性别差异对脓毒症大鼠肺组织Toll样受体4及髓样分化蛋白-2基因表达的影响

目的 探讨性别差异对脓毒症大鼠肺组织Toll样受体4（TLR4）、髓样分化蛋白-2（MD-2）和肿瘤坏死因子-α（TNF-α）基因表达的影响。方法脂多糖（LPS）刺激前后留取雌性和雄性大鼠肺组织标本，提取总RNA，采用半定量逆转录-聚合酶链反应（RT-PCR）测定TLR4、MD-2和TNF-α基因表达情况，采用放射免疫测定法检测大鼠血浆雌二醇（E2）含量。结果正常雌性和雄性大鼠肺组织均可表达少量TLR4、MD-2和TNF-α基因，性别间差异均无显著性（P均〉0．05），但脓毒症雌性大鼠各项指标表达均明显弱于雄性（P均〈0．01）。相关分析表明，雌性和雄性脓毒症大鼠肺组织TLR4、TNF-α mRNA表达与血浆E2含量均呈显著负相关（P均〈0．05）。结论LPS诱导的肺组织TLR4信号转导通路活化存在性别差异，内源性雌激素作用可能导致雌性脓毒症大鼠对LPS的反应性弱于雄性。     

脂多糖结合蛋白抑制肽对内毒素诱导的U937细胞TLR4的影响

目的 研究脂多糖结合蛋白(LBP)抑制肽对内毒素(LPS)诱导的人单核巨噬细胞株U937细胞TLR4的影响。方法 佛波脂(PMA)诱导U937成熟后分为5组，即正常对照组；LPS组；低剂量抑制肽组；中剂量抑制肽组；高剂量抑制肽组。用RT-PCR和蛋白定量方法(Westem blot)测定TLR4 mRNA和蛋白的表达，ELISA测定培养上清液中肿瘤坏死因子-α(TNF-α)的含量。结果 LBP抑制肽组TLR4的mRNA，蛋白的OD值，TNF-α的浓度均较LPS组低，较正常组高。结论 LBP抑制肽通过抑制由TLR4介导的LPS信号跨膜转导和TNF-α的分泌，对LPS所致疾病如脓毒血症、急性肺损伤可能有潜在的治疗作用。     

Toll样受体在脂多糖耐受性机制作用中的研究进展

脂多糖(LPS)耐受性是指机体或细胞经低剂量LPS刺激后，对LPS的再次刺激呈低反应或无反应状态，它与LPS信号转导变化密切相关。LPS细胞外信号转导是通过脂多糖结合蛋白(LBP)呈递给CD4^ 来实现的，但对LPS跨膜转导机制一直不明确。近年来Toll样受体(TLRs)的发现为此提供了新的线索。TLRs表达量和功能下调及TLR4信号通路中各个环节的功能缺陷，     

CD14抑制肽的体外生物活性

目的研究CD14抑制肽(CD14inhibitory peptide,CD14-IP)与CD14的结合活性及对内毒素(LPS)和脂多糖结合蛋白(LBP)诱导的人单核巨噬细胞株U937表达肿瘤坏死因子-α(TNF-α)的影响。方法采用酶联免疫吸附(ELISA)法进行CD14-IP与CD14的结合实验。U937细胞用佛波脂(PMA)诱导成熟后分为五组:正常对照组、LPS组(100ng/mLLPS 100ng/mLrhLBP)、高剂量多肽组、中剂量多肽组及低剂量多肽组,后三组分别给予10μg/mL、1.0μg/mL和0.1μg/mL的CD14-IP。用ELISA测定培养细胞上清TNF-α的含量,用RT-PCR测定U937细胞TNF-αmRNA的表达水平。结果CD14-IP有较强的与CD14结合的能力;CD14-IP组TNF-α和TNF-αmRNA水平均较LPS组低,较正常组高。结论CD14-IP能与CD14结合,并能降低培养细胞TNF-α和TNF-αmRNA的表达,具有抑制CD14生物活性的作用。     

