侵袭性肺曲霉菌病小鼠动物模型建立的实验研究

目的建立免疫抑制小鼠侵袭性肺曲霉菌病(IPA)动物模型,为阐明IPA的发病机制和临床治疗提供基础研究。方法用环磷酰胺造成小鼠免疫抑制,双侧鼻孔滴入烟曲霉菌孢子,分别于24 h、48 h、72 h、96 h不同时间点处死小鼠,通过肺组织病理、肺组织真菌培养确定侵袭性肺曲霉菌病模型是否构建成功。结果 IPA模型组的组织培养可见烟曲霉菌,病理切片模型组小鼠72 h时可见较严重的出血和充血;96 h时肺内有菌丝,肺泡间隔增宽,组织坏死。正常状态感染烟曲霉小鼠72 h可见充血,肺泡弹性纤维被破坏;96 h仅表现为炎症和出血,未见菌丝。结论成功建立了小鼠IPA动物模型,为研究侵袭性曲霉菌病的发病机制、早期诊断和防治奠定了基础。     

中性粒细胞减少小鼠侵袭性肺曲霉病模型的构建

目的构建中性粒细胞减少合并侵袭性肺曲霉病（IPA）的动物模型。方法40只小鼠随机分为4组：A组为模型组，环磷酰胺150mg／kg于接种前4d及接种前1d腹腔注射，接种当天给予1％戊巴比妥45mg／kg腹腔注射麻醉后，1×10^8个／mL烟曲霉孢子悬液40μL滴鼻。B组为非免疫抑制接种对照组，除以生理盐水代替环磷酰胺外，其余操作同模型组。C组为免疫抑制对照组，除以生理盐水代替烟曲霉孢子悬液滴鼻外，其余操作同模型组。D组为空白对照组，以生理盐水代替环磷酰胺腹腔注射并以生理盐水代替烟曲霉孢子滴鼻，其余操作同模型组。模型建立成功的判断标准：肺病理切片见坏死灶、肺泡内出血，PAS染色见45。角分叉的分隔菌丝或孢子；组织培养烟曲霉阳性。结果肺大体观察、病理切片H—E染色及PAS染色和肺组织培养结果表明，A组10只小鼠在接种烟曲霉3d后均发生了IPA，B、C、D组小鼠均无IPA发生。结论本法可成功构建中性粒细胞减少小鼠IPA模型。     

血清BG、GM在不同免疫状态下肺烟曲霉菌感染大鼠中的实验研究

目的 构建不同免疫状态下肺烟曲霉菌感染的大鼠模型,研究免疫缺陷严重程度与肺烟曲霉菌感染的关系。方法 注射生理盐水及不同浓度的地塞米松和环磷酰胺建立不同免疫状态的大鼠模型,经鼻滴1×10⁷CFU/mL烟曲霉菌标准菌株孢子悬液引起肺烟曲霉菌感染,并于感染1、3、5、7、9 d分别随机处死每组1/5大鼠,收集大鼠血液标本,动态观察半乳甘露聚糖(GM)、(1,3)-β-D-葡聚糖(BG)、白细胞计数(WBC)、中性粒细胞计数(NEUT)、CD4⁺T细胞水平变化,同时取肺组织标本进行病理组织检查和真菌培养。150只大鼠均分为6组,组1为正常对照组,组2为低剂量免疫抑制组,组3为高剂量免疫抑制组,组4为正常状态感染组,组5为低剂量免疫抑制感染组,组6为高剂量免疫抑制感染组。结果 与组1比较,组4在感染1～5 d时WBC明显升高,组6则在感染1～7 d WBC明显降低,但在感染9 d时明显升高,差异有统计学意义(P<0.05);吸入烟曲霉菌后,CD4⁺T细胞水平均有降低的趋势,组4在感染7 d、组5在感染3 d、组6在感染5 d...     

