IRAK-4活性在脂多糖预处理减轻肝脏缺血再灌注损害中作用的实验研究

目的:观察脂多糖(LPS)预处理后白细胞介素-1受体相关激酶-4（IRAK-4）表达水平在大鼠肝脏缺血再灌注(I/R)早期的变化，探讨LPS预处理减轻肝脏缺血再灌注损害(I/RI)的相关机制。 方法:雄性SD大鼠，随机分为正常对照组，缺血再灌注组(I/R组)和LPS预处理组（LPS组）。正常对照组未予任何处理；LPS组第 1 d 经尾静脉给予脂多糖0.1mg·kg-1，第2、3、4、5 d给予0.5 mg·kg-1；I/R组给予等体积0.5 mL无菌PBS液。第 8 d，建立肝脏缺血再灌注模型。再灌注后0 min、60 min及180 min, 蛋白免疫印记法及逆转录-聚合酶链式反应测定肝组织的IRAK-4蛋白和mRNA表达水平；酶连免疫吸附法检测肝组织NF-κB活性及血清TNF-α含量。 结果:再灌注0 min, IRAK-4蛋白与mRNA表达水平依次为LPS组>I/R组>正常对照组(P<0.01), NF-κB活性以及TNF-α含量LPS组与I/R组差异无显著(P>0.05)，但均高于正常对照组(P<0.01)；再灌注后60 min及180 min，LPS组的IRAK-4蛋白与mRNA表达水平，NF-κB活性以及TNF-α含量却明显低于I/R组（P<0.01）。 结论:抑制IRAK-4表达是LPS预处理减轻肝脏I/RI的重要机制之一。     

HSP70对大鼠肝脏局部缺血再灌注后炎症反应的影响

目的：通过热应激预处理诱导HSP70表达，探讨其对肝脏缺血再灌注损伤后炎症反应的影响。方法:采用大鼠局部缺血再灌注模型（IR组），并在热应激预处理（H+IR组）及槲皮素＋热应激预处理（Q+H+IR组）条件下观察肝脏缺血再灌注后HSP70、ICAM-1的表达及MPO的活性；测定血清ALT和AST的活性；电镜观察肝细胞结构的改变。结果:在H+IR组检测的各时点HSP70表达均明显高于其它两组、对肝脏进行缺血再灌注后，肝细胞损伤较轻，血清ALT、AST升高不明显(P<0.01)；肝组织中ICAM-1表达增加，以再灌注后6 h最显著，MPO活性升高以12 h最为显著，但两者变化均低于IR组和Q+H+IR组(P<0.01)。结论：〖HTSS〗热应激预处理诱导产生HSP70蛋白能够降低大鼠肝脏缺血再灌注损伤过程ICAM-1的表达和MPO活性的改变，进而抑制炎症反应引起的肝脏损伤。     

低分子肝素对缺血再灌注大鼠肾组织核因子-κB表达的影响

探讨低分子肝素对缺血再灌注大鼠肾组织核因子-κB（NF-κB）表达的影响。建立大鼠IRI模型，健康WistaI大鼠80只随机分为正常对照组、假手术组、模型未治疗组、LMWH治疗组，后两组又分别分为术后1、3、6、24h组。检测各组血清肌酐（Scr）水平及中性粒细胞（PMNs）细胞间黏附分子-1（ICAM-1）表达；通过光镜和免疫组织化学方法观察各组大鼠肾组织形态学及趋化因子NF-κB表达变化。结果表明：（1）肾缺血再灌注未治疗组造模后1h，Scr水平虽然没有明显变化，但ICAM-1、NF-κB表达增多，肾小管坏死积分值亦较假手术组明显增加（P〈0．01）；（2）缺血再灌注6h以后，两组Scr浓度明显增高（P〈0．01），但LMWH治疗组SCr、ICAM-1、NF-κB表达水平及肾小管坏死积分值均明显低于模型未治疗组（P〈0．05）；（3）肾组织中NF-κB表达与肾小管损伤积分值呈现良好的相关性（r=0．71，P〈0、01）；而NF-κB与ICAM-1间则呈现显著正相关（r=0．62，P〈0．05）。由此说明：（1）ICAM-1、NF-κB在肾缺血再灌注早期的瞬时表达，可能参与了炎症早期的白细胞迁移与浸润，与肾损伤的发生密切相关；（2）LMWH可通过减少ICAM-1及NF-κB的表达，阻抑炎症反应过程，减轻肾组织损伤。     

