颗粒溶素和白细胞介素12重组耻垢分枝杆菌对小鼠结核病的治疗作用

Therapeutic effects of granulysin/interleukin-12 recombinant M.smegmatis in a murine model of M.tuberculosis infection

目的探讨携带编码人天然颗粒溶素和小鼠白细胞介素12(IL-12)基因质粒(pZM03)的重组耻垢分枝杆菌对结核分枝杆菌感染的疗效与机制。方法结核分枝杆菌 H_(37)Rv 感染 Balb/c小鼠4周后,分别用生理盐水、耻垢分枝杆菌、pZM03、重组耻垢分枝杆菌治疗6次,于第1次治疗后3个月处死小鼠,检测器官荷菌量、脾淋巴细胞γ干扰素的分泌水平、血清 IL-12和 IgG_(2a)分泌水平和肺、脾组织中颗粒溶素表达,同时观察小鼠肺、脾组织病理改变情况。结果重组耻垢分枝治疗组的肺和脾组织对数荷菌量分别为(4.57±0.28)和(3.47±0.25)CFU/g,比生理盐水组的(5.77±0.12)和(4.66±0.18)CFU/g、载体组的(5.62±0.14)和(4.65±0.26)CFU/g 及质粒组的(5.04±0.22)和(4.25±0.48)显著降低;重组耻垢分枝杆菌组和 pZM03组经结核菌素纯蛋白衍生物刺激后,γ干扰素分泌水平显著高于耻垢分枝杆菌组和生理盐水对照组;重组耻垢分枝杆菌组血清 IL-12水平和IgG_(2a)水平显著高于耻垢分枝杆菌组和生理盐水对照组。用免疫组织化学方法检测到小鼠肺及脾组织中颗粒溶素的表达。生理盐水和耻垢分枝杆菌组的肺组织病理改变以渗出为主,病变广泛,增生改变不明显;重组耻垢分枝杆菌组的病变范围局限,并有类上皮细胞和泡沫细胞。治疗组和对照组的脾脏病理改变不明显。结论携带颗粒溶素和 IL-12的重组耻垢分枝杆菌对小鼠结核病有一定的免疫治疗作用,与增强宿主 Th1型免疫应答和颗粒溶素的抗菌活性有关。

Objective To study the effects and mechanisms of recombinant Mycobacterium smegmatis （M. smegmatis） encoding human granulysin and murine interleukin-12 （IL-12） on murine M. tuberculosis infection. Methods Balb/c mice infected with M. tuberculosis were treated with normal saline, M. smegmatis, pZM03, recombinant M. smegmatis respectively. The numbers of viable bacteria in the lung and the spleen were counted. The levels of IL-12, IgG2a in serum and intederon-γ（IFN-γ） released from spleen lymphocytes stimulated with purified protein derivative （PPD） were detected with enzyme linked immunosorbent assay （ ELISA ）. The expression of granulysin in tissue was detected with immunohistochemistry. Lungs and spleens were prepared for pathological analysis. Results The recombinant M. smegmatis group [ （4. 57 ±0. 28） in lung, （3.47 ±0. 25） in spleen] showed significantly reduced number of colony forming units （log CFU/g） compared with the control [ （5.77 ±0. 12） in lung, （4. 66 ±0. 18） in spleen], M. smegmatis [ （5. 62 ±0. 14） in lung, （4. 65 ±0. 26） in spleen] and pZM03 [ （5.04 ± 0. 22） in lung, （4. 25 ± 0. 48） in spleen ] group. The expression of granulysin in tissue, and increased level of IL-12 and IgG2a in serum were found in recombinant M. smegmatis group. IFN-γ released from spleen lymphocytes stimulated with PPD in recombinant M. smegmatis group and pZM03 group was higher than that of other groups, but the difference between recombinant M. smegmatis group and pZM03 group was not significant. The pathological changes in lungs of the recombinant M, smegmatis group were localized, while those in the control group were extensive. Significant pathological changes were not found in the spleens of all groups. Conclusion The recombinant M. smegmatis had immunotherapeutic effects,which were associated with a switch to Thl response and the antibacterial activity of granulysin.