增强自噬的乙型脑炎重组DNA疫苗促进BALB/c小鼠树突状细胞功能

Autophagy-targeted DNA vaccine against Japanese encephalitis virus promotes dendritic cell function in BALB/c mice

目的探讨自噬介导的乙型脑炎重组DNA疫苗对BALB/c小鼠体内树突状细胞(dendritic cells,DCs)免疫相关功能的影响。方法取健康BALB/c雌性小鼠,随机分配成5组,分别用pcDNA3.1(+)空载体、pJME质粒、pJME-LC3重组质粒肌注免疫小鼠,并使用无菌PBS肌注作为空白对照组,乙型脑炎减毒活疫苗腹腔注射作为阳性对照组,免疫注射共3次,每次间隔2周,末次免疫2周后无菌取小鼠脾脏,研磨后从单细胞悬液中分离DCs,使用免疫荧光技术观察重组质粒在DCs中的表达水平;利用流式细胞术检测DCs表面主要组织相容性复合体Ⅱ(major histocompatibility complex Ⅱ,MHCⅡ)的表达水平以及小鼠脾脏DCs摄取FITC-Dextran后的平均荧光强度;CCK8法检测分析不同免疫组DCs对同种异体小鼠脾脏淋巴细胞的刺激增殖效果。结果相对于其他免疫组,pJME-LC3实验组大量DCs胞质中可见质粒编码蛋白表达;pJME-LC3实验组免疫鼠脾脏DCs摄取FITC-Dextran的能力明显强于其他免疫组(P<0.05);pJME-LC3组小鼠成熟DCs表面MHCⅡ分子的表达水平明显升高(P<0.05);进一步研究发现pJME-LC3组小鼠脾脏DCs对同种异体小鼠脾脏淋巴细胞刺激增殖能力高于其他免疫组(P<0.05)。结论pJME-LC3重组质粒可以促进免疫鼠脾脏DCs抗原提呈以及刺激淋巴细胞增殖的能力,提示自噬介导的乙型脑炎重组DNA疫苗可能通过影响BALB/c小鼠DCs功能促进免疫效应。

Objective To investigate the effects of autophagy-targeted DNA vaccine against Japanese encephalitis virus(JEV)on the immune-related functions of dendritic cells(DCs)in BALB/c mice.Methods Healthy female BALB/c mice were randomly divided into 5 groups and injected with pcDNA3.1(+)empty vector(negative control),pJME plasmid,recombinant pJME-LC3 plasmid muscle,sterile PBS(blank control group)and live attenuated JEV vaccine(positive control group),respectively.The mice were immunized three times with an interval of two weeks.Splenic DCs were isolated two weeks after the last immunization.Immunofluorescence assay was used to observe the expression of the recombinant plasmid in dendritic cells.Expression of major histocompatibility complex Ⅱ(MHC Ⅱ)on DCs and the uptake of FITC-Dextran by DCs were observed by flow cytometry.CCK8 assay was used to detect the effects of DCs on the proliferation of spleen mononuclear cells from allogeneic mice.Results The expression of plasmid-encoded protein in the DCs of the pJME-LC3 group was significantly higher than that of the pJME,pJME-LC3+3-MA and pcDNA3.1(+)empty vector groups.The uptake of FITC-Dextran by DCs was significantly enhanced in the pJME-LC3 group than in the other groups(P<0.05),and the expression of MHC Ⅱ moleculars on DCs was increased in the pJME-LC3 group as well(P<0.05).The splenic DCs from the mice in the pJME-LC3 group had a stronger effect on the proliferation of spleen mononuclear cells from allogeneic mice that those from other groups(P<0.05).Conclusions The recombinant plasmid pJME-LC3 could promote the ability of mouse splenic DCs to present antigen and to stimulate lymphocyte proliferation after immunization,suggesting that the autophagy-targeted recombinant DNA vaccine against JEV could enhance immune responses by affecting the function of DCs in BALB/c mice.