γ干扰素转基因表达对过敏小鼠模型的治疗作用及其机制研究  

The molecular mechanisms of the effects of murine interferon-γ transgenic expression on allergen-induced allergic model via adenoviral vector

作　　者：陈彬[1] 高占成[1] 

机构地区：[1]北京大学人民医院呼吸科,100044

出　　处：《中华结核和呼吸杂志》2005年第5期315-319,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金资助项目(30370609)

摘　　要：目的通过腺病毒载体介导小鼠γ干扰素(mIFNγ)在小鼠肺脏转基因表达,探讨其对卵白蛋白(OVA)诱导过敏小鼠模型的治疗作用及其机制。方法48只BALB/c小鼠,按随机数字表法分为7组,即阴性对照组(A组)、过敏模型组Ⅰ、Ⅱ、Ⅲ组(B、D和F组)、转基因治疗组Ⅰ、Ⅱ、Ⅲ组(C、E和G组)。除A组外,B、C、D、E、F、G各组在第0天和第5天经腹腔给予OVA(每只15μg)致敏,第12～14天经气道吸入0.5%的OVA雾化液激发(20ml/次),建立过敏模型。第15天C、E、G组小鼠经鼻滴入50μl带有mIFNγ基因的复制缺陷型腺病毒(AdCMVmIFNγ)悬液[每只5×109/空斑形成单位(PFU)],A、B、D、F组小鼠经鼻滴入50μl生理盐水作为对照。第18天处死A、B、C组小鼠,第21天处死D和E组小鼠,第25天处死F和G组小鼠,获取支气管肺泡灌洗液(BALF)进行细胞分类计数,其上清液用酶联免疫吸附法测定mIFNγ的浓度,右肺用4%多聚甲醛固定及苏木精伊红(HE)染色,左肺组织提取总RNA,用半定量逆转录聚合酶链反应(RTPCR)了解肺组织中白细胞介素4(IL4)、IL-5、IL-6、IL-10、IL1-2、IL13和IL18的表达。结果(1)经鼻给予AdCMVmIFN-γ后,BALF中可测得mIFNγ的高效表达,C组第3天为(729.0±104.7)pg/ml,E组第6天为(984.5±119.1)pg/ml,G组第10天为(310.6±59.7)pg/ml。(2)B、C、D、Objective To investigate adenoviral vector mediated murine interferon-γ(mIFN-γ) transgene expression and its effect on allergen-induced airway inflammation and multiple interleukin (IL) cytokines (IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and IL-18) expression in a murine allergic model. Methods Forty-eight mice were divided into 7 groups by random digits table: a negative control group (A), allergic model groups Ⅰ, Ⅱ, Ⅲ (B, D, F), gene therapy groups Ⅰ, Ⅱ, Ⅲ (C, E, G). Except for group A, mice of the other groups were peritoneally sensitized with ovalbumin (OVA, 15 μg/mouse) twice on day 0 and day 5, and challenged by inhalation of 0.5% OVA (20 ml per time) twice per day from day 12 to 14. On day 15, AdCMVmIFN-γ(5×10 9 PFU/mouse)solution 50 μl was administrated by nasal drip in groups C, E and G. For groups A, B, D and F, 0.9% NaCl 50 μl was administrated by nasal drip. Groups A, B and C were sacrificed on day 18. Groups D and E were sacrificed on day 21. Groups F and G were sacrificed on day 25. The concentration of mIFN-γ in bronchioalveolar lavage (BALF) was measured by enzyme-linked immunosorbant assay. Accumulation of inflammatory cells and eosinophils (EOS) were quantified by cell count and histopathological analysis. Multi-cytokine expression was tested by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).Results (1) mIFN-γ was efficiently expressed after gene therapy. The concentration of mIFN-γ in BALF was (729.0±104.7)pg/ml 3 days after gene therapy (group C); it was (984.5±119.1)pg/ml after 6 days (group E); and (310.6±59.7)pg/ml after 10 days (group G). (2) The total cell number in BALF of group B and C was (318±41)×10 3/ml and (137±12)× 10 3/ml, respectively(P<0.01); the constituent ratio of EOS in BALF was 0.715±0.054 and 0.452±0.016, respectively(P<0.01). The total cell number in BALF of group D and E was (183±23)×10 3/ml and (92±6)×10 3/ml, respectively(P<0.01); the constituent ratio of EOS was 0.393±0.065 and 0.083±0.038, respectively(P<0

关 键 词：转基因表达 治疗作用 小鼠模型 Γ干扰素 逆转录-聚合酶链反应(RT-PCR) 过敏 机制研究 IFN-γ IL-10 IL-12 IL-13 BALB/C小鼠 复制缺陷型腺病毒 支气管肺泡灌洗液 MRNA丰度 腺病毒载体介导 酶联免疫吸附法 IL-18 空斑形成单位 

分 类 号：R346[医药卫生—基础医学]