睾丸局部感染时Sertoli细胞IL-1、IL-6、TGF-β和FasL的变化

目的：研究大鼠Sertoli细胞在抗感染中的免疫调节作用。方法：以溶脲脲原体(UU)和致病性大肠杆菌(E．coli)直接注入大鼠膀胱模拟上行性感染的途径，分别在1周、2周、3周处死大鼠，分离取得睾丸组织作组织切片观察病理变化及从大鼠睾丸组织中分离获得高纯度的Sertoli细胞，用免疫组化方法比较正常组与UU感染组、致病性大肠杆菌组之间：IL-1,IL-6,TGF-β和FasL表达的差异。结果：与正常组相比，UU和E .coli感染后，其IL-1分泌升高，IL-6分泌下降，TGF-β分泌升高，FasL表达也升高，结论：大鼠Sertoli细胞在抗感染免疫中，可通过IL-1，IL-6，TGF-β和FasL的表达来发挥免疫调节作用。     

Sertoli细胞FasL 和TGF-β mRNA表达在感染时的免疫调节作用

目的 研究大鼠Sertoli细胞在睾丸局部感染时的免疫调节作用。方法 以解脲脲原体（UU）和致病性大肠杆菌，直接注入大鼠膀胱模拟上行性感染的途径，分别在感染后1、2、3wk处死大鼠，从大鼠睾丸组织中分离获得高纯度的Sertoli细胞。然后抽提总RNA，用RT－PCR法比较正常组与UU感染组、致病性大肠杆菌感染组之间FasL mRNA和TGF－β mRNA表达的差异性。结果 与正常组相比较在UU感染后，1－3wk FasL mRNA的表达均升高；TGF－β mRNA的表达在1、2wk时升高，3wk时下降；而致病性大肠杆菌感染后，FasL mRNA和TGF－β mRNA的表达在1－3wk时均升高。结论 大鼠Sertoli细胞在抗感染免疫中，可通过FasL mRNA和TGF－β mRNA表达的变化，调节睾丸局部的免疫功能及有助于睾丸免疫豁免的维持。     

睾丸局部感染时Sertoli细胞IL-1、IL-6 mRNA的表达

目的；研究大鼠Sertoli细胞在抗感染中的免疫调节作用。方法：以溶脲脲原体（ureaplasma urealyicum,UU)和致病性大肠杆务直接注入大肠膀胱模拟上行性感染的途径，分别在1，2和3周处死大鼠，从大鼠睾丸组织分离获得高纯度的Sertoli细胞，然后抽提总RNA，用RT－PCR方法比较正常组与UU感染组，致病性大肠杆菌组之间IL－1，IL－6 mRNA表达的差异。结果：与正常组相比，UU感染后，其IL－1 mRNA在1，2周时升高，3周时下降，IL－6在1，2周时下降，3周时升高；而致病性大肠杆菌感染后，其IL－1 mRNA在1，2周时均升高，3周下降；IL－6在1周时升高，2周时下降，3周又升高，结论：大鼠Sertoli细胞在抗感染免疫中，可能IL－1，IL－6的表达来发挥免疫调节作用。     

脂多糖通过NF-κB途径调节大鼠星形胶质细胞Toll样受体表达

目的 研究脂多糖（LPS）对大鼠星形胶质细胞Toll样受体表达的影响及其机制。方法 在原代培养的第3代星形胶质细胞中加入不同浓度的LPS作用24h,通过免疫荧光、western blot观察星形胶质细胞 Toll样受体的表达和NF-κB p65的表达,同时研究NF-κB通路抑制剂对其的影响。结果 在正常状况下,星形胶质细胞胞浆和胞膜表达大量的TLR3受体,很少的TLR4受体。在LPS的刺激下,星形胶质细胞的TLR3表达保持不变,TLR4受体的表达随予以LPS的量增加而增高。LPS可刺激星形胶质细胞NF-κB的表达升高,抑制NF-κB通路活化抑制TLR4受体的上调。 结论 星形胶质细胞Toll样受体的表达是不同源的,TLR4受体随环境的变化而改变,其分子机制可能与NF-κB信号途径有关。     

