發(fā)布時(shí)間：2019-01-09 09:22

【摘要】：目的研究庫普弗細(xì)胞(Kupffer cell)在小鼠日本血吸蟲(Schistosoma japonicum)肝病發(fā)展過程中的表型變化。方法將20只6周齡的BALB/c雄性小鼠經(jīng)腹部皮膚感染16條日本血吸蟲尾蚴,在感染后0、21、32、42、52 d分別處死小鼠,取肝臟組織,HE染色和Masson三色染色觀察肝臟病理變化,實(shí)時(shí)定量PCR(q PCR)檢測(cè)肝臟Th1型細(xì)胞因子γ干擾素(interferon-γ,IFN-γ)、α腫瘤壞死因子(tumor necrosis factor-α,TNF-α)、Th2型細(xì)胞因子白細(xì)胞介素-4(interleukin-4,IL-4)、IL-13、IL-10)以及庫普弗細(xì)胞的M1型巨噬細(xì)胞分化標(biāo)志物誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthetase,i NOS)、白細(xì)胞分化抗原16(cluster of differentiation 16,CD16)、IL-6和M2型巨噬細(xì)胞分化標(biāo)志物精氨酸酶-1(arginase 1,Arg-1)、CD206、IL-10的表達(dá)。體外培養(yǎng)庫普弗細(xì)胞系,分別給予0、5、25、50 ng/ml IFN-γ或IL-4刺激12 h,或者給予25 ng/ml IFN-γ或IL-4分別刺激0、12、24、36 h后,q PCR檢測(cè)庫普弗細(xì)胞M1型、M2型巨噬細(xì)胞分化標(biāo)志物。結(jié)果HE染色和Masson三色染色結(jié)果顯示,小鼠感染后32 d肝組織中開始有蟲卵沉積,42 d有明顯的肉芽腫和纖維化病變。q PCR結(jié)果顯示,與感染后0 d相比,IFN-γ在32 d表達(dá)水平最高,相對(duì)表達(dá)量為29.243±3.245,52 d時(shí)迅速下降,為8.923±3.002。IL-4在42 d最高,為25.521±4.957。IL-13在52 d最高,為50.793±9.631(均P0.05)。i NOS在42 d表達(dá)水平上升,相對(duì)表達(dá)量為2.950±0.321,在52 d下降,為1.783±0.319。Arg-1在52 d最高,為2.003±0.152(均P0.05)。0、5、25、50 ng/ml IFN-γ刺激庫普弗細(xì)胞12 h后,i NOS、IL-6和CD16的相對(duì)表達(dá)量分別為54.690~68.577、1.887~2.427、2.417~2.787(均P0.05)。25 ng/ml IFN-γ刺激0、12、24、36 h后,i NOS、IL-6和CD16的相對(duì)表達(dá)量分別為34.810~109.210、10.327~15.143、1.887~3.317(均P0.05),而Arg-1與對(duì)照組相比無明顯變化。0、5、25、50 ng/ml IL-4刺激庫普弗細(xì)胞12 h后,Arg-1、IL-10和CD206的相對(duì)表達(dá)量分別為9.153~24.253、1.923~3.687和37.770~72.133(均P0.05);25 ng/ml IL-4刺激0、12、24、36 h后Arg-1、IL-10和CD206的相對(duì)表達(dá)量分別為3.563~12.613、1.637~2.673和19.732~71.943(均P0.05),而i NOS無明顯變化。結(jié)論在日本血吸蟲感染早期,庫普弗細(xì)胞以M1型為主;而在感染中晚期,庫普弗細(xì)胞以M2型為主,表明庫普弗細(xì)胞轉(zhuǎn)變與肝臟免疫微環(huán)境密切相關(guān)。
[Abstract]:Objective to study the phenotypic changes of (Kupffer cell) in mouse Schistosoma japonicum (Schistosoma japonicum) liver disease. Methods Twenty 6-week-old male BALB/c mice were infected with 16 cercariae of Schistosoma japonicum through abdominal skin. The mice were killed on day 0, 21, 32 and 42 respectively. Liver tissue, HE staining and Masson trichrome staining were used to observe the pathological changes of the liver. Real time quantitative PCR (q PCR) was used to detect interferon- 緯 (IFN- 緯), (tumor necrosis factor- 偽 (TNF- 偽) and interleukin-4,IL-4 (Th2 type cytokine) in liver. IL-13,IL-10) and inducible nitric oxide synthase (inducible nitric oxide synthetase,i NOS),) leukocyte differentiation antigen 16 (cluster of differentiation 16 CD16 in Kupffer cells. Expression of IL-6 and M 2 macrophage differentiation marker arginase 1 (arginase 1 Arg 1) and CD206,IL-10. The Kupffer cell line was cultured in vitro and stimulated for 12 h with 0 ~ 5U 2550 ng/ml IFN- 緯 or IL-4, or with 25 ng/ml IFN- 緯 or IL-4 for 36 h., q PCR was used to detect the M1 type of Kupffer cell line. M 2 macrophage differentiation markers. Results the results of HE staining and Masson trichrome staining showed that there was egg deposition in the liver tissue of mice 32 days after infection, and obvious granuloma and fibrosis on 42 days after infection. The results showed that compared with 0 days after infection, there were obvious granuloma and fibrosis in the liver of mice. The expression level of IFN- 緯 was the highest at 32 days, and the relative expression of IFN- 緯 decreased rapidly at 52 days after 29.243 鹵3.245 days, 8.923 鹵3.002.IL-4 at 42 days, 25.521 鹵4.957.IL-13 at 52 days. 50.793 鹵9.631 (P0.05) the expression level of). I NOS increased at 42 days, the relative expression level was 2.950 鹵0.321, decreased at 52 days, 1.783 鹵0.319.Arg-1 was highest at 52 days, 2.003 鹵0.152 (P 0.05). After stimulated by 2550 ng/ml IFN- 緯 for 12 h, the relative expression of I NOS,IL-6 and CD16 in Kupffer cells was 54.690, 68.5771.887 and 2.4272.4172.787, respectively (P0.05). 25 ng/ml IFN- 緯 stimulated, i NOS, for 36 h. The relative expression levels of IL-6 and CD16 were 34.81010 ~ 109.21010 ~ (10.327) ~ 15.143n ~ (1.887) ~ 3.317 (P0.05), respectively, but Arg-1 had no significant change compared with the control group. After 12 h stimulation of Kupffer cells with Arg-1, Arg-1, was observed. The relative expression levels of IL-10 and CD206 were 9.153 and 37.70 respectively (P 0.05). The relative expression levels of Arg-1,IL-10 and CD206 were 3.563 ~ 12.613 ~ 1.637 ~ 2.673 and 19.732n ~ (71.943), respectively, 36 h after 25 ng/ml IL-4 stimulation (P0.05), but I NOS did not change significantly. Conclusion in the early stage of Schistosoma japonicum infection, the Kupffer cells are mainly M1 type, while in the middle and late stage of infection, the Kupffer cells are mainly M2 type, which indicates that the Kupffer cell transformation is closely related to the liver immune microenvironment.
【作者單位】： 徐州醫(yī)科大學(xué)病原生物學(xué)與免疫學(xué)教研室江蘇省免疫與代謝重點(diǎn)實(shí)驗(yàn)室;第二軍醫(yī)大學(xué)熱帶傳染病學(xué)教研室;
【分類號(hào)】：R532.21
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本文編號(hào)：2405437