不同剂量结核病DNA疫苗的电转染免疫原性研究

目的 研究不同剂量结核病DNA疫苗电转染后的免疫原性.方法 40只BALB/c小鼠通过随机数字表法分为8组,每组5只.其中4组分别用100 μl生理盐水和10 μg、50 μg、100 μg结核分枝杆菌Ag85A DNA肌内注射免疫小鼠;另4组用100 μl生理盐水和10 μg、50 μg、100 μgAg85ADNA肌内注射加电转染免疫小鼠.每2周免疫1次,共3次.最后1次免疫后2周杀死小鼠.用ELISA方法检测小鼠脾细胞培养上清液中γ干扰素（IFN-γ）和白细胞介素4（interleukin 4,IL-4）水平;用流式细胞术检测小鼠外周血单个核细胞（PBMC）分泌IFN-γ的辅助性T细胞（helper T cell,Th）1细胞百分比、分泌IL-4的Th2细胞百分比,以及Th1与Th2的细胞比值.用SAS 9.2软件处理数据,实验数据以“-x±s”表示,对有关数据进行两因素析因设计的方差分析,两两比较采用t检验,P＜0.05为差异有统计学意义.结果 免疫结束后2周,小鼠脾淋巴细胞分泌IFN-γ水平,50 μg（646.05±342.53） pg/ml]和100 μgAg85ADNA肌内注射组（738.61±372.68） pg/ml]显著高于生理盐水组（1.73±3.88）pg/ml]（f值分别为4.065、4.647,P值均＜0.05）和10 μg Ag85A DNA组（87.83±120.82）pg/ml]（t值分别为3.513、4.094,P值均＜0.05）;10 μg Ag85A DNA电转染组（357.06±105.18） pg/ml]显著高于生理盐水组（t=2.247,P＜0.05）,高于100 μg Ag85ADNA电转染组（86.08±135.73） pg/ml],但差异无统计学意义（t=1.706,P＞0.05）;50 μg Ag85ADNA电转染组（648.60±439.41）pg/ml]显著高于生理盐水组（t=4.081,P＜0.05）和100 μg Ag85A DNA电转染组（t=3.539,P＜0.05）.与直接肌内注射组IFN-γ水平（87.83±120.82） pg/ml]比较,10 μg Ag85A DNA电转染组增高3倍（357.06 pg/ml）/（87.83 pg/ml）]; 50 μg Ag85A DNA肌内注射组（646.05±342.53） pg/ml]与电转染组（648.60±439.41）pg/ml]比较,差异无统计学意义（t=-0.016,P＞0.05）;100 μg Ag85A DNA电转染组（86.08±135.73）pg/ml]较肌内注射组（738.61±372.68） pg/ml]降低88.35％（t=4.105,P＜0.05）.小鼠PBMC分泌IFN-γ的Th1细胞百分比,不同剂量Ag85A DNA肌内注射组10 μg Ag85A DNA：（1.39±0.84）％;50 μg Ag85A DNA：（1.55±0.33）％;100 μg Ag85A DNA：（2.13±0.47）％]和DNA电转染组10 μg Ag85A DNA：（1.42±0.47）％; 50 μg Ag85A DNA：（1.88±0.51）％; 100 μg Ag85ADNA：（1.43±0.68）％]均高于生理盐水组（0.65±0.31）％]（t值分别为2.002、2.431、4.015、2.084、3.332和2.105,P值均＜0.05）,但不同剂量Ag85A DNA电转染组与肌内注射组之间差异均无统计学意义（t值分别为0.081、0.901和-1.91,P值均＞0.05）.小鼠PBMC分泌IL-4的Th2细胞百分比,不同剂量Ag85A DNA肌内注射组10 μg Ag85A DNA：（1.42±1.18）％; 50 μg Ag85A DNA：（1.14±0.78）％; 100 μg Ag85A DNA：（1.24±0.76）％]和DNA电转染组10 μg Ag85A DNA：（1.19±1.09）％; 50 μg Ag85A DNA：（2.06±0.96）％;100 μgAg85A DNA：（1.47±0.65）％]均显著低于生理盐水电转染组（4.14±2.55）％]（t值分别为-3.392、-3.738、-3.616、-3.676、-2.599和-3.325,P值均＜0.05）,但不同剂量DNA肌内注射组与DNA电转染组之间差异无统计学意义（t值分别为0.284、-1.139和-0.292,P值均＞0.05）.结论 DNA电转染免疫可以增强低剂量DNA疫苗的免疫应答,使用少量的DNA疫苗就可以产生较好的免疫效果.