NBD多肽预处理对局灶脑缺血再灌注大鼠脑内核因子-κB核转位活化的影响

目的探讨NBD多肽预处理对局灶脑缺血再灌注大鼠大脑缺血皮质细胞内核因子-κB活化的影响。方法将SD健康雄性大鼠（280～300g）共36只随机分为假手术组（n=6）、模型组（n=15）、药物组（n=15）。缺血模型制备前2h经右侧侧脑室注射NBD多肽25μl进行预处理。运用改良线栓法制备右侧大脑中动脉闭塞再灌注大鼠模型。运用免疫组化检测再灌注后72hNF-κBp65在胞浆／胞核的蛋白表达变化;运用免疫荧光定位及Western印迹（半定量）检测NF-κB p65及IκBα的蛋白表达情况。结果免疫组化结果显示与假手术组比较,再灌注72h模型组胞浆／胞核内NF-κB p65大量表达（P〈0．05）;NBD多肽预处理后NF-κB p65主要在胞浆表达,胞核内表达明显减少（P〈0．05）;免疫荧光双标定位及Western印迹半定量检测显示模型组胞浆／胞核内NF-κB p65蛋白均大量表达（P〈0．05）,IκBα蛋白呈现低表达（P〈0．05）;NBD多肽预处理后胞核内NF-κB p65蛋白表达明显减少,主要以胞浆表达为主（P〈0．05）,IκBα胞浆／胞核内表达显著增加（P〈0．05）。结论局灶脑缺血再灌注72hNF-κB p65蛋白胞核表达明显增加．NF-κB核转位／活化过程被激活：NBD多肽预处理后通过增加胞核／胞浆内IκBα表达有效阻止NF-κB的核转位／活化过程,从而有效地减轻再灌注后72h局灶脑缺血再灌注对脑组织的损害。     

大鼠局灶性脑缺血-再灌注时ε型蛋白激酶与热休克蛋白70表达变化及相关性

目的探讨大鼠脑缺血—再灌注时ε型蛋白激酶C（PKCε）与热休克蛋白70（HSP70）表达间关系。方法60只大鼠，随机分成缺血-再灌注组及假手术组，根据再灌注时间的不同，分别分为再灌注1、6、12、24、48、72h亚组，应用蛋白印迹法观察缺血皮层、基底节区各时间点胞浆内PKCε及HSP70及胞膜PKCε的表达。结果脑缺血后再灌注1h胞浆内PKCε含量开始下降，至再灌注24h达最低点，持续至再灌注72h。而膜内PKCε变化趋势与此相反，含量逐渐上升，于再灌注24h达到最高。再灌注1hHSP70有少量表达，再灌注6h即增高，至再灌注48h达最高，再灌注72h稍有减弱。假手术组各时间点均低含量表达。脑缺血-再灌注时HSP70与胞膜PKCε表达呈正相关，但无差异性，（r=0．55，P〉0．05）；HSPT0与胞浆内PKCε表达间呈显著负相关（r=-0．672，P〈0．05）。结论脑缺血再灌注时缺血皮层、基底节区PKCε发生了膜转位激活现象，同时有HSP70的表达，两者间呈一定的相关性。     

