枯否细胞在刀豆蛋白A诱导的急性肝损伤中的作用

给小鼠用刀豆蛋白以O，YIAWkg）尾静脉注射，复制实验性急性肝损伤模型，通过检测血浆丙氨酸氨基转移酶（ALT），肿瘤坏死因子．a（’FNF－a）、光镜和电镜观察下肝组织病理学变化评价肝损伤。雄性国出VCjJ’鼠，体重20－3（），6－7周龄，实验前16h禁食，动物由湖北省医科院实验动物中心提供。随机将实验小鼠分为对照组（n＝8）、o）’lrt组（n＝8）和二氧化硅（Stq）＋（ConA组（n＝8）。对照组尾静脉注射无菌磷酸盐缓冲液（PBS）0．3ml/只，ConA组尾静脉注射见onA20mg/kg，SiO2＋ConA组在静脉注射haA20lug／kg前18h分别向尾…     

免疫性肝损伤中诱导型一氧化氮合酶的细胞来源

采用胶原酶-链霉蛋白酶灌流法对免疫性肝损伤大鼠肝实质细胞与枯否细胞进行分离与原代培养,Griess反应法检测细胞培养上清中一氧化氮（NO）生成量的变化。结果显示在刺激条件及细胞数量同等情况下,肝实质细胞NO生成量显著高于枯否细胞;肿瘤坏死因子（TNFα）单克隆抗体可拮抗枯否细胞由细菌脂多糖（LPS）刺激所致NO生成的增加,而对卡介苗（BCG）所致NO生成无显著影响。提示免疫性肝损伤中NO生成主要源于肝实质细胞;LPS通过使枯否细胞释放TNFα对NO生成进行调节。     

微小RNA-7敲减对ConA诱导的小鼠急性肝损伤的影响

目的:研究微小RNA-7(miR-7)敲减(KD)对急性肝损伤(ALI)模型小鼠的影响。方法:野生型(WT)小鼠和miR-7KD小鼠腹腔注射30 mg/kg刀豆蛋白(ConA)建立急性肝损伤模型;48 h后,观察小鼠肝脏的形态、重量及其脏器指数变化;HE染色观察小鼠肝脏组织病理学变化;血清学方法检查血清中谷丙转氨酶(ALT)的水平;ELISA法检测血清中细胞因子IL-4和IFN-γ的水平;流式细胞术检测肝脏组织中CD4~+T细胞的比例及其相关的细胞因子IL-4和IFN-γ的表达变化。结果:与对照组相比,miR-7敲减后急性肝损伤小鼠的肝脏组织颜色变浅,重量减轻,重量指数明显增加(P0.05);HE染色显示miR-7KD小鼠血清炎症细胞浸润显著增多;血清学方法检测发现急性肝损伤小鼠血清ALT的水平明显上升(P0.05);ELISA法检测显示miR-7KD小鼠血清中IFN-γ水平明显升高(P0.01),而IL-4的表达水平则明显降低(P0.01);流式细胞术检测结果显示,miR-7KD小鼠肝脏中CD4+T细胞比例显著升高(P0.01),其相关的细胞因子IFN-γ的表达水平也显著上调(P0.01),而IL-4的水平没有发生明显变化。结论:敲减miR-7基因可明显促进ConA诱导的小鼠急性肝损伤。     

百日咳毒素S1亚单位的分子修饰及在重组杆状病毒中的表达

目的：百日咳毒素既是百日咳杆菌的主要毒性因子,又是重要的保护性抗原,其Ｓ１亚单位具有ＡＤＰ－核糖转移酶活性和免疫保护性决定簇。为高效表达前构建的一个丧失酶活性但仍保持保护决定簇的Ｓ１变异子,开展了本研究。方法：通过添加人流感病毒血凝素基因信号序列或嵌膜序列的方法,对天然和变异Ｓ１亚单位基因片段作了遗传学修饰。然后使用重组杆状病毒技术使这些Ｓ１亚单位在昆虫细胞和昆虫幼虫中实现了高水平表达。结果：这些     

CD24分子的免疫学研究进展

人CD24、鼠CD24a或热稳定抗原是一种重要的糖基磷脂酰肌醇锚定的膜蛋白（GPI）,该蛋白可作为判断多种细胞（如T细胞、B细胞等）成熟与否的标志,参与调节机体的免疫应答,与系统性红斑狼疮、风湿性关节炎等自身免疫性疾病以及肿瘤疾病密切相关,因此引起人们广泛的关注.新近研究发现：CD24可通过CD24-Siglec10/G信号通路能够抑制免疫反应,保护宿主免遭由于细胞死亡所引起的致死反应.     

