壳聚糖-磺酸甜菜碱对人肝癌HepG-2细胞的siRNA递送实验研究

Research on siRNA delivery mediated by chitosan-culfobetaine on human hepatoma HepG-2 cells

目的设计合成新型高分子材料壳聚糖-磺酸甜菜碱(CS-DMAAPS)化合物,并考察该材料与siRNA复凝后对人肝癌HepG-2细胞的转染能力。方法采取Michael加成法将含C=C双键的磺酸甜菜碱化合物(DMAAPS)与壳聚糖(CS)偶联接枝;利用核磁共振氢谱进行结构表征;CCK-8检测材料的细胞相容性;荧光显微镜观察siRNA的转染效率;Real-time PCR检测转染siRNA后对Bcl-2 mRNA的沉默效率。结果核磁共振氢谱数据表明:DMAAPS已成功枝接到CS上。荧光显微镜观察结果显示:质量比(m0/mt:CS-DMAAPS/siRNA)为32、16、8、4、2的复合颗粒分别转染人肝癌HepG-2细胞24 h后,转染率分别为33.78%、45.82%、50.98%、69.89%、81.22%。Real-time PCR检测结果显示:质量比为4的CS-DMAAPS/siRNA复合颗粒转染人肝癌HepG-2细胞48 h后,对人肝癌HepG-2细胞的Bcl-2基因沉默效率为26.8%。结论壳聚糖-磺酸甜菜碱(CS-DMAAPS)化合物能实现siRNA对人肝癌HepG-2细胞的转染,且细胞相容性良好,能实现目标基因的部分沉默。

Objective The aim of this study is to synthesize the chitosan-sulfobetaine（ CS-DMAAPS） copolymer as siRNA delivery vector,and investigate the transfection efficiency on HepG-2 cells. Methods The sulfonic acid betaine（ DMAAPS） with C = C was grafted onto chitosan（ CS） by Michael addition method,and the structure of the copolymer was characterized with1H-NMR. The cytocompatibility of CS-DMAAPS was examined through CCK-8 method. The transfection efficiency of CS-DMAAPS was observed with fluorescence microscopy. And the silencing efficiency of Bcl-2 gene expression was detected after siRNA gene transfection.Results NMR data showed that DMAAPS had been successfully connected to CS. The transfection efficiencies of the copolymers with different CS-DMAAPS/siRNA mass ratio（ m0/mt： 32,16,8,4 and 2） were not same. They were 33. 78%,45. 82%,50. 98%,69. 89% and 81. 22%,respectively. Real-time PCR results showed that the silencing efficiency of Bcl-2 gene expression in HepG-2 cells was 26. 8% after transfection by using nanoparticles（ m0/mt： 4）. Conclusion CS-DMAAPS could be a feasible new gene delivery material with good biocompatibility,which could delivery siRNA into HepG-2 to inhibit the expression of Bcl-2 gene.