替罗非班对大鼠心肌缺血再灌注后无复流及NF-κB激活的影响

目的： 研究血小板膜糖蛋白Ⅱb/Ⅲa受体拮抗剂替罗非班对大鼠心肌缺血再灌注后无复流及NF-κB激活的影响。方法：雄性Wistar大鼠,随机分为假手术组、心肌缺血再灌注组和替罗非班治疗组（60 μg/kg,再灌注前30 min股静脉注射）。心肌缺血再灌注组和替罗非班治疗组采用开胸冠状动脉结扎方法,缺血90 min再灌注120 min建立急性心肌缺血再灌注无复流模型。观察大鼠心肌缺血、梗死及无复流范围;免疫组织化学方法半定量分析心肌细胞及微动脉核因子-κB p65（NF-κB p65）的阳性表达;测定心肌髓过氧化物酶（MPO）活性及丙二醛（MDA）含量。结果： 心肌缺血再灌注组和替罗非班治疗组大鼠缺血区心肌细胞及微动脉NF-κB p65的阳性表达、心肌MPO活性、MDA含量高于假手术组;替罗非班治疗组大鼠缺血区心肌细胞及微动脉NF-κB p65的阳性表达、心肌MPO活性、MDA含量低于心肌缺血再灌注组（P＜0.05）;心肌无复流范围及梗死范围小于心肌缺血再灌注组（34.36%±6.04% vs 52.09%±6.89%, P＜0.01; 80.41%±8.48% vs 90.13%±5.72%, P＜0.05）。结论： 大鼠心肌缺血90 min再灌注120 min时,可发生无复流现象;替罗非班可缩小心肌无复流及梗死范围,抑制NF-κB激活,减少中性粒细胞浸润及氧自由基释放。     

热休克预处理对大鼠缺血再灌注心肌保护作用机制的探讨

目的:观察心肌缺血再灌注时P-选择素（Ps）表达情况；探讨热休克蛋白(HSP)对缺血再灌注心肌Ps及细胞凋亡表达的影响。 方法:成年雌性Wistar（n=40）大鼠随机分为3组。热休克组全麻后高热处理造成热休克动物模型，对照组及假手术组仅予全麻处理。24 h后热休克组及对照组结扎左冠状动脉前降支(LAD)1 h，再灌注2 h造成心肌缺血再灌注动物模型。假手术组只于LAD处穿线而不结扎。术毕测心梗范围、HSP70、Bax、Bcl-2、Ps、凋亡细胞及血清CK-MB。 结果:热休克组HSP70表达高于对照组及假手术组(P<0.05)，后两组无明显差别(P>0.05)；热休克组心梗范围小于对照组(P<0.05)，CK-MB值低于对照组(P<0.01)，凋亡细胞、Bax及Ps表达低于对照组(P<0.05)，两组Bcl-2表达无显著差别(P>0.05)；假手术组无Ps表达。 结论:HSP70可抑制缺血再灌注诱导的心肌细胞凋亡，抑制Bax表达致Bax/Bcl-2比值下降为其机制之一；Ps参与心肌缺血再灌注损伤；HSP70可能有抑制心肌Ps表达的作用，这或许是热休克预处理对大鼠缺血再灌注心肌的另一保护机制。     

虎杖甙下调兔肺缺血再灌注损伤时TLR4的表达

目的： 研究家兔肺缺血再灌注损伤时Toll样受体4(TLR4)信号转导通路变化及虎杖甙(PD)对其影响。方法：复制在体肺缺血再灌注损伤模型。健康日本大耳白免30只, 随机分为对照(C)组、缺血再灌注(IR)组、PD组。鲎试剂试管法检测血浆内毒素(ET); 免疫组化法检测肺组织TLR4、核因子-κB p65(NF-κB p65)和热休克蛋白70(HSP70)的蛋白表达；原位杂交法检测肺组织细胞间黏附分子-1(ICAM-1)mRNA表达；电镜观察肺超微结构变化。结果：IR组、PD组与C组相比血浆ET无显著差异(均P>0.05)。IR组肺组织TLR4、NF-κB p65、HSP70蛋白及ICAM-1mRNA表达较C组显著升高(均P<0.01)；PD组上述改变较IR组明显下降，但仍高于C组(均P<0.01); 电镜见PD组肺超微结构损伤明显轻于IR组。结论：肺缺血再灌注损伤过程中HSP70合成增加，作为内源性配体之一上调TLR4，可能通过激活NF-κB，进而诱导ICAM-1的转录和分泌。PD可以下调该信号转导途径，减轻肺缺血再灌注损伤。     