脂多糖诱导小鼠肺损伤模型巨噬细胞活化及共刺激分子CD40上调研究

目的观察肺泡巨噬细胞（AM）活化过程和共刺激分子CD40表达变化，探讨其在脂多糖（LPS）诱导的小鼠急性肺损伤（ALI）模型中所发挥的作用。方法BALB／c小鼠分为正常对照组和LPS处理组。光镜观察24、48h肺组织病理变化；RT-PCR测定活化巨噬细胞（activated macrophage，AMφ）中TLR4表达；电泳迁移率变动分析（EMSA）检测AMφ核提取物中NF-κB的活性；Northern blot检测CD40 mRNA表达，流式细胞术检测CD40蛋白表达；ELISA测定肺泡灌洗液（BALF）中TNF-α、MIP-2及IL-1β的含量。结果小鼠吸入LPS后，肺泡隔断裂、肺间质充血及肺泡腔中性粒细胞浸润，并检测到AMφ表面TLR4的表达、核转录因子NF-κB活性增强、共刺激分子CD40mRNA和蛋白显著表达，并随着时间延长出现增加趋势，BALF中炎症因子释放增加，正常对照组无明显变化（P〈0．05）。结论LPS诱导的ALI中，AM活化和共刺激分子CD40上调，导致瀑链式炎症反应，造成肺急性炎症损伤。     

乳腺癌中CD44~+/CD24~-表型及乳腺癌分子亚型的分布及意义

目的探讨CD44+/CD24-表型在乳腺癌中的临床病理意义和乳腺癌分子亚型在中国人群中的分布。方法回顾分析217例乳腺癌患者,根据ER、PR、Her-2及CK5/6的水平划分为5个分子亚型。应用双重免疫组织化学检测其CD44/CD24双染的情况,分析CD44+/CD24-表型在乳腺癌中表达情况及其与临床病理的相关性;分析乳腺癌分子亚型在中国人群中的分布情况。结果 217例乳腺癌病例中,luminal A型130例,luminal B型15例,Her-2过表达型21例,basal-like型29例,Normalbreast-like型22例。CD44+/CD24-表型在所有乳腺癌阳性表达率为38%。CD44+/CD24-表型与患者年龄、肿瘤大小、组织分级、淋巴结转移等无关(P0.05)。结论乳腺癌干细胞表型(CD44+/CD24-)在乳腺癌中只占一小部分,在人类乳腺癌中CD44+/CD24-表型无临床病理意义;5种分子亚型中luminal A型所占比例最高,其他亚型所占比例较低。     

大蒜油激活枯否氏细胞分泌一氧化氮的实验研究

一氧化氮(NO)在免疫细胞对肿瘤细胞产生毒性方面具有重要意义，可影响肿瘤的增殖生长及死亡。肝脏枯否氏细胞(Kuppfer cell,KC)是机体巨噬细胞中最大的群体，有学者发现生物反应调节剂能激活枯否氏细胞的抗癌活性。本实验在体外培养中，观察大蒜油刺激小鼠枯否氏细胞前、后NO的分泌功能变化。     

