新型结核病疫苗菌株(B/R菌株)对T细胞免疫记忆的建立及保护效应的研究

Study on the new tuberculosis vaccine strain(B/R Strain)on T lymphocyte immune memory formation and immune protection effect

目的探讨研究新型结核病疫苗菌株(B/R菌株)对T细胞免疫记忆的建立及保护效应的影响。方法C57BL/6小鼠分别皮下接种PBS、BCG、H37Ra和B/R菌株,各组小鼠于免疫后不同时间点,利用ELISA方法检测血清中IL-2、IFN-γ的水平;用流式细胞技术检测小鼠脾脏中央型记忆性淋巴细胞(TCM)及效应型记忆性淋巴细胞(TEM)的数量;检测各组小鼠脾脏和肺脏组织中的菌落数(CFU),以及根据肺脏病理损伤程度评价B/R菌株的保护效果。结果免疫小鼠8周,B/R菌株组小鼠血清中,产生了高于BCG组的IL-2(q=4.709,P<0.05)和IFN-γ(q=5.477,P<0.05),与H37Ra组无统计学差异。免疫小鼠12周,B/R菌株组小鼠血清中IL-2、IFN-γ水平与BCG组和H37Ra组均无统计学差异。免疫小鼠16周,B/R菌株组小鼠血清IFN-γ水平高于BCG组(q=4.243,P<0.05),与H37Ra组无统计学差异;B/R菌株组小鼠血清IL-2水平与BCG组和H37Ra组均无统计学差异。免疫小鼠8、12、16周,与PBS组相比较,除12周时B/R菌株组的CD8^+TCM水平有所降低外,B/R菌株组均可诱导产生较高水平的CD4^+TCM(F=5.178,P<0.05)、CD4^+TEM(F=72.04,P<0.05)以及CD8^+TCM(F=16.86,P<0.05)、CD8^+TEM(F=27.93,P<0.05)。随免疫时间延长,即16周时,与BCG组相比较,B/R菌株组可维持更高水平的CD4^+TEM(q=31.55,P<0.05)和CD8^+TEM(q=16.08,P<0.05),TCM无统计学差异。BCG、H37Ra和B/R菌株免疫小鼠后,各组小鼠脏器病理损伤程度及菌落数,无统计学差异。结论B/R菌株免疫小鼠后,可建立较高水平的T细胞免疫记忆,以TEM为主,其免疫记忆水平可维持近4月,并且与BCG免疫保护效果相当。

In order to investigate the effect of the new tuberculosis vaccine strain(B/R strain)on T lymphocyte immune memory formation and immune protection effect.C57BL/6 mice were subcutaneously inoculated with PBS,BCG,H37Ra and B/R strain respectively.At different times after immunization,the expression levels of IL-2 and IFN-γin serum were detected by ELISA and the levels of central memory T cells(TCM)and effector memory T cells(TEM)in the spleen of mice were detected by flow cytometry.To evaluate the protective effect of the B/R strain,the colony-forming units(CFU)in the spleen and lung tissues were measured,as well as the degree of pathological lung damage.In our results,After 8 weeks of immunization,compared with BCG group,the serum of B/R group produced higher levels of IL-2(q=4.709,P<0.05)and IFN-γ(q=5.477,P<0.05),and there is no statistical difference with H37Ra group.After 12 weeks of immunization,the levels of IL-2 and IFN-γin serum of mice in B/R group were not significantly different from those in BCG group and H37Ra group.After 16 weeks of immunization,the serum IFN-γlevel of the B/R group was higher than that of the BCG(q=4.243,P<0.05),and there is no statistical difference with H37Ra group;The serum IL-2 level of the B/R group was not statistically different from that of the BCG and H37Ra groups.After 8,12 and 16 weeks of immunization,compared with PBS group,B/R strain group induced higher levels of CD4^+TCM(F=5.178,P<0.05)、CD4^+TEM(F=72.04,P<0.05)and CD8^+TCM(F=16.86,P<0.05)、CD8^+TEM(F=27.93,P<0.05),except that CD8^+TCM decreased at 12 weeks.With the extension of immunization time,that is,16 weeks later,compared with BCG group,B/R group can maintain a higher level of CD4^+TEM(q=31.55,P<0.05)and CD8^+TEM(q=16.08,P<0.05),and the TCM showed no statistical difference.After immunizing mice with BCG,H37Ra and B/R strain,there was no statistical significance in organ pathological injury degree and CFU.In conclusion,after immunizing mice with B/R strain,a higher level of T cell immune memory was established,mainly in TEM,and its immune memory level could be maintained for nearly 4 months,and was equivalent to BCG immune protection effect.