乌司他丁对脂多糖诱导的大鼠肺组织Toll样受体4信号通路表达的影响

目的 探讨乌司他丁对脓毒性大鼠肺损伤的保护机制.方法 选健康清洁级SD大鼠30只,随机(随机数字法)分为生理盐水对照组10只、脂多糖(LPS)组10只、脂多糖+乌司他丁(LPS+ UTI)组10只.注药24 h后取肺组织观察形态学改变,分别应用RT-PCR或Western blot 及ELISA方法测定肺组织Toll样受体4(TLR4)、核因子-κB (NF-κB)及肿瘤坏死因子TNF-α mRNA及蛋白表达.结果 脂多糖导致大鼠肺组织损伤,乌司他丁可下调肺组织TLR4 mRNA (t=3.563,P=0.032)、TNF-α mRNA(t=5.147,P=0.028)及TLR4蛋白(t=2.692,P=0.041)、NF-κB蛋白(t=2.459,P=0.024)、TNF-α蛋白(t=3.336,P=0.037)表达,并减轻肺损伤.结论 乌司他丁对脂多糖致肺损伤具有一定保护作用,其机制之一可能与抑制TLR4-NF-κB信号途径转导有关.     

脂多糖受体复合物基因多态性与感染易感性的研究进展

不同个体对感染的易感性是不同的，已经有越来越多的证据表明，遗传的易感性在感染性疾病的发病中起重要作用。基因的多态性即等位基因的变异，通常以一定频率存在于人群中。脂多糖（LPS）激活细胞的胞外信号转导机制是：LPS进入血液循环，首先是与血液中脂多糖结合蛋白（LBP）结合，形成LPS-LBP复合物，再结合到靶细胞的CD14上，然后再与细胞膜上TLR2和TLR4相结合从而激活细胞。LPS激活TLR2和TLR4还必须有另外一种重要的调节分子（MD-2）存在。     

脓毒症大鼠心肌细胞Toll样受体4和炎症因子基因表达的变化及作用机制

目的 观察脓毒症大鼠心肌细胞凋亡的变化及与Toll样受体4(TLR4)、肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)基因表达的关系;同时探讨谷氨酰胺(Gln)在脓毒症心肌损伤治疗中的保护作用.方法 采用内毒素脂多糖(LPS)腹腔注射法制备脓毒症大鼠模型.将大鼠随机分为对照组、LPS组及Gln组,再分为术后0、6、12、24 h亚组,每组6只.于相应时间点取心肌组织,采用逆转录-聚合酶链反应(RT-PCR)检测TLR4、TNF-α及IL-6的mRNA表达,并观察心肌组织病理学改变.结果 LPS组大鼠心肌细胞凋亡程度增高.LPS组、Gln组各时间点心肌细胞TLR4、TNF-α及IL-6的mRNA表达均较对照组明显增加(P均<0.05).而Gln组心肌细胞凋亡程度较LPS组轻,各时间点TLR4 mRNA表达均较LPS组升高幅度明显降低,且于12 h、24 h差异有统计学意义;TNF-α mRNA表达亦降低,于6 h、24 h差异有统计学意义;IL-6 mRNA表达较LPS组升高幅度降低,且于12 h差异有统计学意义(P均<0.05).结论 TLR4信号转导基因及其调控细胞因子TNF-α、IL-6基因表达在脓毒症心肌细胞损伤的发生发展中起重要作用.Gln通过影响相关基因表达干预脓毒症心肌细胞的凋亡.     

内毒素相关受体基因共转染肠上皮细胞的研究

基因转染可将目的基因导入靶细胞进行基因功能和基因表达调控的研究。但转染效率的高低受诸多因素的影响，尤其是多种功能相关基因的共转染，更加困难。既往研究发现，人肠上皮细胞株(HIC)不表达内毒素(LPS)相关受体CD14(cluster of differentiation 14)、Toll样受体4(Toll-like receptor4，TLR4)和MD-2(myeloid differentiation protein-2)。为进一步证实这3种LPS相关受体的不表达是否是肠上皮细胞耐受LPS的关键机制，我们用FuGENE6转染试剂进行CD14、TLR4和MD-2基因共转染人肠上皮细胞的探索。     

