树突状细胞多价核酸疫苗抗血吸虫感染作用及机制研究

目的 探讨基因转染对树突状细胞(DC)功能的影响及DC多价核酸疫苗抗血吸虫感染作用及机制。方法 利用脂质体介导的基因转染技术将Sj26、Sj23和Sj14基因分别转染DC,流式细胞术(FCM)检测DC摄取抗原能力,混合淋巴细胞反应(MLR)检测DC对同种异体T淋巴细胞的刺激作用。Sj26、Sj23和Sj14基因转染DC分别和联合免疫BALB/c小鼠3次,末次免疫2周后,每只小鼠感染日本血吸虫尾蚴40条,小鼠感染血吸虫6周后,计数成虫和虫卵。ELISA法检测血清特异性IgG、血清干扰素-γ(IFN-γ)和白细胞介素-4(IL-4)及脾淋巴细胞培养上清IFN-γ、IL-4水平,噻唑蓝(MTT)法检测脾淋巴细胞的增殖。结果 与真核表达载体pcDNA3转染DC和未处理DC相比较,基因转染DC摄取抗原的荧光强度明显降低(P<0.01),对同种异体T淋巴细胞的刺激指数明显升高(P<0.01)。各免疫组小鼠诱导的减虫率和减卵率均高于对照组(P<0.01),而基因转染DC联合免疫组小鼠抗血吸虫感染作用高于单一基因转染DC免疫组(P<0.001)。基因转染DC免疫组小鼠末次免疫2周后血清特异性IgG水平较免疫前显著升高(P<0.05),血清IFN-γ水平明显升高(P<0.01),而血清IL-4的水平无明显变化。与对照组比较,基因转染DC免疫组小鼠脾淋巴细胞经ConA和SEA刺激后培养上清IFN-γ水平显著增高,IL-4水平显著降低,刺激指数(SI)显著增高(P<0.001)。结论 基因转染能促进DC的成熟,增强DC的活性,DC多价核酸疫苗可增强抗血吸虫感染作用,其作用机制以Th1型免疫应答为主。

Protective immunity and mechanism of dendritic cell multivalent nucleic acid vaccine against infection of Schistosoma japonicum

To study the effect of gene transfer on the functions of dendritic cell (DC) and protective immunity and mechanism of DC multivalent nucleic acid vaccine against infection of Schistosoma japonicum, DCs were transfected with recombinant eukaryotic expression vector pcDNA3-Sj26, pcDNA3-Sj23 and pcDNA3-Sj14. The ability of DC to absorb antigen was determined by flow cytometry (FCM). The mixed lymphocyte reaction (MLR) was used to detect the effect of gene transferred DC on stimulating allogenetic T lymphocyte. BALB/c mice were immunized three times with Sj26, Sj23 and Sj14 gene transferred DC, alone or combination, and infected with 40 cercariae of S. japonicum per mouse 2 weeks after the last immunization. The number of adult worm and the egg number in liver were calculated six weeks after infecting. ELISA was used to detect the levels of specific IgG, IFN-γ and IL-4 in sera from each mice group. The level of IFN-γ and IL-4 in the culture supernatant of spleen lymphocytes stimulated with soluble egg antigen (SEA) and ConA were quantified by ELISA. The proliferation of spleen lymphocytes were measured by the method of MTT. Compared with pcDNA3 transferred DC and untreated DC, the fluorescence intensity of antigen absorbing decreased significantly in S. japonicum encoding gene-transferred DC (P<0.01), and the stimulating index (SI) increased significantly (P<0.01). The rate of worm reduction and egg reduction in each group of immunization were higher than that of control group (P<0.01), while protective immunity induced by gene transfer DC were significantly higher than that of gene transfer DC alone (P<0.001).Compared with previous immunization, the levels of specific IgG increased significantly in sera from group of S. japonicum encoding gene-transferred DC 2 weeks after the last immunization (P<0.05), the levels of IFN-γ increased significantly (P<0.01), while the levels of IL-4 were not significantly different. In response to ConA and SEA, the level of IFN-γ in the culture supernatant of spleen lymphocytes from group of S. japonicum encoding gene-transferred DC increased significantly, the level of IL-4 decreased significantly, while SI increased significantly (compared with control group, P<0.001). Results indicated that S. japonicum encoding gene transfer can promote DC maturation and enhance the biologic activity of DC. DC multivalent nucleic acid vaccine could induce and enhance protective immunity against infection of S. japonicum. Predominant Th1 type immune response might play an important role in the protective immunity induced by gene-transferred DC against infection of S. japonicum.