高脂高果糖诱导的非酒精性脂肪性肝病小鼠枯否细胞及其信号分子的活化CSCD

目的探讨高脂高果糖饮食诱导的非酒精性脂肪性肝病（NAFLD）小鼠枯否细胞（KCs）活化及其信号通路蛋白的变化,了解KCs在非酒精性脂肪性肝病（NAFLD）致病机制中的意义。方法将20只6～8周龄SPF级C3H小鼠随机分为4组（正常组、果糖组、高脂组、高脂果糖组）饲养,每组5只。16周后处死小鼠,做肝脏病理检查,同时使用蛋白免疫印迹（WesternBlot）方法检测肝组织中F4／80、NF-KB、p-AKT、AKT的表达情况。结果与正常组比较,果糖组、高脂组、高脂果糖组肝组织脂质沉积明显,肝脏HE染色存在明显的炎症及肝细胞脂肪变,高脂高果糖组最为严重。与正常组比较,3组模型组小鼠肝组织中F4／80、NF—KB显著升高,果糖组p-AKT（P〈0．01）、高脂高果糖组AKT（P〈0．05）明显降低。结论高脂高糖饮食可使C3H／HeN小鼠出现典型的非酒精性脂肪肝表现,NAFLD形成涉及肝组织中KCs活化及其相关信号通路的激活。     

FasL与ConA诱导的小鼠肝损伤关系的研究

目的：用ＣｏｎＡ诱导的肝损伤模型对ＦａｓＬ与肝损伤的关系进行了观察。方法：用ＴｒｉｔｏｎＸ－１００和８ｍｏｌ／Ｌ尿素对重组ｈＦａｓＬ融合蛋白包涵体进行纯化,免疫家兔制备多克隆抗体,进行免疫印迹实验表明多抗可以识别ｈＦａｓＬ融合蛋白及纯化的胞外区蛋白。用已知ＣｏｎＡ诱导的小鼠肝损伤模型,用制备的重组产物多抗做免疫组化观测肝组织ＦａｓＬ的表达。结果：与对照相比看到损伤肝细胞ＦａｓＬ表达增高;预先腹腔注射该多抗可降低由ＣｏｎＡ诱导的肝损伤时转氨酶的升高。结论：ｒｈＦａｓＬ多抗中和Ｆａｓ从而减轻这种损伤的作用表达ＦｓａＬ参与了ＣｏｎＡ诱导的肝损伤机制,同时指出这种肝损伤主要是由肝细胞自身引起的。     

Mechanisms of pertussis toxin-induced myelomonocytic cell adhesion: role of CD14 and urokinase receptor

Pertussis toxin (PTX) has been shown previously to promote myelomonocytic cell adhesion in serum. The aim of the present study was to identify, using transforming growth factor-beta1 and 1, 25-(OH)2 vitamin D3 (TGF-beta1/D3)-primed U937 cells, the PTX-binding site(s) and the adhesion molecule(s) responsible for PTX-induced myelomonocytic cell adhesion. Monoclonal antibodies (mAbs) directed against CD14, CD11b, CD18 or urokinase receptor (uPAR) significantly inhibited PTX-induced primed U937 cell adhesion in serum in a concentration-dependent manner. However, only anti-CD14 and anti-CD18 mAbs were able to prevent the myeloid cells from binding to PTX-coated plates and significantly inhibited a PTX-induced rise of [Ca2+]i in primed U937 cells. A receptor-isolation study showed that biotinylated PTX recognized a 48 000-molecular weight protein in primed U937 cell lysates, which could be specifically blocked by excess unlabelled PTX or by anti-CD14 mAb. On the other hand, mAb directed against uPAR significantly blocked PTX-induced myeloid cell adhesion to serum and to immobilized vitronectin, a major extracellular matrix protein in serum. Taken together, our data suggest that PTX may bind to cell-surface CD14 to induce myelomonocytic cell adhesion to vitronectin in serum via uPAR activation, which may represent a pathogenetic mechanism for the respiratory tract infection induced by Bordetella pertussis.     

Effect of CD14 blockade on endotoxin-induced acute lung injury in mice

CD14 functions as a cell surface receptor for endotoxin (lipopolysaccharide [LPS]) and is thought to have an essential role in innate immune responses to infection. Previous studies have revealed attenuation of the systemic response after sepsis by blocking CD14. In this study, we tested the hypothesis that CD14 blockade protects against inflammatory responses associated with LPS pneumonia. We examined the effect of an anti-murine CD14 monoclonal antibody (4C1) on the development of acute lung injury induced by intratracheal LPS in mice. We also measured the production of cytokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2) and nitric oxide by murine peritoneal macrophages exposed to LPS in vitro. Nuclear factor (NF)-kappa B translocation was evaluated in nuclear extracts from lung homogenates. 4C1 significantly attenuated pulmonary edema and neutrophil emigration after LPS administration. The production of cytokines and nitric oxide by LPS-stimulated macrophages was significantly decreased by 4C1 treatment. NF-kappa B translocation induced by LPS instillation was also suppressed by 4C1. These results suggest that blockade of CD14 might attenuate acute lung injury after intratracheal instillation of LPS through the suppression of NF-kappa B translocation. The inhibitory effect of CD14 blockade on cytokine production and nitric oxide release of macrophages might contribute to the attenuation of lung injury.     

