本文選題：問號(hào)鉤端螺旋體 + 脂多糖�。� 參考：《浙江大學(xué)》2017年碩士論文

【摘要】：目的 了解問號(hào)鉤端螺旋體(Leptospira interrogans)脂多糖(lipopolysaccharide,L-LPS)誘生人單核細(xì)胞或小鼠單核-巨噬細(xì)胞產(chǎn)生炎性細(xì)胞因子的作用以及問號(hào)鉤端螺旋體感染小鼠內(nèi)臟組織中外周血單核-巨噬細(xì)胞浸潤(rùn)的情況。方法采用酚水法提取問號(hào)鉤端螺旋體黃疸出血群賴型賴株L-LPS,分別用LPS定量試劑盒和鱟試驗(yàn)試劑盒測(cè)定L-LPS濃度和內(nèi)毒素活性。采用人或小鼠細(xì)胞因子抗體芯片,檢測(cè)人THP-1單核細(xì)胞或小鼠J774A.1單核-巨噬細(xì)胞受L-LPS作用或被問號(hào)鉤端螺旋體賴株感染24或48 h后產(chǎn)生細(xì)胞因子情況。采用小鼠細(xì)胞因子抗體芯片檢測(cè)問號(hào)鉤端螺旋體賴株感染C3H/HeJ小鼠血清細(xì)胞因子。以兔抗小鼠外周血單核-巨噬細(xì)胞特異性CD68-IgG為一抗、HRP標(biāo)記驢抗兔IgG為二抗,采用免疫組化法檢測(cè)C3H/HeJ小鼠感染問號(hào)鉤端螺旋體賴株5、7或10 d后肺、肝、腎組織中外周血來源的CD68+單核-巨噬細(xì)胞的浸潤(rùn)情況。結(jié)果1×109問號(hào)鉤端螺旋體黃疸出血群賴型賴株可提取約1μ的L-LPS。L-LPS凝固鱟試劑所需最低濃度為0.5 ng/mL,為大腸埃希菌脂多糖(E-LPS)活性1/5(0.1 ng/mL)。L-LPS作用THP-1細(xì)胞24或48h后有29種細(xì)胞因子水平顯著升高,其中嗜酸細(xì)胞活化趨化因子(eotaxin)、粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)、細(xì)胞間粘附分子-1(ICAM-1)、單核細(xì)胞趨化因子(1-309)、白細(xì)胞介素-1α(IL-1α)、IL-6、IL-12p40、單核細(xì)胞趨化蛋白-2(MCP-2)、腫瘤壞死因子--α(TNF-α)和干擾素γ誘導(dǎo)單核因子(MIG)10種細(xì)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、巨噬細(xì)胞炎性蛋白-1β(MIP-1β)、G-CSF、IL-1β、IL-7、IL-13、IL-15、干擾素誘導(dǎo)蛋白-10(IP-10)、M-CSF、MIP-1δ、TGF-β1、可溶性 TNF 受體Ⅰ(sTNFRI)和血小板衍生生長(zhǎng)因子-BB(PDGF-BB)19種細(xì)胞因子水平升高1.5～5倍(p0.05)。L-LPS作用J774A.1細(xì)胞24或48h后有9種細(xì)胞因子水平升高,其中G-CSF、IL-6、IL-10、TNF-α、T細(xì)胞和單核及嗜酸性粒細(xì)胞CC亞族趨化因子(RANTES)5種細(xì)胞因子水平升高5倍以上(p0.01),MCP-1、IL-12p40、IL-1β和可溶性TNF受體Ⅱ(sTNFRⅡ)4種細(xì)胞因子水平升高升高1.5～5倍(p0.05)。問號(hào)鉤端螺旋體賴株感染細(xì)胞上清以及問號(hào)鉤端螺旋體賴株感染小鼠血清中細(xì)胞因子檢測(cè)結(jié)果與上述L-LPS作用細(xì)胞相似。問號(hào)鉤端螺旋體賴株感染C3H/HeJ小鼠5 d后肺、肝和腎組織中出現(xiàn)CD68+單核-巨噬細(xì)胞浸潤(rùn),感染7或10d時(shí)上述組織內(nèi)CD68+單核-巨噬細(xì)胞數(shù)量不斷增加,肺和腎組織出現(xiàn)結(jié)構(gòu)模糊、細(xì)胞裂解等組織破壞現(xiàn)象。結(jié)論L-LPS具有很強(qiáng)的誘導(dǎo)單核-巨噬細(xì)胞上調(diào)多種炎性細(xì)胞因子和單核-巨噬細(xì)胞趨化因子表達(dá)的作用,其中IL-6和TNF-α為主要炎性細(xì)胞因子,問號(hào)鉤端螺旋體感染小鼠內(nèi)臟組織中有大量單核-巨噬細(xì)胞浸潤(rùn)。
[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 偽 (IL-1a), interleukin-1 偽 (IL-1 偽), monocyte chemoattractant protein (-2MCP-2N), tumor necrosis factor- 偽 (TNF- 偽) 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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