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为探索高效安全的分子免疫增强剂,本实验设计合成含11个CpG基序的寡聚核苷酸(CpG ODN),制备壳聚糖纳米颗粒包裹CpG ODN,研究新型CpG ODN壳聚糖纳米颗粒(CpG-CNP)对人乙型肝炎疫苗的免疫佐剂效应。在肌注乙型肝炎疫苗免疫6周龄昆明小鼠的同时,肌注接种裸CpG ODN 30 pmol/只(A1组)和CpG-CNP 5pmol/只(A2组),口服CpG-CNP 30 pmol/只(B组),并设生理盐水空白疫苗对照组(C组)。在接种后每周采血,Sandwich ELISA检测实验小鼠IL-2、IL-4、IL-6、IgG、IgA和IgM水平以及特异性抗体(HbsAb)含量和免疫细胞数量的变化。结果发现各实验组小鼠的白细胞介素、血清免疫球蛋白、乙肝特异抗体和外周血免疫细胞均较对照组显著增多(P<0.05);CpG-CNP肌注组小鼠血液的免疫球蛋白、特异性乙肝表面抗体和白细胞介素含量、淋巴细胞和单核细胞数量较A1组和B组显著增加(P<0.05)。CpG-CNP口服组与裸CpG肌肉注射组无显著差异(P>0.05)。这些表明CpG-CNP不仅能高效诱导小鼠对乙肝疫苗的体液免疫应答,同时也显著增强其细胞免疫应答;CNP包裹可提高CpG的免疫刺激效应,减少CpG剂量,提示新CpG-CNP肌注或口服可用于HBV的免疫预防。
In order to explore efficient and safe molecular immunostimulants, we designed and synthesized CpG ODN containing 11 CpG motifs, prepared CpG ODN with chitosan nanoparticles, and investigated the effects of novel CpG ODN chitosan nanoparticles (CpG-CNP) Immuno Adjuvant Effect on Human Hepatitis B Vaccine. At the same time, Kunming mice (6 weeks old) were intramuscularly injected with Cp CpG ODN 30 pmol / mouse (Group A1) and CpG-CNP 5 pmol / mouse (Group A2) Only (group B) and normal saline blank vaccine control group (group C). Sandwich ELISA was used to detect the levels of IL-2, IL-4, IL-6, IgG, IgA and IgM and the changes of specific antibody (HbsAb) and the number of immune cells in mice. The results showed that the interleukin, serum immunoglobulin, hepatitis B specific antibody and peripheral blood immune cells of mice in each experimental group were significantly increased compared with the control group (P <0.05); the blood immunoglobulin of CpG-CNP mice , Specific hepatitis B surface antibody and interleukin, lymphocytes and monocytes significantly increased compared with A1 and B groups (P <0.05). CpG-CNP oral group and naked CpG intramuscular injection group no significant difference (P> 0.05). These results indicated that CpG-CNP can not only efficiently induce the humoral immune response to hepatitis B vaccine but also significantly enhance its cellular immune response. CNP encapsulation can enhance the immunostimulatory effect of CpG and reduce the dose of CpG, suggesting that the new CpG-CNP intramuscular injection or Oral can be used for immune prevention of HBV.