【作者】 吉午阳；

【导师】 陈玉祥；

【作者基本信息】 中南大学 ， 药剂学， 2009， 硕士

【摘要】 目的:慢性乙型肝炎（Chronic Hepatitis B,CHB）是一种严重危害人类健康的疾病,目前缺乏有效的药物对其进行治疗。由于它主要通过病毒基因表达引起人体免疫损伤而致病,近年来人们开始了CHB基因治疗研究,纳米载体是基因治疗载体的一种,它具有病毒载体所不具备的优点。本研究旨在研制荧光硅纳米颗粒,探讨其做为一种新型纳米基因载体的可行性和其自发荧光做为一种生物标志物的易于检测性,研究其携带特异性10-23脱氧核酶（10-23Deoxyribozyme,10-23DRz）对HBsAg和HBeAg表达的抑制作用,在细胞和分子水平探讨硅纳米-10-23DRz复合物治疗CHB的可能性。第一章荧光硅纳米的制备及生物学特性方法:微乳液法制备荧光硅纳米颗粒（fluorescent silicananoparticles,FSNPs）,用NaCl对其进行表面修饰。制备的颗粒经干燥后,粒径仪观察其粒径,电位分析仪检测纳米颗粒表面Zeta电位值。将FSNPs溶入生理盐水中,通过尾静脉注入SD大鼠体内。用上下法（up-and-down procedure,UDP）检测其对SD大鼠的急性毒性,并在荧光显微镜下观察其在SD大鼠主要脏器的分布情况。结果:在W/O型微乳液体系中制备出来了粒径在32nm、大小均匀、分散性好、Zeta电位为+53.9mv的硅纳米。在荧光显微镜下此纳米呈红色荧光,各大脏器均有分布,特别在脑和前列腺中有大量分布,说明它可以很好地透过血脑屏障和血前列腺屏障。用上下法估算出此硅纳米的LD₅₀大于2000mg/kg。第二章荧光硅纳米10-23DRz颗粒对HBV的抑制作用方法:针对HBV的S基因和C基因设计特异性的10-23 DRz,10-23DRzHBVs和10-23DRzHBVc。将10-23 DRz与修饰过的硅纳米按一定比例混合,得到复合物后,用15%变性聚丙烯酰胺凝胶电泳检测结合效果。将硅纳米DNA复合物加入胎牛血清中,用15%变性聚丙烯酰胺凝胶电泳检测硅纳米对DNA的保护作用。将硅纳米DNA复合物转染HepG2.2.15细胞,用ELISA法检测其对HBsAg和HBeAg的抑制效果。结果:硅纳米可有效地结合10-23 DRz,在血清中可保护10-23DRz免遭核酸酶的降解。硅纳米-10-23 DRz复合物能有效地抑制HBsAg和HBeAg的表达,抑制率为92.27%,高于脂质体对照组。结论:制得粒径32nm、分散均匀、表面电位+53.9mv、荧光稳定、低毒的硅纳米颗粒。硅纳米-10-23 DRz复合物能有效地抑制乙肝病毒HBsAg和HBeAg的表达。更多还原

【Abstract】 OBJECTIVE:Chronic Hepatitis B（CHB） is a serious disease of harming the human health.At present,there are no effective drugs to cure it.Recently,scientists are trying to carry out gene therapy for its treatment.CHB can cause body immune injury through viral gene expression.The introduction of nanoparticle vector promotes the development of gene therapy.This research aims at preparing the fluorescent silica nanoparticles（FSNPs）,probing into the feasibility of FSNPs as a gene transfer vector and the easy detection of the fluorescence of FSNPs as a biology marker,studying FSNPs-10-23 DRz inhibition of the expression of HBsAg and HBeAg,exploring the possibility of FSNPs-10-23 DRz complex treating CHB at cellular and molecular levels.PartⅠThe preparation and biological characteristics of FSNPsMETHODS:The FSNPs were prepared by microemulsion.NaCl was used to do surface modification on them.After a process of drying, their size was detected by a particle analyzer,and their Surface Zeta Potential was measured by an electrical Potential analysator.The FSNPs were dissolved in the physiological saline and their physiological saline solution was administered of the SD rats through the tail vein.Its acute toxicity to the SD rats was detected by up-and-down procedure and its distribution in the main organs of the SD rats was observed with fluorescence microscope.RESULTS:The FSNPs were successfully prepared in the W/O microemulsion.The nanoparticles were spherical and uniform in size, about 32 nm in diameter,+53.9mv in surface Zeta potential and highly monodispersed.More than 2000mg/kg LD₅₀ of the FSNPs was estimated by up and down procedure.Under the observation of fluorescence microscope,the FSNPs emitted red fluorescence distributed in each of the main organs.This indicated that the FSNPs could successfully pass through the blood-brain and blood-prostate barriers.PARTⅡThe FSNPs deliver 10-23DRz to inhibit the expression of HBVMETHODS:The special 10-23DRzs were designed by the sequence of S and C gene of HBV.The special 10-23DRzs was binded to the modified nanoparticles.The binding effect was tested by 15% denaturing polyacrylamide gel electrophoresis.Adding the FSNPs-DNA complex into fetal bovine serum,the protective effect of nanoparticles was detected by 15%denaturing polyacrylamide gel electrophoresis.The HepG2.2.15 cell was incubated with FSNPs-DNA complex,its inhibitory effect to HBsAg and HBeAg was analyzed by ELISA assay.RESULTS:The FSNPs could combine DNA effectively,protect against nuclease degradation in the serum.The NANO-DNA complex can inhibit the expression of S and C gene of HBV.The inhibition rate was 53.9mv and was higher than the liposomes control group.Conclusion:The FSNPs were successfully prepared,and it could combine special 10-23DRz and this complex could enter HepG2.2.15 cell to inhibit the expression of S and C gene of HBV.更多还原