摘要
目的 研究含非甲基化的胞嘧啶-磷酸-鸟嘌呤（CpG）寡脱氧核苷酸（CpG-ODN）能否预防或减轻慢性支气管哮喘（简称哮喘）小鼠气道重塑的发生。方法 40只雌性清洁级C57BL／6小鼠按随机数字表法分为4组，每组10只。（1）慢性哮喘组（A组）：分别于第1、14天小鼠腹腔注射1ml致敏液[卵蛋白（OVA，10μg）＋氢氧化铝凝胶（100μg）]；从第21天开始雾化吸入2．5％OVA溶液，每次30min，每周3次，连续8周。（2）CpG—ODN干预组（B组）：在OVA致敏的同时给予浓度为60μg／ml的CpG—ODN腹腔注射1ml共2次，以后每2周腹腔注射1次共4次。（3）GpC—ODN对照组（C组）：将CpG替换为鸟嘌呤-磷酸-胞嘧啶（GpC，剂量及用法同CpG-ODN）作为对照。（4）生理盐水（NS）对照组（D组）：给予NS进行致敏（每次1ml腹腔注射）和激发（用NS雾化吸入）。在末次激发24h后所有小鼠取血测嗜酸粒细胞（EOS）数和血清IgE；收集支气管肺泡灌洗液（BALF）做细胞分类计数，用酶联免疫吸附测定（ELISA）法检测上清液中自细胞介素13（IL-13）和转化生长因子β1（TGF—β1）的浓度。取左肺组织行苏木精-伊红（HE）染色和Masson三色染色，用抗α平滑肌肌动蛋白（α—SMA）抗体和抗TGF—β1抗体行免疫组化染色。结果 A组外周血EOS计数为（89±10）×10^4／ml、血清IgE为（279±53）ng／ml，BALF中EOS计数和分类分别为（6．30±1．30）×10^5／ml、0．181±0．030，A组IL-13浓度为（4015±361）pg／ml、TGF—β1浓度为（356±64）pg／ml，以上数值与D组比较差异有统计学意义（t值分别为24．0、15．7、14．7、18．4、12．0和18．9，P均〈0．01）；A组Masson三色染色、α-SMA、TGF-β1阳性染色面积百分比[分别为（29．7±4．2）％、（45±7）％、（34±4）％]与D组比较差异也有统计学意义（t值分别为18．0、15．6和17．9，P均〈0．01）。应用CpG-ODN干预后（B组）的Masson三色染色、α—SMA、TGF—β1阳性染色面积百分比[分别为（13．8±3．2）％、（25±3）％、（18±4）％]与A组比较差异也有统计学意义（t值分别为9．5、8．9、9．8，P均〈0．05）。结论应用CpG-ODN干预慢性哮喘模型，不仅能抑制Th2细胞反应和EOS肺浸润，还能抑制气道重塑的发生和发展，它可能是通过抑制TGF—β1、IL-13等因子而抑制气道重塑的。
Objective To investigate if CpG-oligodeoxynucleotides （CpG-ODN） intervention has inhibitory effects on the development of airway remodeling in an ovalbumin （OVA） -sensitized mouse model of chronic asthma. Methods Forty female C57BL/6 mice were randomly divided into four groups （ n = 10） ： （1）Group A （chronic asthma model） ： mice were sensitized by intraperitoneal injection of OVA （10 μg） precipitated with aluminium hydroxide （ 100μg） on days 1 and 14. From day 21, the mice were challenged by nebulized 2. 5% OVA solution （30 min/d, three times a week for 8 weeks）. （2） Group B （ CpG-ODN intervention group） ： mice were sensitized and challenged as above, and were given 60 p,g CpG- ODN by intraperitoneal injection for once every two weeks. （3）Group C （GpG-ODN control） ： Mice were given GpC-ODN instead of CpG motifs, other treatments same as Group B. （4） Group D （saline control） ： mice were sensitized and challenged by saline. All mice were killed 24 h after the final OVA challenge. Blood was obtained for eosinophil counts and measurement of serum IgE by enzyme-linked immunoabsorbent assay （ELISA）. Bronchoalveolar lavage fluid （BALF） was collected for total and differential counts. The concentration of inteleukin-13 （IL-13） and transforming growth factor-betal （TGF-β1） in BALF was measured by ELISA. The left lung was isolated for pathological examination. Lung sections were stained with hematoxylin and eosin （HE）, and Masson＇s triehrome. Other sections were prepared for immunohistochemistry using monoclonal antibodies against α-smooth muscle actin （α-SMA） and TGF-β1. Results The eosinophil count [ （89 ± 10） × 10^4/ml], serum IgE [ （279 ± 53 ）ng/ml ], BALF eosinophils [ （6. 30 ± 1.30） × 10^5/ml] and the concentrations of BALF IL-13 [ （4 015 ± 361 ） pg/ml] and TGF-β1 [ （356 ±64） pg/ml] in the OVA-sensitized mice （Group A） showed significant difference as compared with those in the NS control group （ Group D,t values are 24. 0, 15.7, 14. 7, 18.4, 12.0 and 18.9 respectively, all P〈 0. 01 ）. In Group A, the percentages of positive staining area in Masson＇ s triehrome, α-SMA staining and TGF-β1 staining were （ 29. 7 ± 4. 2 ） % , （ 45 ± 7 ） % and （ 34 ± 4 ） % respectively. These percentages were significantly different from those in the NS control group （ Group D ,t values are 18.0, 15.6 and 17.9 respectively, all P 〈 0. 01 ）. In mice treated with CpG-ODN （ Group B）, the percentages of positive staining area in Masson＇ s triehrome, α-SMA staining and TGF-β1 staining were （ 13.8 ± 3.2） %, （24.7 ±3.1）%, （18 ±4）% respectively, which were significantly different from those in Group A （t values are 9.5, 8.9 and 9. 8 respectively, all P 〈 0.05 ）. Conclusions This study demonstrated that CpG- ODN could prevent Th2 responses, eosinophilic inflammation and the development of airway remodeling. Its inhibitory effect on airway remodeling might, in part, be due to inhibition of the expression of eytokines such as TGF-β1 and IL-13.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2006年第9期612-616,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
上海市卫生局科技发展基金(034094)