TLR2、TLR4、TLR5和TLR9在小鼠Hp感染模型中的表达

目的:研究BALB/c小鼠在幽门螺杆菌(Hp)感染前后和用抗生素治疗后胃黏膜组织中Toll样受体(TLR)2、TLR4、TLR5、TLR9、IL-1β的表达,探讨Toll样受体家族在Hp感染机制中的作用。方法:60只BALB/c小鼠随机分为3组,第1组不予处理(正常组),第2组予感染Hp(Hp感染组),第3组感染Hp后予抗生素治疗(抗生素治疗组)。RT-PCR和Western blotting法半定量检测小鼠胃内TLR2、TLR4、TLR5、TLR9、IL-1β的表达,Giemsa染色切片计数Hp定植数量,HE染色切片判断黏膜炎症水平。结果:(1)TLR5、TLR9在各组小鼠胃黏膜组织中均无表达。(2)TLR2在Hp感染组胃黏膜组织中表达(PCR:0.13±0.025;Western:1.32±0.27)高于抗生素治疗组(PCR:0.04±0.011;Western:0.43±0.08),正常组无表达。(3)TLR4在Hp感染组胃黏膜组织中表达(PCR:0.22±0.051;Western:0.72±0.17)高于抗生素治疗组(PCR:0.06±0.009;Western:0.21±0.04),正常组无表达。(4)IL-1β在Hp感染组胃黏膜组织中有表达(PCR:0.27±0.038;Western:0.58±0.14),抗生素治疗组和正常组无表达。结论:TLR2、TLR4可能参与了Hp的致病机制,TLR5、TLR9可能未参与Hp的致病机制。     

侵袭性烟曲霉菌病大鼠模型的建立及分析

目的:建立侵袭性肺烟曲霉菌病大鼠模型,并对模型研究价值进行相关分析,为侵袭性肺曲霉菌病的实验研究提供参考.方法:腹腔注射环磷酰胺,动态观察大鼠血常规变化,建立免疫抑制大鼠模型.自制气管插管接种烟曲霉菌孢子悬液引起侵袭性肺烟曲霉菌病,分别于接种后1、3、5、7 d处死大鼠后取肺组织进行病理切片观察及组织匀浆培养.结果:分次不同剂量环磷酰胺腹腔注射后大鼠表现为精神萎靡、体重减轻,血常规示白细胞及分类细胞数量明显减少,中性粒细胞、淋巴细胞不能分类.病理切片可见免疫抑制大鼠感染烟曲霉菌后随着时间延长肺组织病变逐渐加重.肺组织出血、纤维素渗出;烟曲霉菌孢子聚集并长出菌丝,引起肺泡结构破坏、弥漫性肺水肿.结论:成功建立了免疫抑制大鼠侵袭性肺烟曲霉菌病动物模型,为研究免疫抑制宿主侵袭性肺曲霉菌病的发病机制、病理生理、药物疗效提供参照.     

TLR4在博莱霉素诱导的肺纤维化小鼠模型肺组织中的表达

目的探讨Toll样受体4(TLR4)在博来霉素诱导的肺纤维化小鼠模型肺组织中的表达。方法将60只小鼠随机分为两组:对照组(气管内注入生理盐水)和纤维化组(气管内注入博来霉素5 mg/kg)。处理后用实时定量PCR和免疫蛋白印迹法分别在7、14、21 d观察肺组织中TLR4m RNA和蛋白质的表达。此外,通过HE染色判断肺纤维化的程度。所有数据均用均数±标准差(sx±)表示,两组之间比较采用t检验,多组之间比较采用方差分析。结果 14、21 d纤维化组TLR4 m RNA表达明显高于对照组(P<0.05),且在第21天达到高峰。与对照组相比,纤维化组TLR4蛋白表达增加,21 d达高峰。结论在博来霉素诱导的肺纤维化小鼠模型中,TLR4的表达与炎症和肺纤维化均密切相关。     

