丙烯腈对大鼠T细胞亚群及Toll样受体4水平的影响

The effects of acrylonitrile on T lymphocyte subsets and expression of toll-like receptor 4 in rats

目的 探讨丙烯腈亚慢性中毒对大鼠T细胞亚群、Toll样受体4（toll-like receptor 4,TLR4）水平及相关细胞因子的影响.方法 64只SD大鼠随机分成雌雄各4组,每组8只,分别以0、5、10、20 mg/kg剂量丙烯腈灌胃染毒,每日1次,每周5次,连续染毒13周.应用流式细胞术检测大鼠血液和脾脏T淋巴细胞亚群（CD3、CD4、CD8）的变化;采用实时荧光定量多聚酶链式反应（Real-time PCR）测定脾脏TLR4、白细胞介素-1β（ILβ）和肿瘤坏死因子（TNF）-α mRNA表达水平;蛋白免疫印迹法（Western blot）测定大鼠脾脏组织TLR4蛋白表达水平.结果 与对照组比较,雌性大鼠20 mg/kg组血液CD3、CD4和5、10、20 mg/kg组CD4/CD8均明显降低,20 mg/kg组CD8明显升高,差异有统计学意义（P＜0.05或P＜0.01）;雄性大鼠5 mg/kg组CD3,20 mg/kg组CD4和CD4/CD8均明显降低,20 mg/kg组CD8明显升高,差异有统计学意义（P＜0.05或P＜0.01）.与对照组比较,雌性大鼠20 mg/kg组脾脏CD4和CD4/CD8明显降低,20 mg/kg组CD8明显升高,差异有统计学意义（P＜0.05或P＜0.01）;雄性大鼠20mg/kg组CD4和10、20 mg/kg组CD4/CD8明显降低,20 mg/kg组CD3和CD8明显升高,差异有统计学意义（P＜0.05或P＜0.01）;雄性大鼠5、10、20 mg/kg组和雌性大鼠10 mg/kg组脾脏TLR4 mRNA表达明显降低,差异有统计学意义（P＜0.05或P＜0.01）;雌性大鼠5 mg/kg组和雄性大鼠20 mg/kg组脾脏TLR4蛋白表达水平明显降低,差异有统计学意义（P＜0.05）;雌性大鼠5、10、20 mg/kg组和雄性大鼠5、10 mg/kg组脾脏IL-1β mRNA表达量明显降低,差异有统计学意义（P＜0.05或P＜0.01）;雌性大鼠10、20 mg/kg组和雄性大鼠5、10 mg/kg组脾脏TNF-α mRNA表达水平明显降低,差异均有统计学意义（P＜0.01）.结论 丙烯腈可引起血液及脾脏T淋巴细胞亚群CD3、CD4、CD8及CD4和CD8的比值的变化,同时也明显影响TLR4 mRNA和蛋白的表达及相关细胞因子IL-1β、TNF-α分泌,这将会给机体的细胞免疫功能带来影响.

Objective To explore the effects of acrylonitrile on T lymphocyte subsets,expression of toll-like receptor 4 and related cytokines in rats.Methods Sixty-four Sprague-Dawley rats were randomly divided into 4 female groups and 4 male groups,and there were 8 rats in each group.Rats in each group were respectively given a single dose of0,5,10 and 20 mg/kg acrylonitrile by gavage,once a day,5 days a week,for 13 weeks.Blood and spleen T lymphocyte subsets was detected by flow cytometry,the mRNA expression of TLR4,IL-1β and TNF-α was analyzed by real-time quantitative PCR,the protein expression of TLR4 was evaluated by Western blot.Results Compared with control group,the percentages of blood CD3,CD4 T cells in 20 mg/kg female group and CD4/CD8 ratio in 5,10 and 20 mg/kg female groups was significantly decreased,CD8 T cells in 20 mg/kg group was significantly increased （P〈0.05 or P〈0.01）,blood CD3 T cells in 5 mg/kg male group,CD4 T cells and CD4/CD8 ratio in 20 mg/kg male groups were lower than that of control group,CD8 T cells in 20 mg/kg make group was sigmificantly inoreased （P〈0.05 or P〈0.01）.Spleen CD4,CD8 T lymphocyte percentages and CD4/CD8 ratio in 20 mg/kg female group decreased significantly,CD8 T cells in 20 mg/kg male group was significantly increased （P〈0.05 or P〈0.01）,spleen CD3,CD4,CD8 T cells in 20 mg/kg male group and CD4/CD8 ratio in 10,20 mg/kg male groups was also significantly decreased,CD3 T cells in 20 mg/kg and CD8 T cells in 10,20 mg/kg male groups were sigmificantly increased （P〈0.05 or P〈0.01）,TLR4 mRNA was lower expressed in 5,10 and 20 mg/kg male groups and 10 mg/kg female group （P〈0.05 or P〈0.01）,and TLR4 protein in 5 mg/kg female group and 20 mg/kg male group was significantly lower than control group （P〈0.05）.The expression level of IL-1β mRNA was significantly decreased in 5,10 and 20 mg/kg female group and 5,10 mg/kg male group （P〈0.05 or P〈0.01）,TNF-α mRNA was lower expressed in 10,20 mg/kg female groups and 5,10 mg/kg male groups（P〈0.01）.Conclusion Acrylonitrile may lead to the changes of CD3,CD4,CD8 T lymphocyte percentages and CD4/CD8 ratio in rat blood and spleen,and also significantly effected the expression level of TLR4 mRNA and protein together with the secretion of IL-1β,TNF-α.This may cause effects on the cellular immune function.