应用特异性抗原刺激树突细胞增强小鼠抵抗烟曲霉感染的能力

Use of Aspergillus-pulsed DC in mice to enhance their host defense against Aspergillus infection

目的探讨曲霉抗原刺激树突细胞(DC)应用于小鼠后对小鼠抵抗曲霉感染能力的影响。方法小鼠骨髓制备DC,尾静脉接种小鼠;腹腔内注射环磷酰胺后,鼻腔内滴入烟曲霉孢子制备侵袭性肺曲霉病小鼠模型;获取小鼠肺组织并进行匀浆,假丝酵母(念珠菌)显色培养基接种后进行菌落计数,酶联免疫吸附试验(ELISA)检测mγ干扰素(ndFN-γ)含量,部分肺组织进行HE和GMS染色;以反转录．聚合酶链反应(RT-PCR)的方法检测小鼠脾脏中细胞因子IFN-γ的mRNA表达。结果与单纯接种DE和热灭活烟曲霉(HAF)的小鼠相比,烟曲霉抗原刺激DC回输的小鼠存活率显著增高,脾脏内IFN-γ的mRNA表达增加,肺组织烟曲霉负荷明显降低,肺组织匀浆中IFN-γ含量(3．60±1．57ng/ml)亦高于非刺激DC免疫小鼠(HAF．1．35±0．47ngml;单纯DC,1．1±0．42ngml,P＜0．01),接受单纯DC和HAF的小鼠肺组织可见烟曲霉孢子、菌丝生长,有支气管壁的破坏,支气管周围坏死,肺泡和间质内炎症细胞浸润,而接受烟曲霉抗原刺激DC的小鼠肺内浸润炎症细胞减少,未发现坏死和真菌生长。结论应用烟曲霉抗原刺激DC,可以在小鼠体内诱导特异性Th1型反应．增强小鼠抵抗烟曲霉感染的能力。

Objective To study the influence on host defense against Aspergillus infection when immunized with Aspergillus-pulsed DC in mice. Methods DC were generated from mice bone marrow cells and pulsed with the antigen of Aspergillus fumigatus. Then DC were transfered into mice via the lateral tail vein. Immunocompromised mice were made by treatment with cyclophosphamide administered intraperitoneally （i. p. ）. Suspension of conidia was applied to the nostrils of mice to develop the model of invasive pulmonary aspergillosis （IPA）. Lungs were harvested and homogenized. Portions of homogenates were cultured to determine the number of CFU. IFN-γ in lung homogenates was determined by a eytokine-speeifie ELISA kit. Reverse transcription PCR （RT-PCR） analysis was used to determine the mRNA of IFN-γ in spleen cells.Results Compared with naive DC and heat-inactivated Aspergillus fumigatus （HAF） immunized mice, the mice immunized with Aspergillus-pulsed DC demonstrated prolonged survival time, had significantly lower concentration of infectious Aspergillus organisms in their lung tissues,and had higher levels of IFN-γ in spleen cells and lung tissues（3.60 ± 1.57 mg/ml vs 1.10 ± 0.42 ng/ml, 1.35 ± 0.47 ng/ml）. Lung sections from HAF and naive DC-treated mice revealed patterns of lesions characterized by bronchial wall damage, peribronchial necrosis, and the presence of numerous infiltrating inflammatory cells. Conidia and hyphae were seen in these mice. In contrast, these features were not observed in HAF-pulsed DC treated mice whose lung were characterized by few inflammatory cells infiltration, and no evidence of fungal growth after inoculation. Conclusion Transfered into mice, Aspergillus-pulsed DC can induce Thl response to the fungus and enhance their host defense against Aspergillus infection.