PolyI:C介导气道平滑肌细胞TLR3和IL-8、Eotaxin的表达

目的:观察PolyI:C对大鼠气道平滑肌细胞(ASMCs)Toll样受体3(TLR3)及炎性细胞因子表达的影响,探讨ASMCs内TLR3表达与气道炎症间的关系。方法:体外培养大鼠ASMCs,传代培养后分为正常对照组和PolyI:C刺激组,PolyI:C刺激组又分为不同的时间点。用RT-PCR法检测细胞TLR3mRNA的表达;Western blot法检测ASMCs中核因子κB(NF-κB)的蛋白含量;ELISA法检测细胞培养上清Eotaxin和IL-8的浓度。结果:与正常对照组相比较,PolyI:C刺激组ASMCs内TLR3mRNA和NF-κB蛋白表达增加(P0.05);ASMCs培养上清液中IL-8和Eotaxin浓度增加(P0.05),并存在时间依赖性。结论:PolyI:C可以上调ASMCs内TLR3的表达,活化NF-κB,增加趋化因子Eotaxin和IL-8的浓度,参与哮喘的气道炎症反应。     

溶脲脲原体感染对大鼠Sertoli细胞分泌 IL-6和TGF-β1的影响

目的 :研究大鼠Sertoli细胞在感染中的免疫调节作用。方法 :SD大鼠的睾丸经Ⅱ型胶原酶和透明质酸酶二步消化、过滤、离心获得高纯度、高活率的Sertoli细胞。体外培养的Sertoli细胞经溶脲脲原体 (UU)、UU上清和热灭活UU感染或作用 ,用ELISA法分析、观察UU感染时Sertoli细胞分泌IL 6和TGF β1 的变化。结果 :低剂量的活UU和UU上清能明显上调Ser toli细胞分泌IL 6的功能 (P <0 0 1) ,而高剂量时则表现为明显的抑制作用 (P <0 0 1) ;低、高剂量的活UU、UU上清和热灭活UU均能明显抑制Sertoli细胞分泌TGF β1 的功能 (P <0 0 1)。结论 :大鼠Sertoli细胞在睾丸局部感染时通过其分泌的IL 6和TGF β1 可发挥免疫调节作用。     

乙醇性肝病大鼠创伤弧菌脓毒症肝组织Toll样受体及髓样分化蛋白-2的表达变化

目的：探讨乙醇性肝病大鼠创伤弧菌（VV）脓毒症肝组织Toll样受体及髓样分化蛋白-2的表达及其动态变化。 方法:制作乙醇性肝病大鼠创伤弧菌脓毒症模型。大鼠随机分为正常对照组、乙醇性肝病对照组和乙醇性肝病创伤弧菌脓毒症组。观察乙醇性肝病创伤弧菌脓毒症组大鼠在染菌后各时点脓毒症表现,并采用逆转录聚合酶链（RT-PCR）技术检测各时点大鼠肝组织TLR2、TLR4、MD-2 mRNA表达及其动态变化。结果:乙醇性肝病创伤弧菌脓毒症组大鼠染菌后6 h开始出现较明显的毒血症状,随着时间的延长毒血症状加剧。乙醇性肝病大鼠在染菌后2 h,TLR2、TLR4、MD-2 mRNA表达开始升高, 12 h达峰值,染菌后24 h下降。染菌后各时点TLR2、TLR4、MD-2 mRNA表达较正常对照组及乙醇性肝病对照组显著升高（P<0.05,P<0.01）。结论:乙醇性肝病大鼠VV脓毒症肝组织TLR2、TLR4、MD-2 mRNA表达水平随脓毒症病情的进展而升高,其与脓毒症的发生、发展密切相关,动态监测其改变对创伤弧菌脓毒症发病过程具有一定的意义。     