Immunogenicity of different dosage DNA vaccine from Mycobacterium tuberculosis medicated by electroporation

Objective To study the immunogenicity of different dosages DNA vaccines from Mycobacterium tuberculosis medicated by electroporation.Methods Twenty female BALB/c mice were immunized intramuscularly with saline,10 μg Ag85A DNA,50 μg Ag85A DNA and 100 μg Ag85A DNA for three times at two-week intervals,respectively.Twenty female BALB/c mice were medicated intramuscularly by electroporation with saline,10 μg Ag85A DNA,50 μg Ag85A DNA and 100 μg Ag85A DNA for 3 times at two-week intervals,respectively.There were 5 mice each group.Mice were sacrificed at 2 weeks after the final immunization respectively.The levels of IFN-γ and IL-4 in the culture supernatants of splenic lymphocytes were measured with enzyme-linked immunosorbent assay （ELISA）.The ratio of CD4＋ T cells expressing IFN-γ （Th1） and IL-4 （Th2） in whole blood was detected by flow cytometry.Data were expressed as means and standard deviations.Statistical significance between each group was calculated using two factors factorial design ANOVA followed by compared with t test using SAS 9.2 software and a P-value of ＜0.05 was considered to be statistically significant.Results At 2 weeks after the final immunization,IFN-γ levels in splenocyte culture supernatant in 50 μg DNA （646.05 ± 342.53） pg/ml and 100 μg DNA intramuscular injection group （738.61 ± 372.68） pg/ml was significantly higher than that in saline （1.73 ± 3.88） pg/ml （t =4.065 and 4.647,all P＜0.05） and 10 μg DNA intramuscular injection group （87.83±120.82） pg/ml （t=3.513 and 4.094,all P＜0.05） ; IFN-γ level in splenocyte culture supernatant in 10 μg DNA electroporation group （357.06±105.18） pg/ml was significantly higher than that in saline （t =2.247,P＜0.05）,and higher than that in 100 μg DNA electroporation group （86.08± 135.73） pg/ml,but the difference was not statistically significant （t =1.706,P＞0.05）.50 μg DNA electroporation group （648.60±439.41） pg/ml was significantly higher than that in saline（t =4.081,P＜0.05） and 100 μg DNA electroporation group（t =3.539,P＜0.05） ; compared with intramuscular injection group,IFN-γ levels of 10 μg DNA electroporation group increased 3 times （357.06 pg/ml）/（87.83 pg/ml） ; that of 50 μg DNA electroporation group had no significant change （t =-0.016,P＞0.05） ; that in 100 μg DNA electroporation group declined 88.35％ （t =-4.105,P＜0.05）.Percentage of Th1 cells secreted IFN-γ in different dosages of intramuscular DNA （10 μg Ag85A DNA：（1.39 ± 0.84）％,50 μg Ag85A DNA：（1.55 ± 0.33）％,100 μg Ag85A DNA：（2.13±0.47）％） and DNA electroporation groups （10 μg Ag85A DNA：（1.42± 0.47） ％,50 μg Ag85A DNA：（1.88±0.51） ％,100 μg Ag85A DNA：（1.43±0.68） ％） were higher than that in saline group （0.65±0.31）％ （t=2.002,2.431,4.015,2.084,3.332 and2.105,all P＜0.05）,therehas no statistical difference between doages DNA electroporation groups and intramuscular injection groups （t=0.081,0.901 and-1.91,all P＞0.05）.Percentage of Th2 cells secreted IL-4 in different dosages of intramuscular DNA （10 μg Ag85A DNA：（1.42±1.18） ％,50 μg Ag85A DNA：（1.14±0.78） ％,100 μg Ag85A DNA：（1.24±0.76） ％） and DNA electroporation groups （10 μg Ag85 A DNA：（1.19 ± 1.09） ％,50 μg Ag85 A DNA：（2.06 ± 0.96） ％,100 μg Ag85A DNA：（1.47± 0.65） ％） were higher than that in saline electroporation group （4.14±2.55） ％ （t =-3.392,-3.738,-3.616,-3.676,-2.599 and-3.325,all P＜0.05）,there was no statistically significant difference between doages DNA electroporation groups and intramuscular injection groups（t =0.284,-1.139 and-0.292,all P＞0.05）.Conclusion The results suggest that lower doses of DNA immunization by electroporation could improve immune response,using a small amount of DNA vaccine can produce good immune effect.