牛磺酸修饰巯基化壳聚糖季铵盐载体的合成及其体外shTNF-α pDNA递送效果  

The Synthesis of Taurine-Modified Trimethyl Chitosan-Cysteine and Its Effect to Deliver the shTNF-α pDNAin vitro

作　　者：陈瑾轩 印春华[1] CHEN Jinxuan;YIN Chunhua(School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院

出　　处：《复旦学报（自然科学版）》2020年第1期55-63,共9页Journal of Fudan University：Natural Science

摘　　要：本研究合成了牛磺酸修饰壳聚糖季铵盐(TT)和牛磺酸修饰巯基化壳聚糖季铵盐(TTC)聚合物,再利用TT和TTC包载沉默肿瘤坏死因子-α表达的质粒编码短发夹RNA(shTNF-αpDNA),形成TT-shTNF-αpDNA和TTC-shTNF-αpDNA纳米粒,研究了两种纳米粒理化性质、体外摄取和体外基因沉默效果.结果表明:TT-shTNF-αpDNA和TTC-shTNF-αpDNA粒径在180~305 nm之间,电势在12~20 mV之间,分散性良好;TTC-shTNF-αpDNA pH稳定性能优于TT-shTNF-αpDNA;TT-shTNF-αpDNA和TTC-shTNF-αpDNA均先突释后缓释;TT-shTNF-αpDNA和TTC-shTNF-αpDNA均可显著提高pDNA细胞摄取,TTC-shTNF-αpDNA细胞摄取量显著高于TT-shTNF-αpDNA;TT-shTNF-αpDNA和TTC-shTNF-αpDNA均以能量依赖方式入胞,通过巨胞饮和小窝蛋白介导的途径入胞,TTC-shTNF-αpDNA会通过网格蛋白介导的内吞途径入胞,TT-shTNF-αpDNA则不通过此途径入胞;TT-shTNF-αpDNA和TTC-shTNF-αpDNA均可有效递送shTNF-αpDNA入胞,从而抑制TNF-α蛋白表达,TTC-shTNF-αpDNA组基因沉默效果优于TT-shTNF-αpDNA组.因此,TTC有望作为合适的口服基因递送载体.In this paper,taurine-modified trimethyl chitosan(TT)conjugates and taurine modified trimethyl chitosan-cysteine(TTC)conjugates were prepared,and shTNF-αpDNA was encapsulated to form nanoparticles(TT-shTNF-αpDNA and TTC-shTNF-αpDNA).The physicochemical properties,in vitro release and gene silencing effects of TT-shTNF-αpDNA and TTC-shTNF-αpDNA were studied.The results showed that the particle sizes of NPs ranged from 180 to 305 nm,the Zeta potentials were between 12 to 20 mV with good dispersion.The pH stability of TTC-shTNF-αpDNA was greater than that of TT-shTNF-αpDNA.The release model of TT-shTNF-αpDNA and TTC-shTNF-αpDNA were both sudden release followed by slow release.TT-shTNF-αpDNA and TTC-shTNF-αpDNA could increase the cellular uptake of pDNA significantly,and the uptake amount of TTC-shTNF-αpDNA was greatly higher than that of TT-shTNF-αpDNA.Both TT-shTNF-αpDNA and TTC-shTNF-αpDNA entered the cells in an energy-dependent manner,and were introduced into the cells via the giant cytosolic and caveolin-mediated pathways.TTC-shTNF-αpDNA entered the cells via the clathrin-mediated endocytosis,while TT-shTNF-αpDNA not.TT-shTNF-αpDNA and TTC-shTNF-αpDNA both could effectively deliver shTNF-αpDNA into the cell,thus silencing TNF-αprotein expression,and the gene silencing effect of TTC-shTNF-αpDNA was better than that of TT-shTNF-αpDNA.Therefore,TTC was expected to serve as a suitable oral gene delivery vehicle.

关 键 词：牛磺酸修饰 巯基修饰 shTNF-αpDNA 基因递送 基因沉默 

分 类 号：Q595[生物学—生物化学]