转基因小鼠高表达的FasL对Sertoli细胞免疫调节功能的影响

目的 :研究转基因小鼠高表达的FasL对Sertoli细胞在睾丸局部感染时的免疫调节作用的影响。方法 :将溶脲脲原体 (UU)分别注入FasL转基因及野生型小鼠膀胱 ,模拟上行性感染的途径。分别在感染后 1、2和 3wk处死小鼠 ,分离睾丸组织观察其病理变化 ,并用免疫组化染色法比较UU感染前后 ,Ser toli细胞上FasL和TGF β、IL 1α、IL 6的表达及分泌格局的差异。从野生型小鼠睾丸组织中分离高纯度的Sertoli细胞 ,与未感染UU的对照组相比 ,观察UU感染后FasL Sertoli细胞介导Fas Jurkat细胞的凋亡能力的变化。结果 :UU感染组的转基因小鼠 ,睾丸组织发生的病理改变比野生型更为明显 ;两种小鼠感染后Sertoli细胞分泌的调节因子变化格局不同 ;感染后Sertoli细胞对Jurkat细胞的杀伤能力增强。结论 :在抗感染免疫中 ,转基因表达的FasL可影响Sertoli细胞分泌细胞因子的格局 ,进而影响睾丸局部的免疫平衡。过高表达的FasL对机体的抗感染应答并非一定有利。     

乳酸杆菌对溃疡性结肠炎大鼠肠黏膜Toll样受体2及核因子-kappa B表达的影响

目的:观察乳酸杆菌对溃疡性结肠炎大鼠肠黏膜Toll样受体2(TLR2)及核因子-kappa B(NF-κB)表达的影响,探讨乳酸杆菌辅助治疗溃疡性结肠炎的机制.方法:采用三硝基苯磺酸/乙醇法诱导大鼠溃疡性结肠炎模型.观察并评估大鼠一般状态及组织损伤情况,以RT-PCR法和免疫组织化学分别检测TLR2、NF-κB表达.结果:乳酸杆菌组组织损伤情况明显较模型组减轻.模型组大鼠肠黏膜TLR2、NF-κB表达情况明显高于正常对照组和乳酸杆菌组.结论:乳酸杆菌可以通过抑制TLRs/NF-κB通路发挥对溃疡性结肠炎的辅助治疗作用.     

Toll样受体信号通路活化与皮瓣缺血再灌注损伤的关系

目的探讨Toll样受体2(TLR2)、Toll样受体3(TLR3)和Toll样受体4(TLR4)在大鼠皮瓣缺血再灌注损伤模型中的动态表达及介导病程进展中的作用。方法制备大鼠皮瓣缺血再灌注损伤模型,根据再灌注的时间不同分5组(6 h、12 h、24 h、48 h、72 h),同时制备假手术组,每组6只大鼠,采用免疫组化染色和Western blot检测各组大鼠皮瓣组织中TLR2、TLR3和TLR4的表达,同时检测Toll样受体信号通路下游炎性因子TNF-α、IL-6的表达,分析其相关性。结果 HE染色显色在缺血的早期,皮瓣组织呈现不同程度的坏死,在恢复血液灌注后,坏死没有明显好转,大量胶原纤维增生;免疫组化染色显色,与假手术组比较,TLR2、TLR3和TLR4的表达均有不同程度的升高,在再灌注24 h时TLR2和TLR4蛋白表达达高峰,随后逐渐下降,而TLR3在再灌注12 h即达高峰,随后降低,各时间段比较差异有统计学意义(P0.05);炎性因子TNF-α、IL-6的血清浓度明显高于假手术组,且在再灌注24 h时浓度最高(P0.05),随后逐渐下降,到72h没有再次升高趋势。结论在皮瓣缺血再灌注早期,皮瓣组织Toll样受体蛋白即迅速升高,同时通过促进下游炎性因子的分泌而介导组织损伤。     

Expression of IL-1α, IL-6, TGF-β, FasL and ZNF265 During Sertoli Cell Infection by Ureaplasma Urealyficum

To investigate immunoregulatory mechanisms of Sertoli cells in the testis in vitro and in vivo, we utilized our well-characterized Ureaplasma Urealyticum (UU)-induced model. We investigated the expressions of IL-1α, IL-6,TGF-[3, FasL and ZNF265 at the first, second and third weeks post-infection. During recovery from inflammation and with the help of negative regulators TGF-[3 and FasL, the high levels of IL-1α and IL-6 expressions were observed in the early stages of the infection, and decreased gradually in the later weeks both in vitro and in vivo.The trend of varied expression of ZNF265 was similar to those of TGF-β and FasL in vitro and in vivo for Sertoli cells infected with UU. Cellular & Molecular Immunology. 2009;6(3):215-221.     

