人IL-12表达质粒联合含信号肽Mtb8.4基因疫苗对小鼠结核杆菌感染的免疫保护效果  

Plasmid encoding human IL-12 improve protective efficacy of Mtb8.4 gene vaccine with signal sequence against infection of Mycobacterium Tuberculosis

作　　者：李晖[1,2] 李榕 钟森[4] 任红[5] 

机构地区：[1]军事医学科学院野战输血研究所一室 [2]泸州医学院附属医院感染病科,四川泸州646000 [3]江西省南昌市血站 [4]成都中医药大学附属医院 [5]重庆医科大学第二临床学院感染科

出　　处：《细胞与分子免疫学杂志》2007年第4期291-294,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：四川省青年科技基金资助项目(川青科基2002-1)

摘　　要：目的研究结核分枝杆菌含信号肽Mtb8.4(MS)基因疫苗与人白细胞介素12(hIL-12)联合免疫小鼠后,诱导的细胞免疫应答及对小鼠结核杆菌感染的免疫保护效果。方法将40只C57BL/6N小鼠随机分为联合免疫组(MS基因疫苗+hIL-12组)、MS基因疫苗组、卡介苗(BCG)组、空载体组和PBS组。将基因疫苗、空载体和PBS,经肌内注射免疫各组小鼠,每隔3周免疫1次,共免疫3次;BCG组经尾部皮下注射1×106CFU BCG免疫1次。采用ELISA法检测小鼠脾细胞培养上清中细胞因子的水平;用乳酸脱氢酶(LDH)释放法检测免疫小鼠细胞毒性T细胞的杀伤活性。用结核杆菌强毒株H37Rv静脉攻击小鼠后,计数肺和脾组织中结核杆菌的菌落数。对小鼠的部分肺和脾组织作病理切片,经HE染色观察组织病变的程度,经ZN染色检查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果。结果联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液中IFN-γ和IL-2的水平,与BCG组相当,显著高于MS基因疫苗组,IL-4分泌减少,特异性CTL的杀伤活性增强,对小鼠结核杆菌感染有较好的免疫保护效果。表现为小鼠肺和脾组织中结核杆菌的菌落数显著减少,组织病变明显减轻,其效果与卡介苗(BCG)组相当,但优于MS基因疫苗组。结论以hIL-12表达质粒与MS基因疫苗联合免疫后,能显著增强MS基因疫苗对小鼠结核杆菌感染的免疫保护效果。AIM： To study the specific celluar immune response and protective efficacy induced by co-immunization of DNA vaccine of Mtb8.4 with signal sequence （MS） and plasmid encoding human interleukin 12 （hIL-12）. METHODS： Fourty C57BL/6N mice were randomly divided into the following groups： MS gene vaccine plus plasmid of hIL-12, MS gene vaccine, BCG, vacant vector alone and PBS. Mice were immunized intramuscularly in both hind limbs three times at the intervals of three weeks or once subcutaneously with 1×10^6 of viable M. bovis BCG Pasteur at the time of the first DNA immunization （BCG group）. Four weeks after the final inoculation, three mice per group were sacrificed to assess cytokine response by ELISA methods and CTL activities with LDH release assay. The other five mice per group were challenged intravenously in a lateral tail vein with 1×10^6 CFU of virulent M. Tuberculosis H37Rv. Spleen and the left lung were harvested from each mouse at 4 weeks after infection and homogenized in sterile. Serial dilutions of organ homogenates were plated on L-J agar and incubated 37℃ until colonies were visible 4 weeks later. Protective efficacies in each experiment were expressed as reduced CFU and were compared with that in the negative control group. The right lung was obtained from each mouse and inflated with and stored in 10% formalin immediately. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. RESULTS： Co-immunization with MS gene vaccine and plasmid encoding hIL-12 induced the secretion of more of Thl cytokines, but not IL-4, and enhanced CTL activity; while BCG induced the secretion of both types of cytokines （IFN-γ, IL-2 and IL-4）. Co-immunization with MS gene vaccine and plasmid encoding hIL-12 could remarkably reduced CFU counts in organs. CONCLUSION： Plasmid encoding hIL-12 can improve the specific cellular immune response and protective efficacy induced by the MS gene vaccine alone.

关 键 词：含信号肽Mtb8.4(MS) hIL-12 联合免疫 细胞免疫应答 小鼠 

分 类 号：R378.911[医药卫生—病原生物学]