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目的研究库普弗细胞(Kupffer cell)在小鼠日本血吸虫(Schistosoma japonicum)肝病发展过程中的表型变化。方法将20只6周龄的BALB/c雄性小鼠经腹部皮肤感染16条日本血吸虫尾蚴,在感染后0、21、32、42、52 d分别处死小鼠,取肝脏组织,HE染色和Masson三色染色观察肝脏病理变化,实时定量PCR(q PCR)检测肝脏Th1型细胞因子γ干扰素(interferon-γ,IFN-γ)、α肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、Th2型细胞因子白细胞介素-4(interleukin-4,IL-4)、IL-13、IL-10)以及库普弗细胞的M1型巨噬细胞分化标志物诱导型一氧化氮合酶(inducible nitric oxide synthetase,i NOS)、白细胞分化抗原16(cluster of differentiation 16,CD16)、IL-6和M2型巨噬细胞分化标志物精氨酸酶-1(arginase 1,Arg-1)、CD206、IL-10的表达。体外培养库普弗细胞系,分别给予0、5、25、50 ng/ml IFN-γ或IL-4刺激12 h,或者给予25 ng/ml IFN-γ或IL-4分别刺激0、12、24、36 h后,q PCR检测库普弗细胞M1型、M2型巨噬细胞分化标志物。结果HE染色和Masson三色染色结果显示,小鼠感染后32 d肝组织中开始有虫卵沉积,42 d有明显的肉芽肿和纤维化病变。q PCR结果显示,与感染后0 d相比,IFN-γ在32 d表达水平最高,相对表达量为29.243±3.245,52 d时迅速下降,为8.923±3.002。IL-4在42 d最高,为25.521±4.957。IL-13在52 d最高,为50.793±9.631(均P<0.05)。i NOS在42 d表达水平上升,相对表达量为2.950±0.321,在52 d下降,为1.783±0.319。Arg-1在52 d最高,为2.003±0.152(均P<0.05)。0、5、25、50 ng/ml IFN-γ刺激库普弗细胞12 h后,i NOS、IL-6和CD16的相对表达量分别为54.690~68.577、1.887~2.427、2.417~2.787(均P<0.05)。25 ng/ml IFN-γ刺激0、12、24、36 h后,i NOS、IL-6和CD16的相对表达量分别为34.810~109.210、10.327~15.143、1.887~3.317(均P<0.05),而Arg-1与对照组相比无明显变化。0、5、25、50 ng/ml IL-4刺激库普弗细胞12 h后,Arg-1、IL-10和CD206的相对表达量分别为9.153~24.253、1.923~3.687和37.770~72.133(均P<0.05);25 ng/ml IL-4刺激0、12、24、36 h后Arg-1、IL-10和CD206的相对表达量分别为3.563~12.613、1.637~2.673和19.732~71.943(均P<0.05),而i NOS无明显变化。结论在日本血吸虫感染早期,库普弗细胞以M1型为主;而在感染中晚期,库普弗细胞以M2型为主,表明库普弗细胞转变与肝脏免疫微环境密切相关。
Objective To investigate the phenotypic changes of Kupffer cells in the development of Schistosoma japonicum liver disease in mice. Methods Sixteen BALB / c male mice of 6 weeks old were infected with cercariae of Schistosoma japonicum through abdominal skin. Mice were sacrificed at 0, 21, 32, 42 and 52 d after infection. Liver tissue, HE staining and Masson Trichrome staining was used to observe the pathological changes of the liver. Real-time qPCR was used to detect the expression of Th1 cytokine IFN-γ, TNF-α, Th2 cytokines interleukin-4 (IL-4), IL-13, IL-10) and Kupffer cell type M1 macrophage differentiation marker inducible nitric oxide synthase (inducible nitric oxide synthetase (iNOS), cluster of differentiation 16 (CD16), arginase 1 (Arg-1), CD206, IL-10 expression. The Kupffer cell line was cultured in vitro and stimulated with 0, 5, 25, 50 ng / ml IFN-γ or IL-4 for 12 h or 25 ng / ml IFN-γ or IL- After 24 and 36 h, qPCR markers M1 and M2 macrophage differentiation markers were detected by q PCR. Results HE staining and Masson trichrome staining results showed that the mice began to deposit eggs in the liver tissue 32 days after infection and there were obvious granuloma and fibrosis lesions on the 42th day. q PCR results showed that the expression level of IFN-γ was the highest at 32d after infection, and the relative expression level was 29.243 ± 3.245 at 52d, dropping rapidly to 8.923 ± 3.002. The highest level of IL-4 at 42 days was 25.521 ± 4.957. The highest level of IL-13 at 52 d was 50.793 ± 9.631 (all P <0.05). The expression of iNOS increased at 42 d, with a relative expression of 2.950 ± 0.321 and a decrease of 1.783 ± 0.319 at 52 d. Arg-1 was the highest at 52 days, 2.003 ± 0.152 (all P <0.05). The relative expression levels of iNOS, IL-6 and CD16 in Kupffer cells stimulated with 0, 5, 25 and 50 ng / ml IFN-γ for 54 h are respectively from 54.690 to 68.577, 1.887 to 2.427 and 2.417 to 2.787 <0.05). The relative expression levels of iNOS, IL-6 and CD16 after being treated with 25 ng / ml IFN-γ for 0, 12, 24 and 36 h were 34.810-109.210,10.327-15.143 and 1.887-3.317 (all P <0.05) The Arg-1 compared with the control group no significant change. The relative expression levels of Arg-1, IL-10 and CD206 after stimulation with Kupffer cells by 0, 5, 25 and 50 ng / ml IL-4 for 24 hours were 9.153-24.253, 1.923-3.687 and 37.770-72.133 P <0.05). The relative expression levels of Arg-1, IL-10 and CD206 at 25, 24, 36 and 36 h after stimulation with 25 ng / ml IL-4 were 3.563-12.613, 1.637-2.6673 and 19.732-71.943 P <0.05), while i NOS no significant change. Conclusions In the early stage of Schistosoma japonicum infection, the Kupffer cells are predominantly M1. In the late and middle stages of infection, the Kupffer cells are predominantly M2, indicating that the Kupffer cell transformation is closely related to the immunological microenvironment of the liver.