黄芩苷对人淋巴细胞Toll样受体的调节

目的:研究黄芩苷对人淋巴细胞Toll样受体(TLR)的调节,探讨其在免疫调节方面的作用。方法:1.尼龙毛分离法分离人外周血T和B细胞后使用UF-50流式分析寻找最佳黄芩苷作用浓度;2.采用实时定量荧光RT-PCR,分析在最佳黄芩苷作用浓度处理前后TLRs的表达情况;3.通过加入鼠抗人TLR4单抗进行受体抑制实验并应用实时定量荧光RT-PCR探讨黄芩苷对TLRs的调节位点。结果:1.1 mg/ml的黄芩苷在体外可以显著促进人T、B淋巴细胞增殖(P<0.05);2.使用1mg/ml黄芩苷相对于未用黄芩苷处理的T、B细胞,其TLR3、TLR7、TLR8及TLR9 mRNA的表达均有显著增加(P<0.05),T细胞增加倍数分别为21、15、57和66倍,B细胞增加的倍数分别为24、20、61和63倍,而TLR1、TLR2、TLR4、TLR5、TLR6及TLR10 mRNAs表达变化不显著(P>0.05);3.在1 mg/ml黄芩苷作用下人T、B细胞TLR3、7、8和9 mRNA在12小时时表达已开始增加,除T细胞的TLR7 mRNA表达在48小时时略有下降外,其余表达基本一直持续增加,至48小时到达峰值;4.人T、B细胞在被黄芩苷作用前使用鼠抗人TLR4单克隆抗体封闭,会显著降低黄芩苷对TLR3、7、8和9 mRNA表达的诱导作用(P<0.05),T细胞下降比率分别为47.6%、66.7%、50.9%和45.5%;B细胞下降比率分别为50.0%、45.0%、50.8%和50.8%。结论:1.黄芩苷在体外能显著促进T、B淋巴细胞增殖;2.人外周血T、B细胞组成性表达含量差异较大的TLR1-10 mRNA,黄芩苷在体外可以显著上调人T、B淋巴细胞TLR3、TLR7、TLR8及TLR9 mRNAs的表达,但对TLR1、TLR2、TLR4、TLR5、TLR6及TLR10 mRNAs未见明显影响;3.黄芩苷可能通过TLR4发挥对人天然免疫能力的调节。     

Age-dependent activin receptor expression pinpoints activin A as a physiological regulator of rat Sertoli cell proliferation.

It is currently believed that the fertility level of the adult mammalian testis is related to the total number of Sertoli cells, which is established in the early prepubertal life. We have previously reported that, in an in-vitro system, terminal Sertoli cell proliferation is sustained by activin A in concert with FSH. In this paper, we have addressed the question of whether this activin A effect correlates with activin receptor II (ActRII) expression pattern during early post-natal testis development. We first determined the precise developmental interval of activin proliferative effect on Sertoli cells in vitro and then analysed the expression of ActRII in purified testicular cell populations by Northern blot and in-situ hybridization. While the 3 kb ActRII isoform was widely expressed at different ages and in several testicular cells, including Sertoli cells, germ cells and myoid cells, the canonical 6 kb ActRII isoform was specifically and transiently expressed at a high rate in Sertoli cells at 7-9 days after birth, the time when these cells respond to activin A in vitro. In the light of these results, we conclude that activin A regulates terminal Sertoli cell proliferation in the rat testis and that this effect is mediated by the 6 kb isoform of ActRII.     

β2-glycoprotein I,lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction.To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC.Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding.Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals.     

