耻垢分枝杆菌疫苗对小鼠细胞因子产生及Th1/Th2应答的影响

Effects of Mycobacterium smegmatis vaccine on cytokines production and Th1/Th2 responses in mice

目的研究以耻垢分枝杆菌(M.S)制备的M.S疫苗对小鼠免疫应答的影响,验证M.S的免疫原性、探讨M.S疫苗的免疫调节作用。方法将BALB/c小鼠随机分成生理盐水对照组和M.S疫苗低、中、高剂量组,每组6只,分别注射生理盐水和不同剂量的M.S疫苗。取小鼠脾细胞或腹腔巨噬细胞体外培养,用酶联免疫吸附测定(ELISA)法检测培养上清中白细胞介素(IL)-2、IL-4、IL-12和γ干扰素(IFN-γ)的含量,分析M.S疫苗对小鼠Th1/Th2类应答的影响。结果(1)生理盐水对照组和M.S疫苗低、中、高剂量组小鼠产生的IL-12分别为(32.6±22.7)、(58.9±18.6)、(77.3±38.0)、(114.7±9.9)pg/m l,M.S疫苗中、高剂量组与生理盐水对照组比较差异有统计学意义(P<0.05)。(2)生理盐水对照组和M.S疫苗低、中、高剂量组小鼠产生的IL-2分别为(5.0±2.6)、(13.4±9.3)、(15.3±9.7)、(22.6±7.5)pg/m l,M.S疫苗高剂量组与生理盐水对照组比较差异有统计学意义(P<0.01)。(3)体外以ConA刺激时,生理盐水对照组和M.S疫苗中剂量组小鼠产生的IFN-γ分别为(662±279)和(807±163)pg/m l,IL-4分别为(407±127)和(101±26)pg/m l,但M.S疫苗组与生理盐水对照组比较差异均无统计学意义(P>0.05);体外以M.S-纯化蛋白衍生物(PPD)刺激时,生理盐水对照组和M.S疫苗中剂量组小鼠产生的IFN-γ分别为(14.0±6.31)和(55.3±32.4)pg/m l,两组比较差异有统计学意义(P<0.05),各组产生的IL-4均在检测限以下。结论M.S具有良好的免疫原性,以此制备的M.S疫苗具有促进Th1类应答、抑制Th2类应答的免疫调节作用。

Objective To validate the immunogenicity of Mycobacterium smegmatis and to study the immune modulatory function of Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis by analyzing the effects of the vaccine on immune responses in mice. Methods Spleen cells and peritoneal macrophages from BALB/c mice which were randomized into a control group and Mycobacterium smegmatis vaccine groups（ low, middle, and high doses） were cultured in vitro. Then the supernatants were collected and the concentrations of IL-2, IL-4, IL-12, and IFN-γ were analyzed through ELISA. Results （ 1 ） IL-12 produced by the control mice and mice immunized with low, middle, high doses of Mycobacterium smegmatis vaccine was （32. 6 ±22. 7）, （58. 9 ± 18. 6）, （77.3 ±38.0）, （ 114. 7 ±9.9） pg/ml respectively, and the middle and high dose group showed significant difference as compared with the control group （ P 〈 0. 05 ）. （2） IL-2 produced by the control mice and mice immunized with low, middle, high dose of Mycobacterium smegmat/s vaccine was （5.0 ± 2. 6 ）, （ 13.4 ± 9. 23 ）, （ 15.3 ± 9. 7 ）, （ 22. 6 ± 7. 5 ） pg/ml respectively, and the high dose group showed significant difference when compared with the control group （ P 〈 0. 01 ）. （3） When the cells were stimulated with ConA in vitro, IFN-γ, produced by the control mice and mice immunized with middle dose of Mycobactedum smegmatis vaccine was （662 ± 279 ）and （807 ± 163 ）pg/ml, IL-4 produced by the two groups was （407 ± 127）and （101 ± 26） pg/ml, but the differences were not statistically significant （ P 〉 0. 05 ）. When the cells were stimulated with Mycobacterium smegmatis-purified protein derivative （PPD） in vitro, IFN-γ produced by the control mice and mice immunized with middle dose of Mycobacterium smegmatis vaccine was （ 14. 0 ± 6.31 ） and （55. 3 ± 32.4） pg/ml, the difference being statistically significant （P 〈 0. 05）, but IL-4 produced by the two groups was under the limit of detection. Conclusion Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis showed strong immunogenicity promoted Th1 responses and inhibited Th2 response in mice.