全氟化碳通过下调TLR4/NF-κB信号通路抑制脂多糖刺激A549细胞分泌TNF-α的研究

目的探讨全氟化碳对内毒素诱导的人肺泡Ⅱ型上皮细胞(A549细胞)炎症因子TNF-α表达的影响及机制。方法人肺泡Ⅱ型上皮细胞,传代培养后,均分为3组:空白对照组(C组)、脂多糖(LPS)组、全氟化碳+LPS(P+LPS组),P+LPS组在给予LPS同时加入全氟化碳,LPS的终浓度为10μg/ml,全氟化碳的终浓度为12.5%,各组分别孵育30 min、2 h、4 h、12 h、24 h,结束后用蛋白免疫印迹法(Western blot)检测核转录因子-κB(NF-κB)、Toll样受体4(TLR4)蛋白表达水平,酶联免疫吸附法(ELISA)检测细胞培养上清中TNF-α的水平。结果 (1)Western blot结果显示,C组人肺泡Ⅱ型上皮细胞上有TLR4蛋白的表达,但表达量较低;LPS组人肺泡Ⅱ型上皮细胞在30 min、2 h、4 h、12 h、24 h点TLR4及NF-κB蛋白的表达量明显高于C组(P<0.05);与LPS组比较,P+LPS组人肺泡Ⅱ型上皮细胞30 min、2 h、4 h、12 h、24 h点TLR4及NF-κB蛋白表达明显下降(P<0.05),C组人肺泡Ⅱ型上皮细胞的TLR4及NF-κB蛋白表达量与P+LPS组相比,无明显差异(P>0.05)。(2)ELISA结果显示,与C组比较,LPS组人肺泡Ⅱ型上皮细胞30 min、2 h、4 h、12 h、24 h点TNF-α均明显升高(P<0.05)。P+LPS组人肺泡Ⅱ型上皮细胞30 min、2 h、4 h、12 h、24 h点TNF-α较LPS组均明显降低(P<0.05)。结论 (1)TLR4可能介导了LPS诱导的人肺泡Ⅱ型上皮细胞的炎症反应。(2)全氟化碳减轻LPS诱导的人肺泡Ⅱ型上皮细胞的炎症反应,可能与抑制细胞表面TLR4的信号活化,进一步抑制下游NF-κB的转导路径,从而降低TNF-α的产生有关。     

不同免疫抑制状态小鼠鲍曼不动杆菌的致病性差异研究

目的构建正常小鼠、轻度和重度免疫抑制小鼠鲍曼不动杆菌肺炎模型,研究不同免疫状态下鲍曼不动杆菌的致病性。方法 1分别构建正常小鼠、轻度和重度免疫抑制小鼠鲍曼不动杆菌肺炎模型;2144只ICR雌性小鼠随机分为6组,分别为正常对照组(组1)、轻度免疫抑制组(组2)、重度免疫抑制组(组3)、感染组(组4)、轻度免疫抑制感染组(组5)及重度免疫抑制感染组(组6),每组24只小鼠。气管插管后组1、组2、组3经气管插管注射生理盐水10μL,组4、组5、组6注射鲍曼不动杆菌菌液(3.8×108 CFU/mL)10μL;3分别于0、24、48、72、120和168 h 6个时间点每组随机选取4只小鼠,进行血白细胞和中性粒细胞计数、小鼠肺组织细菌计数,以及肺组织病理检查。结果组6有5只小鼠死亡,病死率达到20.8%,余组无死亡。组4为免疫功能正常小鼠,感染鲍曼不动杆菌后2 d肺内细菌全部被清除,白细胞及中性粒细胞计数在感染后1~3 d明显上升,5~7 d后基本恢复正常水平,病理示一过性肺组织损伤包括局部肉芽肿形成及肺间质损伤;组6为重度免疫缺陷小鼠,在感染后肺组织中的细菌量持续增长,第3天上升至感染初期的145倍,之后菌量减少。感染2 d内白细胞及中性粒细胞始终处于极低水平,3 d后逐渐恢复至正常水平,至第7天明显升高至正常水平的2倍以上,组织病理示感染7 d后整叶肺呈实变、坏死,肺泡、支气管及血管结构均被破坏;组5与正常小鼠感染过程类似,细菌在第3天被完全清除。结论正常小鼠感染鲍曼不动杆菌后,肺内细菌可以完全清除,但仍有一过性肺组织病理损伤;重度免疫抑制小鼠感染后细菌会在肺内增殖,肺组织病理损伤随时间延长明显加重;机体不同免疫状态可影响鲍曼不动杆菌感染的发展和转归。     