活脉通络饮对大鼠脑缺血再灌注脑组织NF-κB和IL-1β表达的影响

目的观察活脉通络饮对脑缺血再灌注大鼠脑组织核转录因子-κB(NF-κB)和白细胞介素-1β(IL-1β)蛋白表达的影响,探讨活脉通络饮防治脑缺血的作用机制。方法采用改良的线栓法,建立大鼠大脑中动脉栓塞(MCAO)脑缺血再灌注模型。分别设正常对照组、缺血再灌注组、活脉通络饮低剂量组、活脉通络饮中剂量组、活脉通络饮高剂量组和银杏叶片阳性对照组。分别于脑缺血再灌注术后24h后开始灌胃给药,每d给药2次,连续3 d。采用Western Blot定性和定量法检测脑组织中NF-κB和IL-1β蛋白的表达。结果与对照组相比,缺血再灌注组大鼠脑组织NF-κB和IL-1β蛋白表达增强(P0.01);与缺血再灌注组比较,活脉通络饮低剂量组和中剂量组及银杏叶片阳性对照组大鼠脑组织NF-κB和IL-1β蛋白表达明显减少(P0.01或P0.05),而活脉通络饮高剂量组与缺血再灌注组相比没有统计学差异。结论活脉通络饮对脑缺血再灌注脑损伤的治疗作用可能与其下调大鼠脑组织NF-κB和IL-1β蛋白表达相关。     

腺苷预处理对大鼠脑缺血-再灌注损伤后脑内肿瘤坏死因子α和核因子κB表达的影响

目的 通过观察腺苷预处理对脑缺血-再灌注（IR）损伤后大鼠脑内肿瘤坏死因子α（TNF-α）和核因子κB（NF-κB）表达的影响,探讨腺苷对大鼠脑缺血-再灌注损伤的保护机制。方法 Sprague-Dawley大鼠60只随机分为假手术组（F组）、缺血-再灌注组（IR组）和腺苷预处理缺血-再灌注组（AP组）,每组动物按照缺血-再灌注后2 h、6 h、24 h及48 h这4个时间点随机分组,每组5只。采用大脑中动脉阻塞（MCAO）法制备大鼠大脑中动脉闭塞模型。通过对以上3组脑缺血 再灌注48 h后的大鼠脑组织行头颅MRI、HE染色以及对以上60只大鼠进行神经功能缺损评分,通过免疫组织化学方法观察大鼠脑组织中TNF-α和NF-κB的平均积分吸光度值进一步验证腺苷预处理对大鼠脑缺血-再灌注损伤的脑保护作用,探讨腺苷对大鼠脑缺血-再灌注损伤的保护机制。结果 F组大鼠术后无神经功能缺损体征,AP组和IR组大鼠术后均出现明显神经功能缺损体征。AP组和IR组大鼠神经功能缺损评分显著高于F组,AP组大鼠神经功能缺损评分显著低于IR组,差异均具有统计学意义（P＜0.05）。F组大鼠术后MRI中T1WI、T2WI均未见明显异常,AP组和IR组大鼠脑梗死体积显著高于F组,AP组大鼠脑梗死体积显著低于IR组,差异均具有统计学意义（P＜0.05）。AP组和IR组术后大鼠脑组织中TNF-α和NF-κB的表达显著高于F组,AP组术后大鼠脑组织中TNF-α和NF-κB的表达显著低于IR组,差异均具有统计学意义（P＜0.05）。结论 腺苷预处理可通过减少大鼠缺血-再灌注损伤后脑内TNF-α和NF-κB的表达而减轻神经细胞损伤,达到其脑保护作用。     

Susceptibility of mouse mammary glands to murine gammaherpesvirus 72 (MHV-72) infection: evidence of MHV-72 transmission via breast milk.

Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient small animal model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cells and establishes the latency in B lymphocytes. In this study, we investigated athymic nude and immunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten days following subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs, salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection in former tissues, but increased in mammary glands. Presence of virus DNA sequences was detected in the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42 days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover, virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence) in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days before delivery. We demonstrated the presence of virus DNA also in the milk removed from the stomach of non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2 days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did not become infected from wet-nurses by the intranasal route. This suggests that MHV may be naturally transmitted to newborn mice via breast milk.     

Initiation of the Immune Response by Extracellular Hsp72: Chaperokine Activity of Hsp72

Heat shock proteins exert their beneficial effects via basically two modes of action depending on their relative location within the host. Intracellular heat shock proteins found within cells serve a cytoprotective role by chaperoning na?ve, misfolded and/or denatured proteins in response to stressful stimuli by a process known as the stress response. However, stressful stimuli also induce the release of intracellular heat shock proteins into the extracellular milieu and circulation. The extracellular heat shock protein proteins serve a cytostimulatory role by initiating immune responses designed to fend off microbial infection and destroy neoplastic transformed cells. This review will briefly cover recent advances into elucidating the mechanism(s) by which stress induces the release of heat shock proteins into the circulation, how it initiates immune responses and suggest the possible biological significance of circulating Hsp to the host.     

