Sequence Variants of Toll Like Receptor 4 and Late-Onset Alzheimer's Disease

Background

Toll like receptor 4 (TLR4) has been related to inflammation and beta-amyloid deposition in Alzheimer''s disease (AD) brain. No study has explored the association between haplotype-tagging single nucleotide polymorphisms (htSNPs) of TLR4 and AD risk previously and ApoE e4 status alone showed low sensitivity in identifying late-onset AD (LOAD) patients.

Methods

A total of 269 LOAD patients were recruited from three hospitals in northern Taiwan (2007–2010). Controls (n = 449) were recruited from elderly health checkup and volunteers of the hospital during the same period of time. Five common (frequency≥5%) TLR4 htSNPs were selected to assess the association between TLR4 polymorphisms and the risk of LOAD in the Chinese ethnic population.

Results

Homozygosity of TLR4 rs1927907 was significantly associated with an increased risk of LOAD [TT vs. CC: adjusted odds ratio (AOR) = 2.45, 95% confidence interval (CI) = 1.30–4.64]. After stratification, the association increased further in ApoE e4 non-carriers (AOR = 3.07) and in hypertensive patients (AOR = 3.60). Haplotype GACGG was associated with a decreased risk of LOAD (1 vs. 0 copies: AOR = 0.59, 95% CI = 0.36–0.96; 2 vs. 0 copies: AOR = 0.31, 95% CI = 0.14–0.67) in ApoE e4 non-carriers. ApoE e4 status significantly modified this association (p _interaction = 0.01). These associations remained significant after correction for multiple tests.

Conclusions

Sequence variants of TLR4 were associated with an increased risk of LOAD, especially in ApoE e4 non-carriers and in hypertensive patients. The combination of TLR4 rs1927907 and ApoE e4 significantly increased the screening sensitivity in identifying LOAD patients from 0.4 to 0.7.     

Toll样受体与抗结核感染免疫

结核分枝杆菌(MTB)是结核病的致病菌,其发病机制仍未阐明。Toll样受体(TLR)蛋白家族属于动物模式识别受体家族。研究表明,TLR对先天免疫和获得性免疫都有调控作用,与抗结核感染免疫有关的主要是TLR2和TLR4。对TLR的研究为MTB诱导先天免疫反应机制的阐明以及治疗方法的进步提供了新的思路。     

TAA 致慢性肝病内毒素血症大鼠清道夫受体、CD14 表达变化*

目的:观察清道夫受体(SR)和脂多糖受体CD14在TAA介导的慢性肝病内毒素血症大鼠肝组织中的表达。方法:通过大鼠持续灌胃给小剂量(12mg/kg.d)TAA建立大鼠肝损伤内毒素血症模型,HE染色光镜观察肝脏病理变化;改良赖氏法检测大鼠血清ALT、AST;改良过氯酸法测定血清内毒素含量;酶联免疫法检测大鼠血清CD4+和CD8+;免疫组化染色方法观察大鼠肝组织清道夫受体和CD14的表达。结果:TAA诱导后,大鼠肝脏出现片状坏死并可见灶性炎症;血浆ALT、AST及内毒素水平显著升高(P<0.05或P<0.01);血清CD4+、CD8+T细胞明显降低(P<0.01);肝组织CD14表达上调,清道夫受体表达下调,和正常大鼠相比,差异显著(P<0.05)。结论:肝组织SR表达下降和CD14表达增强可能是TAA介导慢性肝病内毒素血症的重要机制。     

Insulin Hypersensitivity Induced by Hepatic PTEN Gene Ablation Protects from Murine Endotoxemia

Sepsis still remains a major cause for morbidity and mortality in patients. The molecular mechanisms underlying the disease are still enigmatic. A great number of therapeutic approaches have failed and treatment strategies are limited to date. Among those few admitted for clinical intervention, intensive insulin treatment has proven to be effective in the reduction of disease related complications in critically ill patients. Insulin effectively reduces glucose levels and thereby contributes to protection. On the other hand insulin is a potent signaling pathway activator. One of those is the PI3K signaling axis. Activation of PI3K is known to limit pro-inflammatory gene expression. Here we can show that in a mouse model of insulin hypersensitivity induced by the deletion of the PI3K antagonist PTEN, specifically in hepatic tissue, significant protection is conferred in murine models of lethal endotoxemia and sepsis. Acute inflammatory responses are diminished, glucose metabolism normalized and vascular activation is reduced. Furthermore we investigated the hepatic gene expression profile of relevant anti-inflammatory genes in PTEN deficient mice and found marked upregulation of PPARγ and HO-1. We conclude from our data that insulin hypersensitivity via sustained activation of the PI3K signaling pathway exerts protective effects in acute inflammatory processes.     

