载基因壳聚糖纳米粒的免疫增强效应

Immunological enhancement of chitosan nanoparticles as gene carriers

背景：壳聚糖是-种天然的多糖，具有生物降解性、生物相容性及安全无毒等特点，因而成为基因治疗载体研究的热点。目的：将壳聚糖包载含戊型肝炎主要抗原表位基因片段的重组质粒pcHEV23，制备成壳聚糖．pcHEV23纳米粒，并观察其在小鼠体内的免疫增强效果。设计、时间及地点：对比观察实验，于200706／200808在浙江省医学科学院生物工程研究所完成。材料：将等浓度pcHEV23分别与质量浓度为1．4，0．7，0．4，0gm的壳聚糖按1：1体积比混合，复凝聚法制备壳聚糖（高）-pcHEV23、壳聚糖（中）-pcHEVz3、壳聚糖（低）-pcHEV23、空白壳聚糖等4种纳米颗粒。方法：以空质粒pcDNA3、空白壳聚糖纳米粒、质粒pcHEV23为对照组，壳聚糖（高）-pcHEV：3、壳聚糖（中）-pcHEV23、壳聚糖（低）-pcHEV23为实验组，进行免疫小鼠，共免疫3次，每次间隔3周，免疫后2周断尾取血，并于末次断尾采血后进行眼球摘除取血。主要观察指标：检测制备的纳米粒平均粒径、包封率、抵抗核酸酶降解能力；ELISA方法检测免疫小鼠后血清中HEVIgG抗体产生情况；流式细胞仪检测外周血CD4＋、CD8＋T淋巴细胞百分比和CD4＋／CD8＋比值。结果：制备的纳米粒粒径170～470nm，对质粒pcHEV23的包封率均〉95％，能有效抵抗核酸酶降解，起到保护pcHEV23的作用。免疫实验表明壳聚糖-pcHEV23纳米粒可上调小鼠血清HEVkG抗体水平，并显著提高小鼠外周血CD4＋T淋巴细胞百分比及CD4＋／CD8＋比值。结论：壳聚糖能高效装载外源基因pcHEV23，保护其免受核酸酶的降解，有效提高小鼠细胞免疫水平，显示出具有良好的免疫增强作用，作为基因载体具有较大的应用潜力。

BACKGROUND： Chitosan is known as a biocompatible, biodegradable and non-toxic polysaccharide. So it is focused in the research of gene delivery system. OBJECTIVE： To prepare chitosan-pcHEV23 nanoparticles with the chitosan as gene carriers to carry the primary antigen epitope gene of hepatitis E virus （HEV）, and to study its immunological enhancement in mice. DESIGN, TIME AND SETTING： A controlled experiment was performed at the Institute of Bioengineering, Zhejiang Academy of Medical Science from June 2007 to August 2008. MATERIALS： The pcHEV23 was mixed with the chitosan （concentrations 1.4, 0.7, 0.4, 0 g/L） at a ratio of 1 ： 1, and four kinds of chitosan-pcHEV23 nanoparticles were prepared with the method of complex coacervation. METHODS： The blank pcDNA3, blank chitosan nanoparticles and pcHEV23 were taken as the control groups, and chitosan （low）-pcHEV23, chitosan （middle）-pcHEV23 and chitosan （high）-pcHEV23 served as the experimental groups. The mice in immune experiment were processed into three times of immunization at an interval of 3 weeks. At two weeks following the immunization, blood sample was obtained from the amputated tails, and the enucleation of eyeball was performed following the last blood harvesting. MAIN OUTCOME MEASURES： The average particle size, efficiency of the encapsulation, and the ability to protect pcHEV23 from nuclease degradation were determined; Serum HEV IgG contents were assayed with ELISA method; peripheral blood T lymphocyte subsets CD4＋ and CD8＋, as well as the ratio of CD4＋/CD8＋ were detected with flow cytometry. RESULTS： The particle diameter of the nanoparticles was form 170 nm to 470 nm. The efficiency of the encapsulation was all over 95%. The nuclease degradation test confirmed that the pcHEV23 could be protected from DNase Ⅰ degradation. Immunity test proved that the chitosan-pcHEV23 nanoparticles could raise serum HEV IgG contents and increase evidently the percentage of CD4＋ cells and the ratio of CD4＋/CD8＋ in the peripheral blood lymphocytes of immunized mice. CONCLUSION： Chitosan can coacervate pcHEV23 effectively, protect it from DNase Ⅰ degradation and improve the immune enhancing effect of pcHEV23. It shows that this chitosan could be as a potential gene carrier.