Prevalence of antibodies to 72-kilodalton glycoprotein (GP72) in patients with Chagas' disease and further evidence of zymodeme-associated expression of GP72 carbohydrate epitopes

Three competitive inhibition enzyme-linked immunosorbent assays were developed to examine the expression of the 72-kilodalton glycoprotein (GP72) and of a GP72 carbohydrate epitope in Trypanosoma cruzi strains and clones. A total of 148 strains and clones of known isozyme phenotype (principal zymodeme, Z) were tested. With monoclonal antibody 8G2B9 the enzyme-linked immunosorbent assay confirmed that the majority of Z1 strains and clones derived from them had undetectable levels of the carbohydrate epitope identified by antibody 8G2B9. This epitope was, however, readily detectable in all Z2, Z2(h), and Z3 strains and clones (P less than 0.001; 148 strains and clones tested). Zymodeme-associated differences in GP72 expression were not apparent from the enzyme-linked immunosorbent assay with monoclonal antibody WIC 226.4 (raised against periodate-treated GP72) or from that with rabbit anti-GP72 antiserum (84 or 119 strains and clones tested, respectively). Mice infected with culture-form metacyclic trypomastigotes of Z1, Z29, and Z3 or with blood-form trypomastigotes of Z1 and Z3 developed antibodies to affinity-purified GP72, showing that at least some GP72 epitopes are neither zymodeme specific nor stage specific. A total of 128 serum samples from patients with acute or clinically classified chronic Chagas' disease were assayed for immunoglobulin G (IgG) or IgM anti-GP72 antibodies. During the acute phase anti-GP72 IgM antibodies were elevated, whereas anti-GP72 IgG antibodies were low. There were no significant differences in anti-GP72 antibody levels among chronic-phase patient groups. Anti-GP72 antibodies were detected irrespective of the geographical origin of patients and irrespective of whether acute-phase blood parasitemias were due to Z1 (four patients) or Z2 (two patients).     

CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation.

CD72, a B cell surface protein of the C-type lectin superfamily, recruits the tyrosine phosphatase SHP-1 through its ITIM motif(s). Using CD72-deficient (CD72-/-) mice, we demonstrate that CD72 is a nonredundant regulator of B cell development. In the bone marrow of CD72-/- mice, there was a reduction in the number of mature recirculating B cells and an accumulation of pre-B cells. In the periphery of CD72-/- mice, there were fewer mature B-2 cells and more B-1 cells. In addition, CD72 is a negative regulator of B cell activation, as CD72-/- B cells were hyperproliferative in response to various stimuli and showed enhanced kinetics in their intracellular Ca2+ response following IgM cross-linking.     

Heat shock protein 72 (Hsp72) improves long term recovery after focal cerebral ischemia in mice

Many brain protective strategies have been tested over short survival intervals, but few have been examined for long term benefit. The inducible member of the Heat shock protein 70 (Hsp70) family, Heat shock protein 72 (Hsp72), has been widely found to reduce ischemic injury. Here we assessed outcome in Hsp72 transgenic overexpressing mice and wild type littermates for one month following transient focal ischemia. Hsp72 reduced infarct area lost and improved behavioral outcome on rotarod and foot fault at one month. Thus protection by Hsp72 overexpression is long lasting, and includes improved recovery of motor function over one month.     

肝内胆管结石的肝脏病理观察

目的：探讨肝内胆管结石之肝脏病理变化。方法：对７２例肝内胆管结石的肝叶标本进行病理检查，部分尚行粘液及胶原染色。结果：镜一常见的病变是肝间质炎症、胆管上皮及腺体增生和粘液上皮化生（１００％）肝细胞水肿变性及小叶中央性淤胆。胆蛔症。２例伴肝胆管上皮癌，癌旁胆管上皮及腺体异型增生与癌相移行。３３例见局限于门管区的纤维组织增生。结论：肝内胆管结石与胆道炎症、上皮成分增生及粘液化生相关与胆管上皮癌发生有关