细胞自噬参与雷公藤甲素诱导的肝细胞损伤

目的:观察雷公藤甲素引起急性肝损伤时肝细胞自噬的情况。方法:采用LC3-GFP质粒尾静脉高压注射的方法在体监测雷公藤甲素诱导肝细胞自噬的发生;用腺病毒双荧光载体m RFP-GFP-LC3感染体外培养的HepG2细胞,通过激光共聚焦显微镜和蛋白印记的方法检测细胞自噬流的强度。结果:小鼠腹腔注射雷公藤甲素(0.25 mg/kg和0.5 mg/kg)24小时,血清中ALT和AST水平均显著升高;肝组织病理结果显示肝细胞肿胀、空泡化及坏死;激光共聚焦显微镜下可检测到LC3-GFP质粒表达所呈现的绿色荧光;离体细胞实验结果证实雷公藤甲素可诱导肝细胞发生自噬流,且表现为一定程度的剂量、时间依赖性。结论:雷公藤甲素可引发急性肝损伤并伴随明显的细胞自噬。     

D-gal致大鼠急性肝损伤过程中肝卵圆细胞增殖及迁延

目的用D-gal建立大鼠急性肝损伤模型,观察肝损伤后再生过程中肝卵圆细胞的增殖和迁延。方法建立大鼠急性肝损伤模型,于第1、3、7和14天分别取肝组织,分别行病理、免疫组织化学,观察卵圆细胞的分布迁移情况,并取第7天肝组织进行组织电镜观察汇管区新增生细胞超微结构。结果病理切片显示肝细胞变性坏死程度以第7天和第14天为主,出现新增生的细胞。免疫组化示随时间阳性细胞明显增多,分布于汇管区,并向小叶中心迁移,形成大量的胆小管,并有部分向坏死区迁移。透射电镜有新生内源性细胞,小于成熟的肝细胞,细胞器较少,有细胞紧密连接,以数个细胞排列成小胆管,与免疫组化一致。结论在大鼠急性肝损伤时HOC被活化、增殖,并向肝小叶中心迁移,全程参与了肝再生过程。     

骨髓间充质干细胞在氨气致大鼠吸入性肺损伤的疗效观察

目的:观察骨髓间充质干细胞在氨气致大鼠吸入性肺损伤的疗效。方法:随机选取69只普通SD大鼠,随机将这些大鼠分为正常组(n=23)、地塞米松治疗组(n=23)、干细胞治疗组三组(n=23),正常组给予尾静脉注射生理盐水,地塞米松治疗组给予注射地塞米松,干细胞治疗组给予干细胞注射。结果:干细胞治疗组大鼠治疗后1 d、3 d的肺组织病理学评分及肺W/D、BALF白细胞数量及蛋白浓度、TNF-α、IL-8水平均显著低于地塞米松治疗组(P0.05),均显著高于正常组(P0.05)。结论:骨髓间充质干细胞在氨气致大鼠吸入性肺损伤的疗效较地塞米松显著。     

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow

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骨髓问充质干细胞移植对VCD所致卵巢损伤的修复研究

目的：探讨小鼠骨髓间充质干细胞（MSCs）移植对去氧乙烯基环己烯（VCD）所致卵巢早衰治疗的可行性。方法：采用VCD（160mgkg^-l,day^-1）连续腹腔注射来诱导小鼠卵巢早衰。每侧卵巢注射转染了绿色荧光基因小鼠骨髓来源的MSCs,于移植后14、28天及45天,取各组血液标本及卵巢组织,同时观察小鼠动情周期的变化;酶联免疫法检测血清FSH、LH水平,显微镜下观察MSC在卵巢的分布。结果：MSCs移植后各组均可见绿色荧光,并且主要分布于卵巢间质区,卵巢泡膜细胞区也可见绿色荧光细胞。MSCs组动情周期较实验对照组缩短,FSH与LH水平较实验对照组低,差异具有显著性。结论：骨髓间充质干细胞可改善卵巢早衰小鼠的卵巢内分泌功能,并且长时间存在于卵巢组织。骨髓间充质干细胞可能成为卵巢早衰治疗的新方法。     