小鼠感染肺炎衣原体后肺组织TLR2、TLR4基因表达的变化及意义

目的通过建立小鼠肺炎衣原体感染的模型,探讨小鼠感染肺炎衣原体后肺脏组织Toll样受体(TLR)2、4mRNA表达及意义。方法Icr雄性小鼠60只,随机分为实验组、对照组,实验组鼻内接种肺炎衣原体,对照组接种二磷酸蔗糖(2sp)缓冲液,再分别按预定的处死时间1、3、5、7、14d各分为5小组,取肺脏组织。应用逆转录-聚合酶链反应(RT-PCR)半定量方法检测肺组织TLR2mRNA、TLR4mRNA的表达变化。结果小鼠感染肺炎衣原体后,引起TLR表达明显变化。其中TLR2mRNA表达水平在3、5、7、14d其密度值(0.849±0.107、0.981±0.104、0.926±0.116、0.905±0.046)显著高于对照组(0.375±0.149、0.340±0.069、0.314±0.074、0.302±0.010,P<0.01),并在第5天达到高峰;而TLR4mRNA表达水平在第1、3、5、7、14天均低于对照组,但无明显的统计学意义。结论肺炎衣原体感染与TLR的变化有密切的联系,主要在TLR2上表达变化,TLR的变化增强了机体非特异免疫能力,并随着肺炎衣原体感染程度的加重与减轻,TLR2mRNA的表达变化上调或下降。     

急性脑梗塞伴急性肺损害时肺内毒素受体CD14基因的表达

目的 :探讨急性脑梗塞伴肺损害时肺内毒素受体 CD1 4m RNA的表达 ,观察其变化规律与肺损害的关系。方法 :选择急性脑梗塞伴肺损害 87例、急性脑梗塞不伴肺损害 86例、健康对照组 6 0例 ,进行肺组织内毒素 CD1 4m RNA的检测。观察 3组 (12、2 4、36、4 8、72 h)肺内毒素受体 CD1 4m RNA的表达。结果 :1脑梗塞伴肺损害组 12 h肺组织 CD1 4m RNA表达开始升高 ,2 4～ 36 h CD1 4m RNA达高峰 ,72 h后下降 ,各时相点与脑梗塞不伴肺损害组比较有显著差异 (P<0 .0 1) ;2脑梗塞伴肺损害组与健康对照组比较有极显著差异 (P<0 .0 0 1) ;3脑梗塞不伴肺损害组与健康对照组比较有差异 (P<0 .0 5 )。结论 :急性脑梗塞伴肺损害时肺组织内毒素受体 CD1 4m RNA的异常表达 ,是导致急性肺损害的危险因素 ,也是导致多脏器功能障碍的重要危险因素。     

Osteoclastogenesis through TLR/NOD signaling

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) are components of microbial cell walls that cause innate immune responses and inflammation. Toll-like receptor 4 (TLR4) is a receptor for LPS and transduces signals through myeloid differentiation factor 88 (MyD88), which plays essential roles in the TLR/IL-1R signaling and activates NF-kappaB and MAP kinase pathways to induce RANKL expression in osteoblasts. Osteoblasts express NOD2, an intracellular sensor for MDP, in response to LPS, IL-1 and TNF. NOD2 binds RIP2, a serine/threonine kinase which transduces NF-kappaB signaling. Thus MDP synergistically enhances osteoclast formation induced by LPS, IL-1 and TNF through RANK ligand up-regulation in osteoblasts. In summary, innate immune receptors, TLR4 and NOD2, recognize bacterial components on cell surfaces and inside cells, respectively, and these signals cross-talk to induce RANKL expression in osteoblasts, which results in enhancing osteoclast formation and function.     

TLR4 activation mediates kidney ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4(-/-) and MyD88(-/-) mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4(-/-) and MyD88(-/-) TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4(-/-) mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4(-/-) BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.     

慢性阻塞性肺疾病患者外周血单个核细胞中TLR3、TLR7、TLR8及TLR9 mRNA的表达

目的观察慢性阻塞性肺疾病(COPD)患者外周血单个核细胞中TLR3、TLR7、TLR8及TLR9mRNA的表达。方法用密度梯度离心法分离COPD患者和健康人外周血单个核细胞后,提取总RNA并逆转录为cDNA,用实时定量PCR检测TLR3、TLR7、TLR8及TLR9mRNA的拷贝数。结果COPD患者外周血单个核细胞的TLR3和TLR9mRNA表达较正常健康人明显增高(P<0.05),而TLR7和TLR8mRNA的表达在COPD患者和正常健康人之间却无显著差异(P>0.05)。在COPD患者中,急性期和稳定期患者的TLR3、TLR7、TLR8和TLR9无显著差异(P>0.05)。结论TLR3和TLR9的升高可能是COPD患者对病毒敏感性增高的一个重要机制。     

