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目的探讨阳离子脂质体DOTAP(2,3-二油氧基丙基三甲基氯化铵)作为乙肝疫苗佐剂的免疫增强效果。方法薄膜蒸发复水法制备DOTAP阳离子脂质体乙肝疫苗,检测其粒径、Zata电位、包封率;将BALB/c小鼠随机分为5组:DOTAP阳离子脂质体乙肝疫苗组(DOTAP佐剂组)、不加佐剂的乙肝疫苗组(HBS组)、乙肝疫苗加铝佐剂组(铝佐剂组)、乙肝疫苗加CPG-ODN佐剂组(CPG-ODN佐剂组)以及PBS对照组,免疫剂量分别为0.2、2μg/(只·0.1ml),每个剂量10只小鼠,分别于0、28d经肌肉免疫,0、14、28、42d采血,分离血清,ELISA法检测血清中抗HBs特异性IgG抗体、亚型抗体(IgG1、IgG2a)滴度。结果制备的阳离子脂质体乙肝疫苗平均粒径200~300nm,粒径分布均匀,带正电荷,包封率在70%以上。DOTAP佐剂组血清IgG抗体水平明显高于HBS组,其中2μg剂量组差异有统计学意义(P<0.05);与铝佐剂组相比,两个剂量组差异均无统计学意义(P>0.05);0.2μg剂量组显著低于CPG-ODN佐剂组(P<0.05)。DOTAP佐剂组可同时诱导高水平的IgG1、IgG2a型抗体,两个剂量组产生的IgG1型抗体水平均与铝佐剂组相当(P>0.05),但IgG2a型抗体滴度均明显高于铝佐剂组(P<0.05);两个剂量DOTAP佐剂组产生的IgG1、IgG2a型抗体水平与CpG-ODN佐剂组相比,差异均无统计学意义(P>0.05)。结论DOTAP阳离子脂质体作为重组乙肝疫苗的佐剂具有良好的免疫增强效果,其对体液免疫的增强作用不劣于铝佐剂和CPG-ODN佐剂,DOTAP佐剂可同时诱导Th2细胞和Th1细胞介导的免疫应答。
Objective To investigate the immunopotentiating effect of cationic liposome DOTAP (2,3-dioleoxypropyltrimethylammonium chloride) as adjuvant for hepatitis B vaccine. Methods DOTAP cationic liposomes Hepatitis B vaccine was prepared by membrane evaporation and rewatering method. The particle size, Zata potential and entrapment efficiency were measured. The BALB / c mice were randomly divided into 5 groups: DOTAP cationic liposome hepatitis B vaccine group (HBS group), hepatitis B vaccine plus aluminum adjuvant (aluminum adjuvant), hepatitis B vaccine plus CPG-ODN adjuvant (CPG-ODN adjuvant), and PBS control The mice were immunized with 0.2, 2μg / (only 0.1ml) of immunosuppressive drugs at a dose of 10μg / ml for 10,28 days respectively. The blood samples were collected at 0, 28, Anti-HBs-specific IgG antibodies, subtype antibodies (IgG1, IgG2a) titer. Results The average diameter of the prepared cationic liposome hepatitis B vaccine was 200-300nm with uniform particle size distribution and positive charge, and the entrapment efficiency was above 70%. Compared with HBS group, the level of IgG antibody in DOTAP adjuvant group was significantly higher than that in HBS group (P <0.05), but there was no significant difference between two dose groups (P> 0.05); 0.2μg dose group was significantly lower than CPG-ODN adjuvant group (P <0.05). DOTAP adjuvant group could induce high levels of IgG1 and IgG2a antibodies at the same time. The levels of IgG1 antibody produced by the two dosage groups were similar to that of aluminum adjuvant (P> 0.05), but the titer of IgG2a antibody was significantly higher than that of aluminum adjuvant (P <0.05). The levels of IgG1 and IgG2a antibody produced by two doses of DOTAP adjuvant group were not significantly different from those of CpG-ODN adjuvant group (P> 0.05). CONCLUSION DOTAP cationic liposomes have good immune enhancement effect as adjuvant of recombinant hepatitis B vaccine. The enhancement of humoral immunity is not inferior to aluminum adjuvant and CPG-ODN adjuvant. DOTAP adjuvant can induce Th2 cells and Th1 Cell-mediated immune response.