Lactobacillus casei Zhang 对扑热息痛诱导的小鼠急性肝损伤的保护作用

目的:研究益生菌Lactobacillus casei Zhang(Lcz)对扑热息痛(APAP)所致小鼠急性肝损伤的保护作用及其机制。方法:C57BL/6 小鼠被随机分为空白组(Ctrl)、APAP 诱导急性肝损伤模型组(Acetaminophen ,APAP)、阳性药物组(N-Acetylcysteine,NAC)、Lcz 预防组(Lcz/ APAP)和Lcz 对照组(Lcz)。Lcz(1伊109 CFU/ ml)连续灌胃30 d 后,NAC 组在APAP 处理前1 h 腹腔注NAC(150 mg/ kg)。APAP、NAC 以及Lcz/ APAP 组均腹腔注射APAP(300 mg/ kg)。APAP 作用18 h 后,采集血液和收集肝脏组织,检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的水平。通过Western blot 检测肝脏组织中血红素氧化酶(HO-1)、超氧化物歧化酶2(SOD2)、Bcl-2 以及TLR4 的表达水平。结果:Lcz 能抑制APAP 诱导的急性肝损伤血清中ALT 和AST 水平。Lcz 提高了HO-1、SOD2 和Bcl-2 的蛋白表达水平,而降低了APAP 诱导的TLR4 的表达。结论:益生菌Lcz对APAP 诱导的小鼠急性肝损伤有保护作用,其保肝作用机制可能与其抗氧化和抗炎活性有关。     

刀豆蛋白A诱导急性肝损伤的病理学观察

目的：探讨刀豆蛋白Ａ（ＣｏｎＡ）诱导的小鼠急性肝损伤的病理机制，方法：应用ＣｏｎＡ诱导建立小鼠实验性急性肝损伤模型，并进行光镜和电镜观察。结果：小鼠早期肝损在出表现为肝细胞凋亡，结论，凋亡可能为刀豆蛋白Ａ诱导的肝细胞死亡的主要机制，ＣｏｎＡ性肝损伤为研究肝炎等发病机制和病理生理提供了一种较理想的实验动物模型。     

Mechanisms of pertussis toxin-induced myelomonocytic cell adhesion: role of Mac-1(CD11b/CD18) and urokinase receptor (CD87).

Stimulation of monoblastic U937 cells with transforming growth factor beta 1 and 1,25-(OH)2 vitamin D3 (TGF-beta 1/D3) upregulates urokinase receptor (uPAR) and confers urokinase-dependent adhesiveness to the cells for serum- or vitronectin-coated surfaces. Recent studies show that uPAR itself is a high-affinity adhesion receptor for vitronectin and that urokinase (uPA) is an activator of this adhesive function. In the course of exploring possible G-protein involvement in this adhesion it was observed that TGF-beta 1/D3-primed U937 cells became adhesive to vitronectin in an uPAR-dependent manner when exposed to pertussis toxin (PTX). The adherent response is concentration- and time-dependent, and was not due to the ADP-ribosyltransferase activity of the toxin because the purified B-subunit of PTX was equally effective. Although promoting adhesion to serum- or vitronectin-coated surfaces, PTX blocked spontaneous cell adhesion to fibrinogen, an endogenous ligand for the Mac-1 receptor (CD11b/CD18). Flow cytometry study showed that expression of the alpha-subunit of Mac-1 (CD11b) on primed cells was increased by nearly threefold. Monoclonal antibody to CD11b abolished the PTX-induced cell adhesion and the binding of the primed cells to PTX-coated plates. Activation of Mac-1 receptor by its endogenous ligand fibrinogen induced cell adherent response similar to PTX. PTX, but not uPA, triggered a rapid rise in [Ca2+]i in primed U937 cells, and PTX-induced adhesion was significantly attenuated by 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy-methyl ester (BAPTA/AM), a selective membrane-permeant [Ca2+]i chelator. PTX-induced cell adhesion was also prevented by antibodies to uPAR and by conditioned medium containing soluble uPAR. Together these data indicate that PTX B-subunit may bind to Mac-1 integrin, which leads to a rapid rise in [Ca2+]i and subsequent activation of uPAR for adherence to vitronectin, suggesting a functional link between Mac-1 and activation of uPAR important to cellular trafficking and host defence in response to Bordetella pertussis infection.     

