NOD/SCID小鼠胎盘细胞内细胞因子表达及其与妊娠转归的关系

目的：研究Th1和Th2型细胞因子在胎盘淋巴细胞内的表达状况及其与NOD/SCID孕鼠妊娠转归的关系。 方法： 比较BALB/c和NOD/SCID小鼠孕13.5 d胚胎吸收率(RR), 采用4色流式细胞术检测胎盘淋巴细胞内Th1型(TNF-α和IL-2)和Th2型(IL-10)细胞因子表达率。 结果： 虽然NOD/SCID小鼠存在多重免疫缺陷，但BALB/c和NOD/SCID小鼠孕13.5 d RR无显著差异；NOD/SCID×NOD/SCID母-胎界面CD8+IL-10+/CD8+细胞百分率显著高于BALB/c×BALB/c妊娠模型(P<0.01), 而CD4和CD8阳性细胞内TNF-α和IL-2表达水平均无显著差异。 结论： 母-胎界面CD8+IL-10+/CD8+细胞百分率自发性升高可能与NOD/SCID小鼠基本正常的生育力有关。     

LPS对MRL／MpJ小鼠脾T细胞表达TLR4和TNF-a的影响

研究Toll样受体4(TLR4)在MRL／MpJ小鼠脾细胞的表达状况和革兰阴性菌脂多糖(LPS)刺激后TLR4的表达变化以及对TNF-a。产生的影响。MRL／MpJ小鼠经腹腔注射LPS后在不同时间取脾细胞．用三色荧光标记流式细胞仪技术检测小鼠脾T细胞TLR4的表达情况，并与BALB／c小鼠作对照；通过RT-PCR观察LPS刺激前后脾T细胞TNF-dmRNA的表达变化。结果表明TLR4在MRL／MpJ小鼠CD3^ CD4^ T细胞和CD3^ CD8^ T细胞中均有表达；但TLR4^ ／CD4^ 细胞表达仅BALB／c小鼠的33％，在受LPS刺激后，MRL／MpJ鼠CD4^ T细胞TLR4升高时间较BALB／c小鼠延迟；CD8^ T细胞TLR4在LPS刺激前后无明显变化；脾细胞TNF-amRNA水平升高亦延迟。通过对干燥综合征样自身免疫病鼠模型MRL／MpJ小鼠脾T细胞TLR4的表达及功能研究，将有助于认识天然免疫和获得性免疫间的联系和感染在自身免疫病发病中的作用。     

LPS对MRL/MpJ小鼠脾T细胞表达TLR4和TNFα的影响

研究Toll样受体 4 (TLR4 )在MRL/MpJ小鼠脾细胞的表达状况和革兰阴性菌脂多糖 (LPS )刺激后TLR4的表达变化以及对TNF α产生的影响。MRL/MpJ小鼠经腹腔注射LPS后在不同时间取脾细胞 ,用三色荧光标记流式细胞仪技术检测小鼠脾T细胞TLR4的表达情况 ,并与BALB/c小鼠作对照 ;通过RT PCR观察LPS刺激前后脾T细胞TNF αmRNA的表达变化。结果表明TLR4在MRL/MpJ小鼠CD3+ CD4 + T细胞和CD3+ CD8+ T细胞中均有表达 ;但TLR4 + /CD4 + 细胞表达仅BALB/c小鼠的 33% ,在受LPS刺激后 ,MRL/MpJ鼠CD4 + T细胞TLR4升高时间较BALB/c小鼠延迟 ;CD8+ T细胞TLR4在LPS刺激前后无明显变化 ;脾细胞TNF αmRNA水平升高亦延迟。通过对干燥综合征样自身免疫病鼠模型MRL/MpJ小鼠脾T细胞TLR4的表达及功能研究 ,将有助于认识天然免疫和获得性免疫间的联系和感染在自身免疫病发病中的作用。     

通过TLR9途径活化巨噬细胞导致小鼠流产的机制研究

为研究低甲基化型CpG与TLR9相互作用激活巨噬细胞并诱发小鼠妊娠失败的机制,本研究模拟特异性配体CpG活化TLR9的过程,并比较CpG对NK1.1+CD3-细胞和CD11b+F4/80+细胞数量、TNF-α表达水平以及妊娠结局的影响。研究发现,在孕6.5d腹腔注射CpG可显著提高非肥胖型糖尿病(non-obese diabetic,NOD)小鼠胚胎吸收率。相反,相同剂量对野生型BALB/c小鼠则无此影响。胚胎吸收率的增高伴有CD11b+F4/80+巨噬细胞相对数量增多和血清TNF-α水平增高,但是NK细胞构成比无显著改变。采用F4/80中和抗体抑制CD11b+F4/80+巨噬细胞可显著降低血清TNF-α水平,并降低胚胎吸收率。这些证据表明,CpG通过激活TLR9,并提高蜕膜CD11b+F4/80+巨噬细胞相对数量、增强TNF-α表达,进而导致胚胎吸收率增高。     

