钩端螺旋体脂多糖诱生炎性细胞因子及小鼠感染组织中单核—巨噬细胞浸润

发布时间：2018-05-25 06:43

  本文选题：问号钩端螺旋体 + 脂多糖　； 参考：《浙江大学》2017年硕士论文

【摘要】：目的 了解问号钩端螺旋体(Leptospira interrogans)脂多糖(lipopolysaccharide,L-LPS)诱生人单核细胞或小鼠单核-巨噬细胞产生炎性细胞因子的作用以及问号钩端螺旋体感染小鼠内脏组织中外周血单核-巨噬细胞浸润的情况。方法采用酚水法提取问号钩端螺旋体黄疸出血群赖型赖株L-LPS,分别用LPS定量试剂盒和鲎试验试剂盒测定L-LPS浓度和内毒素活性。采用人或小鼠细胞因子抗体芯片,检测人THP-1单核细胞或小鼠J774A.1单核-巨噬细胞受L-LPS作用或被问号钩端螺旋体赖株感染24或48 h后产生细胞因子情况。采用小鼠细胞因子抗体芯片检测问号钩端螺旋体赖株感染C3H/HeJ小鼠血清细胞因子。以兔抗小鼠外周血单核-巨噬细胞特异性CD68-IgG为一抗、HRP标记驴抗兔IgG为二抗,采用免疫组化法检测C3H/HeJ小鼠感染问号钩端螺旋体赖株5、7或10 d后肺、肝、肾组织中外周血来源的CD68+单核-巨噬细胞的浸润情况。结果1×109问号钩端螺旋体黄疸出血群赖型赖株可提取约1μ的L-LPS。L-LPS凝固鲎试剂所需最低浓度为0.5 ng/mL,为大肠埃希菌脂多糖(E-LPS)活性1/5(0.1 ng/mL)。L-LPS作用THP-1细胞24或48h后有29种细胞因子水平显著升高,其中嗜酸细胞活化趋化因子(eotaxin)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、细胞间粘附分子-1(ICAM-1)、单核细胞趋化因子(1-309)、白细胞介素-1α(IL-1α)、IL-6、IL-12p40、单核细胞趋化蛋白-2(MCP-2)、肿瘤坏死因子--α(TNF-α)和干扰素γ诱导单核因子(MIG)10种细胞因子水平升高5倍以上(p0.01),eotaxin-2、IFN-γ、IL-3、IL-10、IL-11、IL-12p70、MCP-1、巨噬细胞炎性蛋白-1β(MIP-1β)、G-CSF、IL-1β、IL-7、IL-13、IL-15、干扰素诱导蛋白-10(IP-10)、M-CSF、MIP-1δ、TGF-β1、可溶性 TNF 受体Ⅰ(sTNFRI)和血小板衍生生长因子-BB(PDGF-BB)19种细胞因子水平升高1.5～5倍(p0.05)。L-LPS作用J774A.1细胞24或48h后有9种细胞因子水平升高,其中G-CSF、IL-6、IL-10、TNF-α、T细胞和单核及嗜酸性粒细胞CC亚族趋化因子(RANTES)5种细胞因子水平升高5倍以上(p0.01),MCP-1、IL-12p40、IL-1β和可溶性TNF受体Ⅱ(sTNFRⅡ)4种细胞因子水平升高升高1.5～5倍(p0.05)。问号钩端螺旋体赖株感染细胞上清以及问号钩端螺旋体赖株感染小鼠血清中细胞因子检测结果与上述L-LPS作用细胞相似。问号钩端螺旋体赖株感染C3H/HeJ小鼠5 d后肺、肝和肾组织中出现CD68+单核-巨噬细胞浸润,感染7或10d时上述组织内CD68+单核-巨噬细胞数量不断增加,肺和肾组织出现结构模糊、细胞裂解等组织破坏现象。结论L-LPS具有很强的诱导单核-巨噬细胞上调多种炎性细胞因子和单核-巨噬细胞趋化因子表达的作用,其中IL-6和TNF-α为主要炎性细胞因子,问号钩端螺旋体感染小鼠内脏组织中有大量单核-巨噬细胞浸润。
[Abstract]:Objective to investigate the effect of Leptospira interrogansa lipopolysaccharide (LPP) on the production of inflammatory cytokines by human monocytes or mouse mononuclear macrophages and the mononuclear cells in peripheral blood of mice infected with Leptospira interroganss. The infiltration of macrophages. Methods L-LPSs were extracted from Leptospira interrogans by phenol-water method. The concentration of L-LPS and the activity of endotoxin were determined by LPS quantitative kit and Limulus test kit respectively. Human or mouse cytokine antibody microarrays were used to detect cytokines produced by human THP-1 monocytes or murine J774A.1 mononuclear macrophages exposed to L-LPS or infected with Leptospira interrogans for 24 or 48 hours. The serum cytokines of C3H/HeJ mice infected with Leptospira interrogans were detected by mouse cytokine antibody chip. Rabbit anti-mouse mononuclear macrophage specific CD68-IgG was used as the first anti-HRP-labeled donkeys anti-rabbit IgG as the second antibody. The lung and liver of C3H/HeJ mice infected with Leptospira interrogans were detected by immunohistochemical method after 57th or 10d. Infiltration of CD68 mononuclear macrophages from peripheral blood in renal tissue. Results the minimum concentration of 1 脳 10 ~ 9 question mark Leptospira icterus haemorrhage group Lai Lai can extract about 1 渭 L-LPS.L-LPS coagulative Limulus reagent. The activity of 1 / 5 ~ 0. 1 ng/mL).L-LPS of Escherichia coli lipopolysaccharide E-LPSs could significantly increase the level of 29 cytokines in THP-1 cells 24 or 48 h after treatment with Leptospira interrogans (Leptospira interrogans) for 24 or 48 hours. Among them, eotaxin, granulocyte-macrophage colony stimulating factor (GM-CSFN), intercellular adhesion molecule-1 (ICAM-1), monocyte chemokine 1-309 (monocyte chemokine), interleukin-1 伪 (IL-1a), interleukin-1 伪 (IL-1 伪), monocyte chemoattractant protein (-2MCP-2N), tumor necrosis factor- 伪 (TNF- 伪) and interference The levels of 10 cytokines in mononuclear cytokines were increased by more than 5 times, and the levels of IL-10 IL-10 IL-11 and IL-12p70 MCP-1, macrophage inflammatory protein 1 尾, G-CSFIL-1 尾, IL-7IL-13 IL-15, interferon inducible protein -10 IP-10CS-FU MIP-1 未 TGF- 尾 1, soluble TNF 鈪，

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