摘要目的 探讨CpG寡脱氧核苷酸(CpG-ODN)增强小鼠体内结核分枝杆菌清除能力的可能机制.方法 将雌性BALB/c小鼠随机分为免疫组36只和对照组24只,并分别腹腔注射CpG-ODN 30 μg(溶于200 μl生理盐水)和200 μl生理盐水.2周后每只小鼠经尾静脉注射结核分枝杆菌H37Rv 1×106 CFU.感染后3周和4周每组分别处死12只小鼠,免疫组小鼠饲养至感染后6周处死.肺和脾组织进行菌落计数,观察肺和脾组织的病理学变化.用实时定量聚合酶链反应方法检测肺和脾组织γ-干扰素、白细胞介素(IL)-4、IL-10、IL-12、IL-18和诱导型一氧化氮合酶(iNOS)mRNA表达的相对含量.组间比较采用t检验.结果 感染后3周,免疫组小鼠的肺和脾组织经培养后无结核分枝杆菌生长.感染后4周,免疫组小鼠的肺和脾组织培养后有结核分枝杆菌生长,但明显少于对照组;肺组织中IL-18、γ-干扰素和iNOS的mRNA表达的相对含量分别为(3.6±0.5、0.32±0.14和23.2±4.7),均明显高于对照组的(1.6±1.1、0.20±0.10和16.2±5.1),IL-12p40 mRNA表达的相对含量(5.7±0.6)明显低于对照组(14.5±1.9),IL-4和IL-10 mRNA表达的相对含量(0.30±0.09和0.28±0.05)与对照组(0.26±0.05和0.29±0.08)无明显差别;脾组织中IL-18、γ-干扰素和iNOS mRNA表达的相对含量(5.5±1.3、0.52±0.07和9.1±1.8)明显高于对照组(0.8±0.4、0.21±0.06和6.0±1.4),IL-12p40、IL-4和IL-10 mRNA表达的相对含量(2.1±0.3、0.23 ±0.10和0.10±0.04)明显低于对照组(5.1±0.4、1.21±0.26和0.57±0.13).与感染后4周比较,免疫组小鼠感染后6周,肺组织γ-干扰素mRNA表达的相对含量(0.95±0.27)明显增高,IL-18、IL-4和IL-10 mRNA表达的相对含量(3.51±0.86、0.45±0.35和0.24±0.21)无明显变化,IL-12p40和iNOS mRNA表达的相对含量(1.72±1.41和1.1±0.5)明显降低;脾组织IL-12p40和IL-18 mRNA表达的相对含量(0.08±0.02)和(0.11±0.03)明显降低,IL-10 mRNA表达的相对含量(0.39±0.11)明显增高,γ-干扰素、IL-4和iNOS mRNA表达的相对含量(0.63±0.32、0.30±0.16和8.4±2.7)无明显变化.结论 CpG-ODN增强小鼠清除体内结核分枝杆菌的能力与IL-18、γ-干扰素和iNOS的表达增强及IL-4和IL-10的表达抑制密切相关.

abstractsObjective To investigate the possible mechanisms of mycobacterial clearance induced by CpG-oligodeoxynucleotides(CpG-ODN)in mice. Methods Eight-week-old female BALB/c mice were treated with intraperitoneal CpG-ODN(30 μg),while the control mice with normal saline. After 2 weeks,the control mice and the CpG-treated mice were infected with Mycobacterium tuberculosis (1 × 106 colonyforming units,H37 Rv strain) through the tail vein. At 3 weeks,4 weeks and 6 weeks after mycobacterial infection,the lung and the spleen tissues were examined for histopathological changes. Real time-PCR was performed to measure the messenger RNA(mRNA) of interleukin(IL)-12,IL-18,interferon(IFN)-γ,IL-4,IL-10 and inducible nitric oxide synthase(iNOS) in the tissues. The homogenates of lungs and spleens were cultured, and the colonies were counted after a 4 weeks incubation period at 37℃. Results At 3 weeks and 4 weeks of mycobacterial infection, CpG-ODN-pretreated mice showed less mycobacterial burden in the lungs and the spleens than that in the control mice [(0±0)×106 CFU/g vs (32±11)×10°CFU/g,(0±0)×10°CFU/g vs(10±4)×106 CFU/g;(26±4)×106 CFU/g vs (56±8)×106 CFU/g,(4±3)×106 CFU/g vs(27±8)×10° CFU/g]. At 4 weeks of mycobacterial infection,CpG-ODN-pretreated mice displayed increased levels of IL-18 mRNA,IFN-γ mRNA,iNOS mRNA[(3.6±0.5,1.6±1.1,0.32±0.14)vs (0.20±0.10, 23.17±4.72, 16.18±5.09)], and decreased level of IL-12p40 mRNA(5.66±0.64 vs 14.54±1.89), but there was no difference in the levels of IL-4 mRNA and IL-10 mRNA in the lungs between CpG-ODN-pretreated mice and the control mice [(0.30±0.09 vs 0.26±0.05),(0.28±0.05 vs 0.29±0.08)]. CoG-ODN-pretreated mice displayed increased levels of IL-18 mRNA,IFN-γ mRNA,iNOS mRNA [(5.54±1.29 vs 0.79±0.36), (0.52±0.07 vs 0.21±0.06),(9.07±1.81 vs 5.97±1.44)],and decreased levels of IL-12p40 mRNA,IL-4 mRNA and IL-10 mRNA [(2.10±0.27 vs 5.07±0.39),(0.23±0.10 vs 1.21±0.26),(0.10±0.04 vs 0.57±0.13)] in the spleens as compared with the control mice. In CoG-ODN-pretreated mice, expression level of IFN-γ mRNA in the lungs at 6 weeks post-infection was higher than that at 4 weeks post-infection(0.95±0.27 vs 0.32±0.14).Conclusion The activation of Toll-like receptor-9(TLR-9) with CpG-ODN could enhance murine mycobacterial clearance in vivo. TLR-9-induced anti-mycobacterial activity involves increased expression of IL-18, IFN-γ, and iNOS but decreased expression of IL-4 and IL-10.