Cholera toxin-induced tolerance to allografts in mice.

When C3H/HeN (C3H) mice were primed with viable C57BL/6 (B6) spleen cells and treated with cholera toxin (CT) on the same day, a profound tolerance to tumour allografts of B6 origin was induced. The tolerant state was sustained for as long as 6 weeks or more. Skin allografts of B6 were rejected by such tolerant C3H mice, although the survival times were prolonged very slightly. Generation of cytotoxic T lymphocytes was reduced markedly in the tolerant mice, whereas delayed footpad reaction to B6 cells was maintained at the normal immune level or higher. There is a possibility that a T-cell subset responsible for delayed footpad reaction is resistant to CT-induced tolerance and participates in the rejection of skin allografts in tolerant mice.     

Involvement of CD14 in lipopolysaccharide- induced liver injury in mice pretreated with Propionibacterium acnes.

OBJECTIVE: The aim of this study was to elucidate the role of CD14 in the Propionibacterium acnes-lipopolysaccharide (LPS) system. METHODS AND RESULTS: CD14 transgenic mice (M14M), which expressed heterotopic CD14 and showed decreased responses to LPS in vivo, were used. Seven days after priming, the size of granulomas induced by an intraperitoneal administration of P. acnes in the M14M mice was smaller than that in the nontransgenic mice. The number of CD14-positive cells in granulomas was also decreased in the M14M mice compared to the nontransgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in the nontransgenic mice but not in the M14M mice. Seven days after priming, tumor necrosis factor-alpha expression was found in monocytic cells in granulomas and Kupffer cells in the nontransgenic mice and was significantly upregulated after LPS injection, whereas the expression was very weak in these cells in the M14M mice. CONCLUSIONS: CD14 plays a role in the P. acnes-LPS system in both priming and induction phases.     

Roles of CD14 in LPS-induced liver injury and lethality in mice pretreated with Propionibacterium acnes

The mechanism of the liver damage and lethality in Propionibacterium acnes (P. acnes)-LPS system remains obscure. To examine the role of CD14 in the system, M14M mice, in which CD14 was expressed heterotopically under the control of the metallothionein promoter were used. The production of soluble CD14 (sCD14) was increased by both P. acnes - priming and LPS challenge (1 microg per mouse) in both nontransgenic and M14M mice, although the plasma level was much higher in M14M nontransgenic than mice. The size of granulomas induced by an intraperitoneal administration of P. acnes in M14M mice 7 days after priming was smaller than that in nontransgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in nontransgenic mice but not in M14M mice. The challenge dose resulted in almost 90% lethality in nontransgenic mice but not in M14M mice 24h after challenge. TNF-alpha, IFN-gamma, IL-12, IL-18 and inducible nitric oxide synthase (iNOS) mRNA expressions produced by LPS challenge in M14M mice were low compared with those in nontransgenic mice. IL-18 mRNA expression was upregulated in P. acnes-primed nontransgenic mice but not in M14M mice. These results suggest that the high sCD14 concentration may account for less marked liver damage in M14M mice. Increase in the challenge dose of LPS (2 microg per mouse) resulted in increased lethality of M14M mice without liver damage. The levels of endothelial cell leukocyte adhesion molecule (ELAM)-1 mRNA expression in several organs in M14M mice 1-3h after LPS challenge were, however, lower than those in nontransgenic mice. The high sCD14 concentration may stimulate endothelial cell activation, which may account for lethality without liver damage in M14M mice. Thus, CD14 is involved in both the priming and induction phases as well as lethality in P. acnes-LPS system.