CD40对烟草烟雾暴露肺气肿小鼠肺部CD8＋T细胞毒性功能的影响

Effect of CD40 knock out on cytotoxic effector function in CD8 ＋ T cell of mice with cigarette smoke- induced emphysema

目的探讨CD40对烟草烟雾暴露肺气肿小鼠肺部CD8^＋T细胞毒性功能的影响。方法40只雄性C57小鼠按随机数字表法随机分为CD40^＋/＋对照组、CD40^＋/＋烟熏组、CD40^-/-对照组、CD40^-/-烟熏组4组，烟熏组采用烟草烟雾暴露24周建立小鼠肺气肿模型。各组肺组织分别采用HE染色观察肺部病理改变并计算肺泡平均内衬间隔；免疫组织化学法检测CD8^＋T细胞和穿孔素^＋、颗粒酶B^＋细胞百分比；实时荧光定量PCR（RT—PCR）检测穿孔素、颗粒酶B、白细胞介素（IL）-27mRNA表达；酶联免疫吸附（ELISA）法检测IL-27细胞因子水平。结果CD40^＋/＋烟熏组肺组织明显破坏，可见大量炎症细胞浸润；CD40^-/-烟熏组肺组织破坏及炎症细胞浸润程度较CD40^＋/＋烟熏组轻。CD40^＋/＋烟熏组肺泡平均内衬间隔显著高于CD40^＋/＋对照组、CD40^-/-对照组和CD40^-/-烟熏组[（37．2±3．6）比（24．0±3．4）、（22．5±2．4）、（29．9±1．7）μm]（均P〈0．05）；CD40。一烟熏组高于CINO“’对照组及CD40。一对照组（均P〈0．05）。CD40“’烟熏组CD8^＋、穿孔素^-、颗粒酶B^＋细胞百分比[（16．3±2．3）％、（11．4±2．1）％、（10．7±1．9）％]均显著高于CD40^＋/＋对照组[（8．3±1．6）％、（5．1±1．2）％、（4．6±1．0）％]、CD40^-/-对照组[（6．4±1．5）％、（4．3±1．0）％、（4．2±1．0）％]、CI40^-/-烟熏组[（8．6±1．7）％、（5．6±1．3）％、（5．5±1．3）％]（均P〈0．05）。CD40^＋/＋烟熏组穿孔素、颗粒酶B、IL-27mRNA相对表达量[（20．3士7．3）、（18．3±12．3）、（2．24-0．7）]均显著高于CIMO^＋/＋对照组[（9．4士4．8）、（10．6±3．8）、（1．3±0．6）]、CD40^-/-对照组[（8．1±3．1）、（7．7±3．5）、（1．1±0．5）]、CD40^-/-烟熏组[（12．94-6．2）、（10．4±4．6）、（1．5±0．4）]（均P〈0．05）。CD^＋/＋烟熏组细胞因子IL-27水平显著高于CD40^＋/＋对照组、CD40^-/-对照组、CD40^-/-烟熏组[（3242±754）比（1627±710）、（1600±680）、（1850±583）ng／L]（均P〈0．05）。结论CD40基因敲除可以抑制烟草烟雾暴露肺气肿小鼠肺部CD8^＋T细胞毒性功能，减轻肺气肿程度。

Objective To explore the effect of CIM0 knock out on the cytotoxic function of CD8 ~ T cell of mice with eigarette smoke-induced emphysema. Methods A total of 40 male C57 mice were divided into four groups according to the random number table, including CD40 ^＋/＋ control group, CD40^＋/＋ smoke- exposure group, CD40^-/-control group, CD40 ^-/-smoke-exposure group. The smoke-exposure groups were exposed to cigarette smoke for 24 weeks to establish emphysema model. Morphological changes were evaluated by linear intercepts. The percentages of CD8, perforin, granzyme B positive eells were evaluated by immunohistochemistry. The mRNA expressions of perforin, granzyme B, interleukin （IL） -27 were measured by fluorescent real time quantitative polymerase chain reaction （RT-PCR）. The IL-27 cytokine level was tested by enzyme-linked immunosorbent assay （ELISA）. Results The mean linear intercepts in CD40^＋/＋ smoke-exposure group was significantly higher than CD40^＋/＋ control group, CD40^-/- control group, and CD40^ -/- smoke-exposure group [ （ 37.2 ± 3.6 ） vs （ 24. 0 ± 3.4 ）, （ 22. 5± 2.4 ）, （ 29. 9 ±1.7） μm] （ all P 〈 0. 05 ） . CD40^-/- smoke-exposure group was higher than CD40^＋/＋ control group, CD40^ -/ -control group （all P 〈 0. 05 ）. The percentages of CD8 positive, perforin positive and granzyme B positive cells in CD40 ＋/＋ smoke-exposure group [ （ 16. 3 ± 2. 3 ） %, （ 11.4 ± 2. 1 ） %, （ 10. 7 ± 1.9 ） % ] were significantly higher than CD40^＋/＋ control group [ （8.3 ± 1.6）%, （5.1 ± 1.2）%, （4. 6 ± 1.0）% ], CD40^ -/- control group [ （6.4 ± 1. 5 ） % , （4. 3 ± 1.0） % , （4. 2 ± 1.0） % ] and CD40^ -/- smoke-exposure group [（8.6±1.7）%, （5.6±1.3）%, （5.5±1.3）%] （allP〈0.05）. RT-PCR results showed that the mRNA expressions of perforin, granzyme B and IL-27 in CD40 ^＋/＋ smoke-exposure group [ （20. 3 ± 7.3）, （ 18.3 ± 12. 3）, （2.2 ±0. 7） ] were significantly higher than CD40 ^＋/＋ control group [ （9.4±4. 8）, （10.6±3.8）, （1.3 ±0.6）], CD40^-/- control group [ （8.1 ±3.1）, （7.7±3.5）, （1.1 ±0.5）] and CD40 ^-/-smoke-exposuregroup [（12.9±6.2）, （10.4±4.6）, （1.5±0.4）] （allP〈0.05）. ELISA results showed that the level of IL-27 in CD40^-/- smoke-exposure group was significantly higher than CD40 ^＋/＋ control group, CD40^＋/＋ control group and CD40^-/- smoke-exposure group [ （ 3 242 ± 754） vs （1 627±710）, （1 600±680）, （1 850±583） ng/Ll （allP〈0.05）. Conclusion Knockout the CD40 gene can inhibit the cytotoxic effector function in CD8 ^＋ T cells of mice with cigarette smoke-induced emphysema, and alleviate the degree of emphysema.