NOD/SCID孕鼠脾脏和胎盘CD45+细胞内细胞因子的表达

目的研究NOD/SCID孕鼠胚胎吸收率（Resorption rate,RR）与母-胎界面局部免疫状况的关系。方法计算并比较孕13.5天同基因妊娠NOD/SCID×NOD/SCID小鼠和非免疫缺陷的BALB/c×BALB/c小鼠的RR,并采用4色流式细胞术检测NOD/SCID小鼠非孕期和孕13.5天脾脏和胎盘细胞内细胞因子的表达状况,以明确其与妊娠耐受相关的淋巴细胞功能亚群。结果NOD/SCID小鼠的RR与对照组BALB/c小鼠相比无显著差异。与此相应,虽然能够证实NOD/SCID小鼠具有多重免疫缺陷,但是孕期母-胎界面多种功能性细胞亚群的百分率发生自发性改变。结论NOD/SCID小鼠脾脏和胎盘某些细胞百分率的自发性改变可能对妊娠结局有利。     

双链RNA和咪喹莫特联合刺激的协同作用研究

目的 研究双链RNA(double-stranded RNA,dsRNA)和咪喹莫特联合刺激是否具有协同效应.方法 在Toll样受体3(TLR3)的特异性刺激剂dsRNA、TLR7的特异性刺激剂咪唪莫特(R837)单独刺激或二者联合刺激条件下,检测BALB/c×C57BL/6和NH细胞缺陷的非肥胖型糖尿病(NOD)×C57BL/6小鼠胚胎吸收率.采用小鼠体内注射上述刺激剂的方法 ,流式细胞术检测子宫CD45+细胞内细胞因子表达水平.为进一步鉴定CD45+细胞身份,在体外培养系统中采用dsRNA和咪喹莫特刺激胎盘和底蜕膜来源的子官CD3+T细胞和CD49b+NK细胞,并检测细胞内细胞因子表达水平.采用丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)抑制剂SP600125和PD98059阻断细胞因子表达水平的增加.结果 此 dsRNA和咪喹莫特联合刺激对胚胎吸收率的增高具有协同作用,同时对CD45+细胞内TNF-α和IFN-γ的表达增强具有协同刺激作用.进一步细胞鉴定研究显示,虽然在BALB/c小鼠CD3+细胞和CD49b+NK细胞中均可发现这种协同效应,但是在NOD小鼠,这种细胞因子水平的增高应主要归因于CD3+T细胞,因为在CD49b+NK细胞不显示这种细胞因子增高趋势.上述刺激剂合用的协同效应町部分地被JNK(Jun N-terminal kinase)MAPK抑制剂SP600125阻断,而几乎被ERK(extracellular signal.regulated kinase)MAPK抑制剂PD98059所完全阻断.结论 增强的TLR3和TLR7联合信号可能是通过NOD小鼠Th1型T细胞,而不是NK细胞所传导.ERK MAPK途径可能在TLR3和TLR7参与的细胞信息传递过程中发挥关键性作用.     

NOD/SCID小鼠胎盘DX5~+CD25~+细胞水平

为了研究胎盘DX5+CD25+细胞在维持母-胎耐受状态中的潜在作用。采用流式细胞术检测免疫功能正常的BALB/c和免疫缺陷的NOD/SCID小鼠外周血和胎盘DX5+CD25+细胞,评价这些细胞与小鼠妊娠预后的关系。结果:虽然NOD/SCID小鼠存在严重的免疫缺陷,但是其胚胎吸收率和平均每窝产仔数等生育力指标均基本正常,并且其孕13 d胎盘DX5+CD25+/DX5+细胞百分率与免疫功能正常小鼠相比无显著性差异,并均呈高百分率分布特征(约占DX5+细胞总数的90%)。NOD/SCID小鼠胎盘中高百分率的DX5+CD25+细胞可能是正常生育力预后的免疫学基础。     

iNKT细胞在脂多糖诱导的早产中的作用

目的探讨恒定自然杀伤T(iNKT)细胞在脂多糖(LPS)诱导的早产中的作用。方法野生型C56BL/6小鼠和缺乏iNKT细胞的Jα18-/-小鼠腹腔注射LPS建立炎症诱导性早产小鼠模型;计算早产率和死胎率;流式细胞术检测蜕膜活化型免疫细胞百分率和树突状细胞共刺激分子的表达。结果与野生型小鼠比较,LPS处理的Jα18-/-小鼠早产率和死胎率显著降低,蜕膜树突状细胞共刺激分子CD40、CD80和CD86的表达显著下降,蜕膜活化型树突状细胞、T细胞和NK细胞百分率明显减少。结论 iNKT细胞可能在LPS诱导的早产发生中具有重要作用。     

