阳离子化白及多糖作为非病毒基因载体的初步研究  

Preliminary study on cationic Bletilla striata polysaccharide as a non viral gene vector

作　　者：王小旭 杜昊洋 姜美如 林锋 WANG Xiao-xu;DU Hao-yang;JIANG Mei-ru;LIN Feng(Department of Organic Chemistry,College of Pharmacy,Harbin Medical University,Harbin 150081)

机构地区：[1]哈尔滨医科大学药学院有机化学教研室,哈尔滨150081

出　　处：《中南药学》2023年第3期612-617,共6页Central South Pharmacy

基　　金：黑龙江省科学基金项目(No.LH2020H026);2021年哈尔滨医科大学大学生创新创业训练计划项目(No.202110226159)。

摘　　要：目的 构建高效低毒的非病毒基因载体,负载组成型光形态发生因子9信号复合体(CSN)的第六亚基CSN6的siRNA干扰片段,考察其对人肝癌细胞的生物学作用。方法 利用白及多糖和聚乙烯亚胺合成阳离子聚合物,其结构经红外光谱、核磁共振、Zeta电位和粒径进一步表征确证;利用其负载DNA及siRNA进行凝胶阻滞实验检测其对上述分子的结合和保护能力;利用MTT法检测其在不同细胞中的细胞毒性;利用阳离子聚合物将荧光标记的siRNA转染至人肝癌细胞中,荧光显微镜下观察;将聚合物与CSN6 siRNA干扰片段的复合物转染至人肝癌细胞,并利用Western blot法进一步对CSN6 siRNA的干扰效率进行检测;抑制CSN6基因的表达,采用MTT法和克隆形成实验对人肝癌细胞增殖作用的影响进行检测。结果红外光谱、核磁共振氢谱、Zeta电位和粒径检测结果显示,白及多糖接枝聚乙烯亚胺载体构建成功;凝胶阻滞结果显示,载体能够成功与核酸分子相结合;Western blot结果显示,将CSN6 siRNA干扰片段转染至人肝癌细胞后,能够有效下调细胞中CSN6基因的蛋白表达水平;MTT和克隆形成实验结果显示,抑制CSN6基因的表达能够抑制人肝癌细胞增殖。结论 利用白及多糖接枝聚乙烯亚胺构建了一种高效低毒的非病毒基因载体,利用其负载CSN6 siRNA干扰片段能够有效抑制人肝癌细胞的增殖作用。Objective To construct a non viral gene vector with high efficiency and low toxicity,load the siRNA interference fragment of CSN6,the sixth subunit of the constitutive light morphogenetic factor 9 signal complex(CSN),and determine its biological effect on human hepatoma cells.Methods The cationic polymer was synthesized from polysaccharides and polyethyleneimine,whose structure was further characterized by IR,NMR,Zeta potential and particle size.Gel retardation experiments were conducted with DNA and siRNA loaded on it to test its binding and protective ability against the above molecules.MTT assay was used to detect its cytotoxicity in different cells.Fluorescently labeled siRNA was transfected into human hepatoma cells with cationic polymers and observed witha fluorescence microscope.The complex of the polymer and CSN6 siRNA interference fragments was transfected into human hepatoma cells,and the interference efficiency of CSN6 siRNA was further detected by Western blot.The expression of CSN6 gene was inhibited and the effect on the proliferation of human hepatoma cells was detected by MTT assay and clonogenic assay.Results The IR,1 H NMR,Zeta potential and particle size showed that the polyvinylimine grafted with polysaccharide was successfully constructed.The gel retardation showed that the carrier successfully combined with nucleic acid molecules. Western blot showed that the CSN6 siRNA interference fragment transfected into human hepatoma cells effectively knocked down the protein expression level of CSN6 gene in the cells. MTT assay and clonogenic assay showed that inhibiting the expression of CSN6 also inhibited the proliferation of human hepatoma cells. Conclusion A non viral gene vector with high efficiency and low toxicity is constructed by grafting polyvinylimine onto leucorrhea polysaccharides. CSN6 siRNA interference fragments can effectively inhibit the proliferation of human hepatoma cells.

关 键 词：白及多糖 聚乙烯亚胺 CSN6 siRNA 基因载体 

分 类 号：R285[医药卫生—中药学] TQ460.1[医药卫生—中医学]