染效率为31.9%,脂质体的转染效率为35.1%,两者差别无统计学意义(P>0.05)。与对照组相比,纳米壳聚糖组和脂质体组Ⅰ型胶原α1链mRNA表达水平明显下降,分别为29%和32%,差别有统计学意义(P<0.05)。结论:纳米壳聚糖载体具有较高的转染效率,携带miR-29b具有较好的抗肝纤维化效果。我们的研究为肝纤维化基因治疗提供了一种新型载体。

Objective：To explore the transfection efficiency and antifibrotic effect of miR -29b expression plas- mid in transfecting cultured rat hepatic stellate cells （HSCs） with nanochitosan as carrier. Methods： Using nanoch- itosan as transfection reagents, miR -29b expression plasmid was transfected into HSCs. Transfection efficiency was observed under fluorescence microscope and flow cytometry. Collagen al （I） mRNA expression were analyzed by re- al -time RT- PCR. Results： UV spectrophotometric analysis indicated that loading efficiency of nanochitosan was 91.56%. Gel retardation experiments showed that nanochitosan and miR- 29b expression plasmid were well com- bined. Using nanochitosan and liposome as transfection reagents, the transfection efficiencies of miR -29b expres- sion plasmid were about 31.9% and 35.1%, respectively. There was no significant difference. Compared with the control group, Collagen otl（I） mRNA expressions in nanochitosan group and liposome group decreased remarkably, about 29% and 32%, respectively. There was statistical significance. Conclusion： Nanochitosan has a relatively high transfection efficiency. MiR- 29b expression plasmid transfected into HSCs using Nanochitosan demonstrated a good antifibrotic effect. The study provided a new vector for gene therapy of liver fibrosis.