淋巴细胞缺陷对内毒素血症小鼠腹腔巨噬细胞活化的影响

目的观察淋巴细胞缺陷对内毒素血症小鼠腹腔巨噬细胞活化的影响。方法采用腹腔注射脂多糖(LPS)建立Balb/c小鼠和T、B细胞缺陷的重症联合免疫缺陷(SCID)小鼠内毒素血症模型,ELISA检测2种小鼠腹腔灌洗液TNF-α和IL-10水平,实时荧光定量PCR检测腹腔巨噬细胞(peritoneal macrophage,PMa)TNF-α、IL-10及丝裂原蛋白激酶磷酸酶-1(mito-gen-activated protein kinase phosphatase-1,MKP-1)mRNA表达;ELISA检测Balb/c及SCID小鼠PMa体外刺激后细胞因子分泌情况。结果 LPS注射后1 h,SCID小鼠腹腔灌洗液TNF-α水平高于Balb/c小鼠,注射后3 h,IL-10水平低于Balb/c小鼠;LPS注射前及注射后,SCID小鼠PMa TNF-αmRNA表达高于Balb/c小鼠PMa,IL-10 mRNA表达低于Balb/c小鼠PMa;体外实验LPS刺激下,SCID小鼠PMa较Balb/c小鼠PMa分泌更多的TNF-α,IL-10的分泌却偏少。LPS注射前及注射后,Balb/c小鼠PMa MKP-1 mRNA表达均明显高于SCID小鼠。结论淋巴细胞缺陷导致内毒素血症小鼠腹腔巨噬细胞活性的增加,淋巴细胞抑制巨噬细胞的活化并可能调控其发育及成熟;MKP-1表达的减少可能是淋巴细胞缺陷导致腹腔巨噬细胞活性增加的分子机制之一。     

阻断TLR2结合降低β2GP1-抗β2GP1复合物刺激的小鼠腹腔巨噬细胞TNF-α的表达

目的探讨β2糖蛋白1(β2GP1)-抗β2GP1复合物诱导小鼠腹腔巨噬细胞表达肿瘤坏死因子α(TNF-α)中Toll样受体2(TLR2)的作用。方法用β2GP1-抗β2GP1复合物、TLR2激动剂Pam3CSK4及TLR4激动剂脂多糖(LPS)、TLR2阻断剂抗小鼠TLR2-Ig G(m TLR2-Ig G)及TLR4阻断剂TAK-242对BALB/c小鼠腹腔巨噬细胞进行体外处理。用实时荧光定量PCR检测TNF-αmRNA水平,Western blot法及免疫荧光细胞化学方法检测TNF-α蛋白表达;采用流式细胞术检测小鼠腹腔巨噬细胞表面TLR2的水平。结果β2GP1-抗β2GP1复合物、Pam3CSK4及LPS均能够显著增加BALB/c小鼠腹腔巨噬细胞表达TNF-αmRNA及蛋白水平,抗m TLR2-Ig G能够抑制上述刺激物促进细胞TNF-α表达的效应,但弱于TAK-242的抑制效应,抗m TLR2-Ig G与TAK-242联合使用未显示更强的抑制效应。流式细胞术结果显示,β2GP1-抗β2GP1复合物、Pam3CSK4及LPS均能增加小鼠腹腔巨噬细胞TLR2的表达,而抗m TLR2-Ig G、TAK-242以及二者联合使用均未显示对TLR2表达的抑制效应。结论TLR4、TLR2都增强β2GP1-抗β2GP1复合物对小鼠腹腔巨噬细胞TNF-α的刺激作用。     

TSLP-induced placental DC activation and IL-10(+) NK cell expansion: comparative study based on BALB/c x C57BL/6 and NOD/SCID x C57BL/6 pregnant models

Thymic stromal lymphopoietin (TSLP)-TSLP receptor (TSLP-R) interactions activate CD11c(+) dendritic cells (DCs) and increase epithelial cell Th2-type cytokine production. We detected intracellular TSLP expression on CK7(+) trophoblast cells and TSLP-R expression on placental DCs from pregnant BALB/cxC57BL/6 and NOD/SCIDxC57BL/6 mice on gestational day 12.5. Murine recombinant TSLP activated DCs from BALB/c mice, with increased CD80 and CD83 expressions; TSLP-activated DCs induced IL-10-producing NK cell expansion. This was abrogated by anti-TSLP Ab or by culturing CD49b(+) NK cells alone. No TSLP-DC-induced IL-10(+)CD49b(+) cell expansion occurred when DCs and CD49b(+) cells were cultured separately. Although TSLP-induced DC activation occurred in NOD/SCID mice, the IL-10(+) NK cell percentage was unchanged. CK7(+) trophoblast cells may activate placental DCs via a TSLP-TSLP-R interaction and induce DC-dependent placental NK cell IL-10 production. TSLP-DC and NK cell contact appears necessary for IL-10(+)CD49b(+) cell expansion. Placental NK cells from NOD/SCIDxC57BL/6 mice appear less sensitive to TSLP-DC induction.     

