Flt3L与CCL5细胞因子佐剂对HBc抗原DNA疫苗特异性免疫应答的促进作用

观察真核重组表达质粒pFlt3L及pCCL5对携带HBc抗原的DNA疫苗诱导的抗原特异性免疫应答的促进作用。将pFlt3L及pCCL5分别用脂质体的方法转染Hep G2细胞,然后采用骨髓细胞增殖及趋化小室实验检测细胞上清Flt3L及CCL5两种细胞因子的生物学活性。将pFlt3L和pCCL5两种重组质粒单独或联合使用与携带HBc抗原的DNA疫苗经肌内注射法免疫小鼠,采用MTT法检测脾淋巴细胞增殖、流式细胞仪检测脾CD8+T淋巴细胞中IFN-γ表达、ELISA法检测脾淋巴细胞培养上清IL-4含量及乳酸脱氢酶(LDH)释放法检测特异性CTL杀伤活性。结果:骨髓细胞增殖及趋化实验证实了Flt3L及CCL5均有生物学活性。pFlt3L和pCCL5单独或联合使用均可促进特异性淋巴细胞增殖反应(P<0.05),提高小鼠脾脏CD8+T淋巴细胞中IFN-γ表达量(P<0.05或P<0.01),IL-4表达水平在各组无显著区别(P>0.05),Flt3L+CCL5组小鼠脾细胞特异性CTL活性显著高于其他各组(P<0.05)。pFlt3L和pCCL5表达质粒联用可显著促进小鼠Th1型细胞因子的表达,并对带HBc抗原的DNA疫苗的免疫应答具有促进作用。

Ag-specific immune enhancement in mice induced by co-inj ection recom-binant plasmids pFlt3L and pCCL5 as adjuvant on DNA vaccine

To investigate the immune enhancement effect of recombinant plasmids pFlt3L and pCCL5 as the adjuvant on DNA vaccine,the plasmids of pFlt3L and pCCL5 were transfected into Hep G2 cells by means of lipid mixtures respectively,and the biologic activity of Flt3L and CCL5 in supernatant were detected by bone marrow cell proliferation experiment or chemotaxis assay for transwell migration of splenic lymphocytes.Mice were coimmunized with HBcAg DNA vaccine and the two DNA constructs by intramuscular injection.Lymphocytes proliferation response and the number of IFN-γ-producing cells in splenocytes were measured by MTT assay or flow cytometry respectively.The levels of IL-4 in supernatant of spleen-lymphocyte cultures were measured by ELISA,and the CTL activity of spleno-lymphocyte was detected with LDH release assay.Compared with the negative control,the product of Flt3L could induce the proliferation of mice bone morrow cells(P<0.05),while CCL5 induced splenic lymphocytes to migrate(P<0.01).The numbers of IFN-γ-producing cells in all immunization groups was significantly higher than that of negative control(P<0.05 or P<0.01);but the levels of IL-4 showed no differences in all groups(P>0.05).CTL activities in group of HBcAg DNA vaccine combined with Flt3L and CCL5 were stronger than that of other groups(P<0.05).It is concludel that HBcAg DNA vaccine combined with Flt3L and CCL5 can induce Th1 type immune response and were effective in enhancing Ag-specific immune response.