骨髓间充质干细胞移植对VCD 所致卵巢损伤的修复研究

目的:探讨小鼠骨髓间充质干细胞(MSCs)移植对去氧乙烯基环己烯(VCD)所致卵巢早衰治疗的可行性。方法:采用VCD(160mg kg-1,day-1)连续腹腔注射来诱导小鼠卵巢早衰。每侧卵巢注射转染了绿色荧光基因小鼠骨髓来源的MSCs,于移植后14、28天及45天,取各组血液标本及卵巢组织,同时观察小鼠动情周期的变化;酶联免疫法检测血清FSH、LH水平,显微镜下观察MSC在卵巢的分布。结果:MSCs移植后各组均可见绿色荧光,并且主要分布于卵巢间质区,卵巢泡膜细胞区也可见绿色荧光细胞。MSCs组动情周期较实验对照组缩短,FSH与LH水平较实验对照组低,差异具有显著性。结论:骨髓间充质干细胞可改善卵巢早衰小鼠的卵巢内分泌功能,并且长时间存在于卵巢组织。骨髓间充质干细胞可能成为卵巢早衰治疗的新方法。     

骨髓间充质干细胞对大鼠急性肾损伤的修复作用

目的：观察外源性骨髓间充质干细胞（Mesenchymal stem cells,MSCs）对庆大霉素（Gentamycin,GM）诱导的大鼠急性肾损伤是否具有治疗作用,并初探其机制。方法：建立腹腔注射庆大霉素致大鼠急性肾损伤模型。实验分为舡常对照组、模型组、MSCs治疗组（模型＋MSCs）、生理盐水组（模型＋生理盐水）。于不同处理后4d分别检测血尿素氮（BUN）和肌酐（Scr）水平,观察肾组织病理改变,免疫印迹及RT-PCR法检测肾组织肝细胞生长因子（Hepatocyte growth factor,HGF）水平。结果：模型组大鼠的BUN及Scr较正常对照组显著升高,且肾小管组织病理损伤严重;而MSCs治疗组大鼠的BUN及Scr水平较生理盐水组显著降低,肾小管组织病理损伤明显减轻。此外。促肾小管损伤修复的肝细胞生长因子（HGF）表达在MSCs治疗组显著高于生理盐水组。结论：MSCs输注可促进庆大霉素所致急性肾小管损伤的修复,改善肾功能,其作用机制可能是与上调肾组织中肝细胞细胞生长因子的表达有关。     

骨髓间充质干细胞对大鼠急性肾损伤的修复作用

目的:观察外源性骨髓间充质干细胞(Mesenchymal stem cells,MSCs)对庆大霉素(Gentamycin,GM)诱导的大鼠急性肾损伤是否具有治疗作用,并初探其机制。方法:建立腹腔注射庆大霉素致大鼠急性肾损伤模型实验分为正常对照组、模型组、MSCs治疗组(模型+MSCs)、生理盐水组(模型+生理盐水)。于不同处理后4d分别检测血尿素氮(BUN)和肌酐(Scr)水平,观察肾组织病理改变,免疫印迹及RT-PCR法检测肾组织肝细胞生长因子(Hepatocyte growth factor,HGF)水平。结果:模型组大鼠的BUN及Scr较正常对照组显著升高,且肾小管组织病理损伤严重;而MSCs治疗组大鼠的BUN及Scr水平较生理盐水组显著降低,肾小管组织病理损伤明显减轻。此外,促肾小管损伤修复的肝细胞生长因子(HGF)表达在MSCs治疗组显著高于生理盐水组。结论:MSCs输注可促进庆大霉素所致急性肾小管损伤的修复,改善肾功能,其作用机制可能是与上调肾组织中肝细胞细胞生长因子的表达有关。     

骨髓间充质干细胞移植在小鼠急性肾损伤肾内的存活与分化情况研究

为探讨急性肾损伤后,移植骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs在损伤肾内的存活能力及分化情况,通过复苏、扩增培养已完成鉴定、经慢病毒EGFP转染的MSCs,观察备用MSCs的EGFP(enhanced green fluorescent protein)表达情况,采用雄性C57BL/6J小鼠12只建立小鼠肾脏缺血再灌注损伤模型后(结扎双侧肾蒂40 min后开放血流并经尾静脉注射MSCs),分别于第1、3、7、14 d处死3只小鼠,采集小鼠左侧肾脏制作石蜡切片,倒置荧光显微镜下观察MSCs在肾内的存活及分化情况,定量分析每个时间点存活于肾内的数目的差异.结果表明复苏、扩增培养出的MSCs增殖活力旺盛,成功建立小鼠肾脏缺血再灌注损伤模型.移植的细胞随时间的延长,存活于肾脏内的数量显著减少(第14 d存活于肾内的细胞数量仅为移植后第1d的1/5),第1、3、7、14 d表达EGFP的MSCs主要分布于肾小球周围、肾脏小血管内壁、肾小管与肾小管之间的间质而肾小管内壁未见到表达EGFP的MSCs分布.这说明移植的MSCs在缺血再灌注损伤后肾脏内能够存活,但肾脏内的微环境限制了移植细胞的存活能力.在肾小管内壁未观测到表达EGFP的MSCs,提示MSCs对肾脏修复的途径不是直接向肾小管内皮细胞分化而另有其它途径.     