氟伐他汀对人THP-1细胞TLR4及下游信号转导通路的影响

目的探讨氟伐他汀对脂多糖(LPS)刺激的人急性单核细胞白血病细胞系(THP-1)Toll样受体4(TLR4)及下游信号转导通路的干预作用。方法采用不同剂量的氟伐他汀、LPS处理THP-1细胞,MTT法检测细胞增殖情况,实时荧光定量RTPCR(qRT-qPCR)检测细胞中TLR4和肿瘤坏死因子-α(TNF-α)mRNA的表达,western blot检测TLR4及下游磷酸化胞外信号调节激酶(ERK)、磷酸化核因子κB(NF-κB p65)蛋白的表达水平。ELISA法测定细胞培养上清中TNF-α蛋白的含量。结果氟伐他汀(10 mg/L)降低LPS刺激的THP-1细胞TLR4(蛋白质和mRNA)的表达(t分别为7.55、7.80,P均0.05),1、5、10mg/L氟伐他汀能显著抑制LPS刺激的下游分子ERK的磷酸化(q分别为11.78、18.15、18.88,P0.05);亦能抑制LPS刺激的NF-κB p65的磷酸化(q分别为5.13、6.87、11.68,P0.05)。同时,氟伐他汀(10 mg/L)能显著干预LPS刺激的细胞TNF-α(蛋白质和mRNA)的表达(q分别为4.23、3.01,P0.05)。结论氟伐他汀可通过干预LPS刺激的TLR4表达及下游分子ERK、NF-κB p65的磷酸化,抑制THP-1细胞TNF-α的表达,可能为氟伐他汀的抗炎机制之一。     

失血性休克鼠肺组织Toll样受体基因的表达

目的探讨单纯性失血性休克（无复苏）对肺组织中Toll样受体（TLR）mRNA表达的影响及意义。方法C57BL／6小鼠45只。随机分为失血组、脂多糖（LPS）组（阳性对照组。尾静脉注射LPS5mg／kg）、假手术组（阴性对照组）。每组15只。心脏穿刺复制失血性休克模型，在不同时间点取出肺组织。提取总RNA，通过逆转录-聚合酶链反应（RT—PCR）半定量方法检测肺组织TLR2 mRNA和TLR4 mRNA的表达水平。结果在失血性休克和LPS刺激后肺出现明显的中性粒细胞浸润、红细胞渗出。正常肺组织中有TLR2 mRNA、TLR4 mRNA表达；住失血性休克和LPS刺激后0、1、2、4和6h肺组织TLR2 mRNA、TLR4 mRNA表达均逐渐增加；而假手术组未发生明显改变。结论失血性休克后肺组织TLR2 mRNA、TLR4 mRNA表达增加与急性肺损伤（ALI）的发生有密切关系。除增强了机体非特异性免疫能力外，同时增加了宿主对随后各种刺激的易感性。过度表达的TLR2、TLR4可能造成组织、器官结构和功能损害。     

JAK/STAT通路对烫伤脓毒症大鼠Toll样受体基因表达的调节作用

目的：探讨Janus激酶／信号转导和转录激活子（JAK／STAT）通路在烫伤脓毒症大鼠Toll样受体2（TLR2）基因表达调控中的意义。方法：Wistar大鼠38只随机分为正常对照组（n=6)、烫伤后金黄色葡萄球菌（金葡菌）感染组（n=12)、AG490拮抗组（n=20)和雷帕霉素（Rapamycin,RPM)拮抗组（n=10), 检测动物肝、肾、肺组织中TLR2和肿瘤坏死因子－α（TNF－α）基因表达的改变。结果：烫伤合并金葡菌攻击后0．5h和2h，动物肝、肾、肺组织中TLR2 mRNA表达显著增高（P＜0．05或P＜0．01）。RPM干预可有效抑制动脉肝、肾组织TLR2 mRNA表达，但对肺脏TLR2 mRNA表达无明显影响；而AG490拮抗组动物各组织中TLR2 mRNA表达与未干预组相比均无明显差异。烫伤合并金葡菌感染后2h大鼠肝、肺、肾组织TNF－α基因表达均显著升高（P均＜0．01）。给予RPM可明显抑制各组织中TNF－α mRNA表达（P＜0．05或P＜0．01），AG490拮抗组大鼠肝、肾组织中TNF－α表达亦均显著低于未拮抗组（P均＜0．01）。结论：烫伤后金葡菌感染可促进体内TLR2基因表达，STAT可能直接或通过其诱生的炎症介质参与了对TLR2 mRNA表达的调控。     

