摘要
从百日咳杆菌中分离出非甲基化寡核苷酸单链(CpG-ODNs),建立卵白蛋白(OVA)致敏的小鼠急性哮喘与慢性哮喘模型,经急性哮喘模型从百日咳杆菌CpG-ODNs与9条相似度高且含有CG片段的seq A～seq I序列中筛选出有效序列(seq A),以seq A腹腔注射(ip)及灌胃(ig)对急、慢性哮喘小鼠分别进行干预。检测支气管肺泡灌洗液(BALF)中白细胞(WBC)总数和嗜酸性粒细胞(EOS)数,酶联免疫吸附法(ELISA)检测BALF中细胞因子及血管内皮生长因子(VEGF)水平。逆转录聚合酶链式反应(RT-PCR)检测小鼠脾细胞TLR-9 mRNA表达量;蛋白印迹(Western blotting)法检测小鼠肺组织TLR-9蛋白表达。结果表明,小鼠急性哮喘模型经腹腔注射及灌胃seq A预处理,可明显抑制BALF中WBC总数增加,显著抑制EOS增加;百日咳杆菌、CpG+及seq A～seq D灌胃组亦显著抑制EOS增加,而seq E～seq I作用不明显;上述各序列干预组以腹腔注射途径抑制EOS作用较为显著。对小鼠慢性哮喘模型,CpG+和seq A腹腔注射预处理均能明显上调BALF中γ干扰素(IFN-γ)①空白对照组:(176.45±23.46)pg.mL-1;②模型组:(174.11±22.71)pg.mL-1;③CpG+(ip):(220.56±15.42)pg.mL-1;④seq A(ip):(225.23±21.60)pg.mL-1;并下调白介素-4(IL-4)①空白对照组:(66.91±5.81)pg.mL-1;②模型组:(81.02±11.24)pg.mL-1;③CpG+(ip):(63.99±6.09)pg.mL-1;④seq A(ip):(62.75±10.03)pg.mL-1;各序列组对VEGF无显著影响(P>0.05);seq A腹腔注射亦能上调小鼠脾细胞TLR-9 mRNA(TLR-9/GAPDH)①空白对照组:0.62±0.13;②模型组:0.66±0.17;③CpG+(ip):1.46±0.26;④seq A(ip):1.42±0.34及肺组织TLR-9蛋白表达(TLR-9/β-actin)①空白对照组:0.63±0.16;②模型组:0.61±0.07;③CpG+(ip):1.15±0.25;④seq A(ip):1.03±0.29;但经灌胃途径无明显影响。结果显示,CpG-ODNs和获得的CpG序列seq A可显著抑制哮喘模型的气道炎症反应以及相应炎症细胞因子水平的改变,其作用机制可能与调节Th1/Th2平衡、调控Toll样受体9(TLR-9)表达有关。
This study focused on prevention and treatment of acute and chronic asthma by oligonucleotides containing unmethylated CpG motifs（CpG-ODNs）.Acute and chronic asthma models of mice were made by sensitizing and inhaling ovalbumin（OVA）;The number of white blood cells（WBC） and eosnophils（EOS） in bronchoalveolar lavage fluid（BALF） was counted and the concentration of cytokines and vascular endothelial growth factor（VEGF） was examined in BALF by ELISA kit.After that,TLR-9 mRNA was detected in mice spleen cells by reverse transcription polymerase chain reaction（RT-PCR） and TLR-9 protein was determined in mice lung tissues by Western blotting.Compared with acute asthma models of mice,WBC in BALF decreased obviously in the groups of Bordetella pertussis,CpG-ODNs and seq A to seq I which were administrated by both of intragastric（ig） and intraperitoneal（ip） injection group,EOS decreased obviously in Bordetella pertussis,CpG＋ and seq A to seq D ig groups,and in all ip administrating groups,although it was not effective in the groups of seq E to seq I.In chronic asthma models of mice,IFN-gamma increased（① control： 176.45 ± 23.46 pg·mL-1;② model： 174.11 ± 22.71 pg·mL-1;③ CpG＋ ip： 220.56 ± 15.42 pg·mL-1;④ seq A ip： 225.23 ± 21.60 pg·mL-1） and IL-4 decreased obviously（① control： 66.91 ± 5.81 pg·mL-1;② model： 81.02 ± 11.24 pg·mL-1;③ CpG＋ ip： 63.99 ± 6.09 pg·mL-1;④ seq A ip： 62.75 ± 10.03 pg·mL-1） in the BALF of CpG＋ and seq A ip group,although VEGF was not changed in this research.And also,TLR-9 mRNA in spleen cells（TLR-9/GAPDH： ① control： 0.62 ± 0.13;② model： 0.66 ± 0.17;③ CpG＋ ip： 1.46 ± 0.26;④ seq A ip： 1.42 ± 0.34） and TLR-9 protein in lung tissues（TLR-9/β-actin： ① control： 0.63 ± 0.16;② model： 0.61 ± 0.07;③ CpG＋ ip： 1.15 ± 0.25;④ seq A ip： 1.03 ± 0.29） both increased in ip groups,but the change was not significant in ig group.The study confirms that CpG-ODNs and seq A could inhibit airway inflammation remarkably,this mechanism might be related with regulating Th1/Th2 balance and controlling the expression of TLR-9.
出处
《药学学报》
CAS
CSCD
北大核心
2011年第3期285-292,共8页
Acta Pharmaceutica Sinica