TLR在小鼠胸腺中的表达及其在胸腺细胞发育中的可能作用

检测体外培养和体内发育过程中,胎鼠胸腺处于不同发育阶段时Toll样受体(TLR)的表达,阐明TLR表达量与胸腺细胞发育相关性,为TLR和胸腺细胞发育分化相关研究提供基础数据。无菌取15d胎龄胎鼠胸腺进行体外培养(FTOC),在培养不同时间点(2d,4d,6d),检测处于不同发育期胸腺TLR的表达;同时在孕期不同天数(15~19d),分别取胎鼠胸腺,检测在体内发育过程中胸腺TLR的表达;在FTOC中加入二脱氧鸟苷培养6d以制备胸腺基质细胞,检测基质细胞与胸腺细胞TLR表达情况。结果:小鼠胸腺中检测到多种TLR。FTOC培养中:培养第2天(F2)开始检测到各种TLR,到培养第6天(F6),TLR1,TLR3,TLR6,TLR7,TLR8明显上调,而TLR4,TLR5保持低水平,TLR4在培养第6天又下降;体内发育过程中:TLR6表达量随胎龄增加有较明显上调,TLR1,TLR3-8保持低水平表达;TLR2,TLR9体内体外都未检测到明显表达。在对胸腺细胞与基质细胞TLR表达比较中发现TLR1,TLR5,TLR6,TLR7高表达于胸腺细胞。胎鼠胸腺表达某些TLR,并且在发育不同阶段表达量有所改变,提示TLR可能参与胸腺细胞的发育过程。     

Klebsiella pneumoniae Increases the Levels of Toll-Like Receptors 2 and 4 in Human Airway Epithelial Cells

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IκBα-dependent NF-κΒ pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.     

假体置入患者外周血白细胞Toll样受体2和4的表达

背景：研究证实全髋关节置换后松动假体表面生物膜中表达Toll样受体,但其变化规律尚不清楚。 目的：观察关节假体置入患者外周血白细胞Toll样受体2,4的表达。 方法：选择关节置换患者和同期10例行关节镜检查患者,于入院次日和假体置入后第3天早晨空腹抽取肘静脉血,流式细胞术分析外周血白细胞中Toll样受体2和Toll样受体4的阳性表达,同时送血样至检验科检查白细胞、血沉、C-反应蛋白水平。 结果与结论：两组白细胞、血沉、C-反应蛋白均在正常范围内。Toll样受体2,4均主要表达于外周血单核细胞,而在外周血淋巴细胞和中性粒细胞的表达率较低;关节置换组术后外周血单核细胞Toll样受体2阳性表达率明显低于术前(P < 0.05);关节置换组Toll样受体4、关节镜组Toll样受体2,4手术前后阳性表达率差异无显著性意义(P > 0.05)。关节置换组手术前后外周血单核细胞Toll样受体2阳性表达率低于关节镜组(P < 0.05),两组Toll样受体4表达率无差异。提示在无感染的情况下,手术本身应激和局部损伤不影响Toll样受体2,4的表达,假体的置入下调了外周血Toll样受体2的表达。     

Associations between toll-like receptors and interleukin-4 in the lungs of patients with tuberculosis

Toll-like receptors (TLRs) are implicated in the intracellular killing of Mycobacterium tuberculosis, and their expression is modulated by interleukin-4 (IL-4) in vitro. Our aim was to examine the expression of TLRs at the site of pathology in tuberculous lung granulomas and to explore the effect of the immune response on TLR expression. Immunohistochemistry was performed on lung granulomas from nine patients with tuberculosis undergoing lobectomy for haemoptysis. All nine patients expressed all of the TLRs studied (TLRs 1-5 and 9), whereas only five out of the nine patients had any granulomas positive for IL-4. Statistical analysis of TLR and cytokine staining patterns in 183 individual granulomas from the nine patients revealed significant associations between pairs of receptors and IL-4. A positive association between TLR2 and TLR4 (P < 0.0001) and a negative association between TLR2 and IL-4 (P < 0.0001) was observed. The associations between TLRs 1, 5, and 9 were significantly different in IL-4-negative compared with IL-4-positive patients. In conclusion, TLRs are expressed by various cell types in the human tuberculous lung, and their expression patterns are reflected by differences in the immune response.