严重腹腔感染大鼠组织Toll样受体2/4基因表达及其调节机制

目的 :研究腹腔感染后主要脏器 Toll样受体 (TL R) 2 / 4 m RNA表达的变化规律及组织分布特点 ,并对其诱生机制进行初步探讨。方法 :采用大鼠盲肠结扎穿孔 (CL P)造成严重脓毒症模型。动物分为正常对照组(10只 )、假手术组 (10只 )、CL P组 (6 0只 )及杀菌 /通透性增加蛋白 (BPI)治疗组 (2 0只 )。分别检测肝、肺、肾、小肠组织 TL R2 / 4 m RNA表达及血浆肿瘤坏死因子α(TNFα)和白介素 10 (IL 10 )水平。结果 :CL P后 2 h肝、肺、肾及小肠组织中 TL R2 / 4 m RNA表达开始增高 ,伤后 6～ 12 h各组织 TL R2 / 4 m RNA表达迅速达峰值。 TL R4 m RNA表达于伤后 2 4～ 4 8h开始下降 ,CL P后 72 h趋于伤前范围甚至阴性 ,而 TL R2 m RNA表达则持续增高至 72 h。早期给予 BPI治疗后 ,动物 12～ 2 4 h肝、肺、肾及小肠组织 TL R2 m RNA水平均显著降低(P<0 .0 5或 P<0 .0 1) ,同时 CL P后 12 h各组织 TL R4 m RNA表达亦不同程度下调 (P<0 .0 5或 P<0 .0 1)。BPI治疗组伤后 12 h血浆 TNFα水平显著降低 (P<0 .0 5 ) ,恢复至伤前正常范围 ,但伤后 2 4 h血浆 IL 10水平显著升高 (P<0 .0 1)。结论 :严重腹腔感染可迅速上调体内多器官 TL R2 / 4的基因表达 ,诱导促炎细胞因子产生 ,TL Rs表达与内毒素的直接刺激作用密     

Stimulation of adenosine A2A receptor inhibits LPS-induced expression of intercellular adhesion molecule 1 and production of TNF-alpha in human peripheral blood mononuclear cells

LPS stimulates CD14/Toll-like receptor (TLR) 4, leading to induce TNF-alpha production. Cell-to-cell interaction through the engagement between intercellular adhesion molecule (ICAM) 1 on monocytes and its ligand on T cells has been suggested to play a role in the TNF-alpha production by LPS-treated human peripheral blood mononuclear cells (PBMCs). Adenosine is reported to inhibit LPS-induced TNF-alpha production. However, little is known about the mechanism of the inhibitory effects induced by adenosine on the LPS-induced immune responses. We found that adenosine inhibited the expression of ICAM-1 and the production of TNF-alpha by human PBMC via adenosine A2A receptor in the presence of LPS. However, the stimulation of A1R or A3R enhanced the actions of adenosine. Adenosine had no effect on the expression of CD14 and TLR-4, suggesting that the inhibitory effects of adenosine on the LPS actions might be independent of the expression of CD14 and TLR-4. Thus, adenosine differentially regulates the expression of ICAM-1 and the production of TNF-alpha through plural subtypes of receptors.     