山丹黄参多糖对四氯化碳致小鼠急性肝损伤的影响

为了研究山丹黄参多糖对四氯化碳(CCl4)致小鼠(Mus musculus)急性肝损伤及相关酶活性的影响,对50只小鼠分别灌胃0.2 ml山丹黄参多糖(12.5、25.0、37.5 g/L)或生理盐水7 d,最后一次灌胃1 h后腹腔注射1%的CCl4橄榄油溶液0.2 ml,16 h后处死小鼠,比色法检测血浆丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)活力的变化,显微镜观察肝组织结构的变化,免疫组织化学法检测肝组织中转化生长因子-β_1(TGF-β_1)表达的变化。与正常对照组相比,实验对照组小鼠体重减轻,血浆丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活力显著升高(P0.01),肝明显肿胀,肝组织结构不清,肝细胞出现炎性坏死,转化生长因子-β_1(TGF-β_1)阳性表达明显增强(P0.01)。与实验对照组相比,山丹黄参多糖各组小鼠体重增加,血浆丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活力明显降低(P0.01),肝无明显肿胀,肝索清晰,炎性坏死很少,细胞结构清晰,转化生长因子-β_1阳性表达明显减少(P0.01)。表明一定剂量的山丹黄参多糖能增强机体活力,促进机体新陈代谢,降低正常细胞的凋亡,对四氯化碳造成的肝组织损伤有一定的保护作用。     

Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

Toll-like receptor (TLR) activation has been implicated in acetaminophen (APAP)-induced hepatotoxicity. Herein, we hypothesize that TLR3 activation significantly contributed to APAP-induced liver injury. In fasted wildtype (WT) mice, APAP caused significant cellular necrosis, edema, and inflammation in the liver, and the de novo expression and activation of TLR3 was found to be necessary for APAP-induced liver failure. Specifically, liver tissues from similarly fasted TLR3-deficient (tlr3^−/−) mice exhibited significantly less histological and biochemical evidence of injury after APAP challenge. Similar protective effects were observed in WT mice in which TLR3 was targeted through immunoneutralization at 3 h post-APAP challenge. Among three important death ligands (i.e. TNFα, TRAIL, and FASL) known to promote hepatocyte death after APAP challenge, TNFα was the only ligand that was significantly reduced in APAP-challenged tlr3^−/− mice compared with APAP-challenged WT controls. In vivo studies demonstrated that TLR3 activation contributed to TNFα production in the liver presumably via F4/80⁺ and CD11c⁺ immune cells. In vitro studies indicated that there was cooperation between TNFα and TLR3 in the activation of JNK signaling in isolated and cultured liver epithelial cells (i.e. nMuLi). Moreover, TLR3 activation enhanced the expression of phosphorylated JNK in APAP injured livers. Thus, the current study demonstrates that TLR3 activation contributes to APAP-induced hepatotoxicity.     

KLF4在内毒素血症小鼠中的表达模式及其意义

目的探讨Kruppel样因子4（Kruppel-like factor 4,KLF4）在内毒素血症小鼠中的表达模式及意义。方法运用实时荧光PCR技术和Western blot技术,分别从mRNA水平和蛋白水平探讨内毒素血症小鼠肝脏和肺脏中KLF4的表达;运用生物信息学技术,对启动子区含有KLF4的结合位点的炎症介质基因进行了预测;运用RT-PCR技术,从mRNA水平探讨内毒素血症小鼠肝脏和肺脏中IL1β的表达模式。结果内毒素血症小鼠肝脏和肺脏中KLF4 mRNA的表达下凋,KLF4蛋白的表达先下凋后升高;IL-18、IL-15、IL-12、IL-18、IL-10等炎症介质基因的启动子区均含有KLF4的结合元件,这些炎症基因的表达可能直接受到KLF4的调控;内毒素血症小鼠肝脏和肺脏中IL-IB的表达模式与KLF4的表达模式呈相反趋势。结论内毒素血症小鼠肝脏和肺脏中KLF4表达下调,KLF4在炎症介质基因表达调控中可能具有重要作用。