结核菌Mtb8.4基因疫苗对结核杆菌感染小鼠的免疫保护效果研究

Study on the immunogenicity and protective efficacy of Mtb8.4 gene vaccine

为研究Mtb8.4基因疫苗的免疫原性及对小鼠结核杆菌感染的免疫保护效果,将雌性C57BL/6N小鼠32只,随机分为4组,即Mtb8.4基因疫苗组、BCG组、pcDNA3.1(+)组和PBS组。小鼠脾细胞培养上清检测细胞因子水平;并按效靶比例分别为100∶1、50∶1、10∶1进行CTL杀伤活性检测。用结核杆菌H37Rv强毒株静脉攻击小鼠,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果。结果表明,Mtb8.4基因疫苗对小鼠结核杆菌感染有一定的免疫保护效果,能够诱导较强的抗原特异性Th1型细胞免疫应答,细胞因子IFN-γ和IL-2分泌增加,IL-4分泌减少,特异性CTL活性增加;使小鼠肺和脾组织中的结核杆菌菌落数较空载体组显著减少,组织病变明显减轻。

To study the immunogenicity and protective efficacy of Mtb8.4 gene vaccine. C57BL/6N mice were vaccinated with Mtb8.4 gene vaccine for three times at 3 weeks intervals. Four weeks after the final inoculation, three mice were sacrificed to assess cytokine response and CTL induction and the other five mice 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 saline. 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 reduction of CFU and were compared with the negative control group. The right lung was obtained from each mouse and inflated with and stored in 10% formalin saline immediately. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Mtb8.4 gene vaccine induced the secretion of more Th1 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）. Mice immunized with MtbS.4 gene vaccine had fewer and smaller tubercles than control groups. As expected, control mice had the highest bacterial loads in both lung and spleen. Immunization with Mtb8.4 gene vaccine could remarkably reduced CFU counts in organs. Mtb8.4 gene vaccine could induce the secretion of Th1 cytokines and the higher protective efficacy against TB, but the immune response of Mtb8.4 gene vaccine have to be improved in the future.