In vivo diabetogenic action of CD4+ T lymphocytes requires Fas expression and is independent of IL-1 and IL-18

CD4(+) T lymphocytes are required to induce spontaneous autoimmune diabetes in the NOD mouse. Since pancreatic β cells upregulate Fas expression upon exposure to pro-inflammatory cytokines, we studied whether the diabetogenic action of CD4(+) T lymphocytes depends on Fas expression on target cells. We assayed the diabetogenic capacity of NOD spleen CD4(+) T lymphocytes when adoptively transferred into a NOD mouse model combining: (i) Fas-deficiency, (ii) FasL-deficiency, and (iii) SCID mutation. We found that CD4(+) T lymphocytes require Fas expression in the recipients' target cells to induce diabetes. IL-1β has been described as a key cytokine involved in Fas upregulation on mouse β cells. We addressed whether CD4(+) T cells require IL-1β to induce diabetes. We also studied spontaneous diabetes onset in NOD/IL-1 converting enzyme-deficient mice, in NOD/IL-1β-deficient mice, and CD4(+) T-cell adoptively transferred diabetes into NOD/SCID IL-1β-deficient mice. Neither IL-1β nor IL-18 are required for either spontaneous or CD4(+) T-cell adoptively transferred diabetes. We conclude that CD4(+) T-cell-mediated β-cell damage in autoimmune diabetes depends on Fas expression, but not on IL-1β unveiling the existing redundancy regarding the cytokines involved in Fas upregulation on NOD β cells in vivo.     

Defect of toll-like receptor 9-mediated activation in NC/Nga mouse macrophages

Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). In this study, we examined TLR9-mediated activation in NC/Nga mice, an animal model for human atopic dermatitis. NC/Nga mouse macrophages produced significantly less TNF-alpha than did BALB/c mouse macrophages in response to CpG oligonucleotide (ODN). In addition to defective TLR9-mediated TNF-alpha production, phosphorylation of ERK1,2 and p38 was rapidly diminished after 60 min of CpG ODN stimulation, whereas phosphorylation of these molecules was sustained until 60 min in BALB/c mice. Furthermore, phosphorylation of c-Jun N-terminal kinase (JNK) was not observed in NC/Nga mouse macrophages. In contrast, B cells and dendritic cells (DCs) from NC/Nga mice showed normal responses to CpG ODN stimulation. The expression level of TLR9 in NC/Nga mouse macrophages was significantly lower than that in BALB/c mouse macrophages, whereas levels of TLR9 expression in B cells and DCs in NC/Nga mice were the same as those in BALB/c mice. These results suggest that defective TLR9-mediated activation in NC/Nga mouse macrophages contributes to the reduction of TLR9 expression levels.     

Dysregulated Toll-like receptor expression and signaling in bone marrow-derived macrophages at the onset of diabetes in the non-obese diabetic mouse

The expression, responsiveness and regulation of mouse Toll-like receptors (TLRs) in bone marrow-derived macrophages (BM-?) were investigated prior to and following the development of diabetes. Expression of TLR3 and TLR5 was significantly higher in newly diabetic non-obese diabetic (NOD) mice when compared with pre-diabetic and control strains of mice. The TLR3 ligand poly(I)poly(C) triggered up-regulation of its own receptor in NOR and pre-diabetic NOD, but TLR3 was already highly expressed in diabetic NOD mice. Expression levels of TLR3 correlated with poly(I)poly(C)-triggered IFN activity. LPS triggered down-regulation of TLR4 in pre-diabetic NOD, NOR and BALB/c, while levels of TLR4 remained consistently elevated in type 1 diabetic NOD and type 2 diabetic NZL mice. Dysregulation of TLR4 expression in the diabetic state correlated with increased nuclear factor kappa B (NF-kappaB) activation in response to the TLR4 ligand LPS and higher expression of IL-12p40, tumor necrosis factor alpha (TNFalpha), IL-6 and inducible nitric oxide synthase but lowered expression of IL-10. Exposure of bone marrow precursor cells from NOD mice to a hyperglycemic environment during differentiation into macrophages resulted in elevated levels of TLR2 and TLR4 and the cytokine TNFalpha. The results indicate that macrophage precursors are influenced by systemic changes in diabetes favoring altered TLR expression and sensitivity that may influence susceptibility to macrophage-mediated diabetes complications and explain inappropriate responses to infection in diabetes.     

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.