乙型肝炎表面抗原与粒细胞巨噬细胞集落刺激因子融合表达质粒免疫乙型肝炎病毒转基因小鼠的研究

Antivirus effect of plasmid co expressing hepatitis B surface antigen and granulocyte macrophage-colony stimulating factor

目的 研究乙型肝炎表面抗原(HBsAg)和粒细胞巨噬细胞集落刺激因子(GM-CSF)的融合表达质粒免疫乙型肝炎病毒(HBV)转基因小鼠及其病毒清除作用。方法 将小鼠随机分为8组,分别注射以下质粒:A组pcDNA3.1-S 100μg;B组:pcDNA3.1-GM-CSF-S 100μg;C组:pcDNA3.1-S-GM-CSF 100μg;D组:pcDNA3.1-S 50μg+pcDNA3.1-GM-CSF 50μg;E组:pcDNA3.1-GM-CSF 100μg;F组:重组酵母乙型肝炎疫苗1μg;G组:pcDNA3.1 100μg;H组:磷酸盐缓冲液(PBS)100μl。于质粒注射前4d肌肉注射0.25％bupivacaine溶液100μl,诱导骨骼肌细胞变性。以HBsAg和GM-CSF的融合乙型肝炎表达质粒免疫HBV转基因小鼠,检测HBV转基因小鼠血清HBsAg表达水平及肝细胞HBsAg的表达,检测脾细胞白细胞介素(IL)-2、IL-4及γ干扰素(IFN-γ)的分泌水平及淋巴细胞增殖情况,并进行肝组织学检查。结果 质粒加强免疫4周后血清HBsAg检测结果A组为28.28±15.32、B组为10.77±9.18、C组为44.16±8.30、D组为21.93±13.28、E组48.04±3.60、F组为38.3±11.66、G组为47.03±3.96、H组为51.46±4.70,F=11.262,P<0.01,其中B组血清HBsAg滴度显著低于其余各组。IL-2分泌水平(pg/ml)A组为25.5±7.5、B组为48.5±11.3、C组为4.0±2.5、D组为38.0±8.5、E组为3.7±2.1、F组为6.5±23.

Objective To investigate whether GM-CSF can enhance the antiviral effect of HBsAg DNA vaccine. Methods Divided animals into 8 groups. Group A: pcDNA3.1-S 100 μg; Group B: pcDNA3.1-GM-CSF-S 100 μg; Group C: pcDNA3.1-S-GM-CSF 100 μg; Group D: pcDNA3.1-S 50 μg + pcDNA3.1-GM-CSF 50 μg; Group E: pcDNA3.1-GM-CSF 100 μg; Group F: recombinant HBsAg vaccine 1 μg; Group G: pcDNA3.1 100 μg; Group H: PBS 100 μl. Serum HBsAg level and concentration of IL-2, IL-4 and IFN- f33 were examined using commercial EL1SA kit. The [1H] thymidine incorporation into DNA of Spleen cells was measured; HBsAg expression of hepatocytes from HBV-transgenic mice was assessed using immunohistochemical analysis. Results Serum HBsAg level was lower and concentration of IL-2. IFN-Y and SI was higher in mice immunized with pcDNA3.1-GM-CSF-S than those from mice immunized with pcDNA3.l-S and other groups (F = 11.262, P<0.01, F= 8.147, P < 0.01, F = 62.275, P < 0.01, F = 1 16.28, P < 0.01). Less Hepatic HBsAg expression and decline of pcDNA3.1-GM-CSF-S of mice immunized with pcDNA3 were observed in comparison with control groups (F = 41.439, P < 0.01). Liver histological analysis showed no evidence of necrosis or inflammation in various groups. Conclusion The plasmid co expressing GM-CSF and HBsAg could improve HBsAg-specific humoral and cellular immune responses induced by plasmid encoding HBsAg alone and enhance HBsAg DNA vaccine antivirus effect.