Geranylgeranylacetone ameliorates inflammatory response to lipopolysaccharide (LPS) in murine macrophages: inhibition of LPS binding to the cell surface

We investigated whether pretreatment with geranylgeranylacetone (GGA), a potent heat shock protein (HSP) inducer, could inhibit proinflammatory cytokine liberation and nitric oxide (NO) production in lipopolysaccharide (LPS)-treated murine macrophages. The levels of NO and tumor necrosis factor-alpha (TNF-alpha) released from murine macrophage RAW 264 cells were increased dose- and time-dependently following treatment with LPS (1 microg/ml). GGA (80 microM) treatment 2 h before LPS addition significantly suppressed TNF-alpha and NO productions at 12 h and 24 h after LPS, respectively, indicating that GGA inhibits activation of macrophages. However, replacement by fresh culture medium before LPS treatment abolished the inhibitory effect of GGA on NO production in LPS-treated cells. Furthermore, GGA inhibited both HSP70 and inducible NO synthase expressions induced by LPS treatment despite an HSP inducer. When it was examined whether GGA interacts with LPS and/or affects expression of Toll-like receptor 4 (TLR4) and CD14 on the cell surface, GGA inhibited the binding of LPS to the cell surface, while GGA did not affect TLR4 and CD14 expressions. These results indicate that GGA suppresses the binding of LPS to the cell surface of macrophages, resulting in inhibiting signal transduction downstream of TLR4.     

Quantitative expression of Toll-like receptor-2, -4, and -9 in dendritic cells generated from blasts of patients with acute myeloid leukemia

BACKGROUND: Dendritic cells (DCs) generated from leukemic blasts constitute a promising tool in immunotherapy for acute myeloid leukemia patients (AML-DCs), because AML-DCs express human leukocyte antigens and costimulatory molecules such as CD40, CD80, and CD86 at a higher level than leukemic blasts. Potentiation of AML-DC vaccine might become feasible by the addition of adjuvants such as lipopolysaccharides (LPS) or CPG-rich oligodeoxyribonucleotides binding to Toll-like receptors (TLR) and inducing a stronger Type 1 T-cell response. STUDY DESIGN AND METHODS: mRNA and protein expression of TLR-2, -4, and -9 were analyzed with quantitative real-time polymerase chain reaction, Western blot, and flow cytometry for mature monocyte-derived DCs generated from 14 AML patients versus 14 healthy volunteers (HV-DCs), and the response of the AML- and HV-DCs to different microbial TLR ligands was determined by enzyme-linked immunosorbent assay for the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, inducible protein (Ip)-10, and interleukin (IL)-6. RESULTS: AML-DCs and HV-DCs strongly expressed TLR-2 and TLR-4, while TLR-9 was expressed at a lower level in both groups. There was no significant difference in TLR expression between the two groups of AML-DCs and HV-DCs. In accordance with the TLR expression levels, DCs generated from both AML patients and HVs responded to the known microbial ligands peptidoglycan (PGN) and lipoteichoic acid for TLR-2 and LPS as ligand for TLR-4, by producing TNF-alpha and IL-6. A response to the ODNs 2006 and 2216 binding to TLR-9 was only detected in AML-DCs. CONCLUSION: Microbial ligands like ODNs and LPS constitute promising adjuvants for enhancing (AML-) DC vaccines.     

Hepatocytes enhance effects of lipopolysaccharide on liver nonparenchymal cells through close cell interactions

The response to lipopolysaccharide (LPS) in the liver is complex, requiring cell-to-cell interactions between hepatocytes and liver nonparenchymal cells (NPC), in particular, Kupffer cells. Previous studies show that cytokines produced by Kupffer cells stimulated with LPS can, in turn, activate hepatocytes. In the present study, we sought to examine whether the reverse, hepatocyte (HC)-NPC interactions, is important in cytokine production in mixed cell cocultures. LPS-stimulated production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 from NPC was augmented in mixed HC-NPC cocultures, as compared with NPC monocultures. This HC-NPC interaction was not observed when hepatocytes were cocultured with NPC from TLR4-mutant (C3H/HeJ) mice or CD14-deficient mice. The effect was partially lost when hepatocytes from lipopolysaccharide-binding protein (LBP)-deficient mice were cocultured with wild-type mice. These data indicate that functional TLR4 and CD14 are required for NPC production of cytokines and that at least one of the critical components from hepatocytes is LBP. The augmented cytokine production by mixed HC-NPC cocultures was abrogated when the cells were separated by a filter system, indicating that close cell interactions are also required for this interaction. Thus, interaction between hepatocytes and NPC are critical for cytokine secretion by NPC.