慢性阻塞性肺疾病患者外周血单个核细胞中论著TLR3、TLR7、TLR8及TLR9 mRNA的表达

目的 观察慢性阻塞性肺疾病(COPD)患者外周血单个核细胞中TLR3、TLR7、TLR8及TLR9 mRNA的表达.方法 用密度梯度离心法分离COPD患者和健康人外周血单个核细胞后,提取总RNA并逆转录为cDNA,用实时定量PCR检测TLR3、TLR7、TLR8及TLR9 mRNA的拷贝数.结果 COPD患者外周血单个核细胞的TLR3和TLR9 mRNA表达较正常健康人明显增高(P＜0.05),而TLR7和TLR8 mRNA的表达在COPD患者和正常健康人之间却无显著差异(P＞0.05).在COPD患者中,急性期和稳定期患者的TLR3、TLR7、TLR8和TLR9无显著差异(P＞0.05). 结论TLR3和TLR9的升高可能是COPD患者对病毒敏感性增高的一个重要机制.     

Bupivacaine inhibits the TLR4- and TLR2-Myd88/NF-κB pathways in human leukocytes

Local anesthetics have anti-inflammatory effects. Because most previous experiments were performed with supra-therapeutic concentrations, we measured the effects of clinically relevant concentrations of bupivacaine on the Toll like receptor 4 (TLR4)- and TLR2-myeloid differentiation primary response 88 (MyD88)-nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathways. We measured tumor necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) release, p38 mitogen-activated protein kinase (MAP-kinase) phosphorylation and translocation of NF-κB in human peripheral blood mononuclear cells (hPBMCs) and human monocytes challenged with lipopolysaccharide (LPS) or tripalmitoylated lipopeptide Pam3CysSerLys4 (Pam3CSK4) in the presence or absence of bupivacaine. Similarly, we measured the effect of bupivacaine on HEK293 cells expressing the hTLR4 and the hTLR2 genes and challenged with LPS or Pam3CSK4. Finally, molecular docking simulations of R(+)- and S(−)-bupivacaine binding to the TLR4-myeloid differentiation protein 2 (MD-2) complex and to the TLR2/TLR1 heterodimer were performed. In PBMCs, bupivacaine from 0.1 to 100 μM inhibited LPS-induced TNF-α and PGE2 secretion, phosphorylation of p38 and nuclear translocation of NF-κB in monocytes. Bupivacaine similarly inhibited the effects of Pam3CSK4 on TNF-α secretion. Bupivacaine inhibited the effect of LPS on HEK293 cells expressing the human TLR4 receptor and the effect of Pam3CSK4 on HEK293 cells expressing the human TLR2 receptor. Molecular docking showed that bupivacaine binds to the MD-2 co-receptor of TLR4 and to the TLR2 receptor. Contrary to numerous experiments performed with supratherapeutic doses, our results were obtained with concentrations of bupivacaine as low as 0.1 μM. We conclude that bupivacaine modulates the inflammatory reactions such as those observed after surgery or trauma, at least partly by inhibiting the TLR4- and TLR2-NF-κB pathways.     

Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney

TLRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2-/- and TLR2+/+ mice, TLR2 antisense oligonucleotides, and chimeric mice deficient in leukocyte or renal TLR2. Tubular cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfunction in TLR2-/- mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury.     

Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells

Imiquimod (IMQ), a synthetic agonist to Toll-like receptor (TLR) 7, is being successfully used for the treatment of certain skin neoplasms, but the exact mechanisms by which this compound induces tumor regression are not yet understood. While treating basal cell carcinoma (BCC) patients with topical IMQ, we detected, by immunohistochemistry, sizable numbers of both myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) within the inflammatory infiltrate. Surprisingly, peritumoral mDCs stained positive for perforin and granzyme B, whereas infiltrating pDCs expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The biological relevance of this observation can be deduced from our further findings that peripheral blood-derived CD11c(+) mDCs acquired antiperforin and anti-granzyme B reactivity upon TLR7/8 stimulation and could use these molecules to effectively lyse major histocompatibility complex (MHC) class I(lo) cancer cell lines. The same activation protocol led pDCs to kill MHC class I-bearing Jurkat cells in a TRAIL-dependent fashion. While suggesting that mDCs and pDCs are directly involved in the IMQ-induced destruction of BCC lesions, our data also add a new facet to the functional spectrum of DCs, ascribing to them a major role not only in the initiation but also in the effector phase of the immune response.