T-bet基因传递对支气管哮喘模型小鼠T淋巴细胞亚群的免疫调节作用  

Immunoregulation effect of transmission of T-bet gene on T cell subgroups in asthmatic Balb/c mice

作　　者：潘小晶[1] 王锁英[1] 许化溪[2] 王胜军[2] 

机构地区：[1]江苏大学附属医院儿科,江苏镇江212001 [2]江苏大学检验医学研究所

出　　处：《中华实用儿科临床杂志》2013年第4期281-284,共4页Chinese Journal of Applied Clinical Pediatrics

摘　　要：目的应用携带T-bet基因的重组腺相关病毒载体（rAAV-T-bet）经鼻干预支气管哮喘（哮喘）小鼠模型，探讨T-bet基因传递对哮喘小鼠T淋巴细胞亚群的免疫调节及改善炎症的作用。方法采用随机数字表法将40只健康的6～8周龄SPF级Balb／c小鼠随机分为正常对照组（A组）、哮喘模型组（B组）、rAAV介导的增强型绿色荧光蛋白（rAAV．eGFP）干预对照组（C组）及rAAV-T-bet干预组（D组），每组10只。B、c、D组以卵清蛋白（OVA）致敏和激发，建立哮喘小鼠模型，其中C、D组在第18、19天分别经鼻滴入等剂量rAAV-eGFP、rAAV．T-bet进行干预；B组处理方法同上，以等剂量的9g／L盐水干预。于最后-次激发24h后处死小鼠并取材。应用免疫组织化学方法观察小鼠肺组织中转录因子T-bet、GATA-3和Foxp3水平；计数BALF中细胞总数并分析其细胞类型；ELISA法测定BALF中细胞因子IFN-γ、IL-4、IL-5表达水平。结果1．B组、C组中T-bet表达细胞的阳性率均明显低于A组（P均〈0．05），GATA-3表达细胞的阳性率明显高于A组（P均〈0．05），D组中T-bet表达明显增强，GATA-3表达则显著下降（P均〈0．05），Foxp3的表达阳性率在各组间的差异与T-bet的表达类似。2．D组BALF中细胞总数为（0．35±0．11）×10^9／L，其中EOS比例为0．04±0．02，虽高于A组，但明显低于B、C组（P均〈0．05）；IL-4和IL-5分别为（158±55）ng／L、（68±22）ng／L，显著低于B、C组（P均〈0．05），高于A组但差异无统计学意义（P均〉0．05），而IFL-γ水平[（113±35）ng／L]明显低于B、C组（P均〈0．05），与A组比较水平相近（P〉0．05）。结论在建立小鼠哮喘模型的基础上，经鼻应用rAAV-T-bet干预可使T-bet基因顺利定向插入到受转染细胞的基因组中，成功编码T-bet蛋白，并进-步对哮喘小鼠模型发挥相应的免疫调节作用。Objective To construct the adeno-associated virus （AAV）vector of mouse carrying T-bet gene, which was applied to mouse model of asthma by nose, and to investigate the immunomodulatory effects of T-bet delivery on asthma. Methods Forty healthy Balb/c mice （ aged 6-8 weeks） were randomly divided into 4 groups ： control group （group A）, asthma model group （group B） , model/control recombinant adeno-assoeiated virus carrying enhanced green fluorescent protein（ rAAV- eGFP） intervention group （group C） and model/rAAV-T-bet virus intervention group （group D） , with 10 mice in each group. In group B, group C, and group D, Balb/e mice were sensitized by intraperitoneal injection with OVA and challenged by nebulized OVA. In group C and group D, Balb/e mice were intervened with the isodose rAAV-eGFP and rAAV-T-bet, while in group B, the mice were intervened with the same amount of saline, both of them were dropped into the nasal cavity. Twenty-four hours after the last injection ,the mice were sacrificed and the samples were obtained. The total cells in bronchoalveolar lavage fluid （BALF） were counted, and the types of cells were analyzed by Wright-Giemsa staining. The levels of interferon-γ（ IFN-γ ）, IL-4 and IL-5 in BALF were determined by enzyme-linked immunosorbent assay. The expressions of T-bet, GATA-3 and Foxp3 protein in the lungs of asthmatic mice were detected with immunohistochemical technique. Results 1. The level of specific transcription factor T-bet in group B and group C were distinctly lower than that of group A （ all P 〈 0.05 ）, while the level of GATA-3 was significantly higher than that of group A （ all P 〈 0.05 ）. In group D, the expression of T-bet protein was stronger than that in group B and group C, however, the expression of GATA-3 decreased obviously （ all P 〈 0.05 ）. The results of Foxp3 were similar to T-bet. 2. In group D, the mice had suppressed levels of IL-4 [ （158 ± 55 ） ng/L] and IL-5 [ （68 ± 22） ng/L ] compared with those

关 键 词：支气管哮喘 T-BET GATA-3 FOXP3 免疫调节 

分 类 号：R285.5[医药卫生—中药学]