LPS/D-GalN诱发NF-κB转基因小鼠急性致死性肝损伤模型的建立

目的以NF-κB转基因BALB/c小鼠建立一个LPS/D-GalN诱发的急性致死性肝损伤模型。方法采取腹腔注射高剂量的LPS/D-GalN建立急性致死性肝损伤小鼠模型,观察模型小鼠的促炎症细胞因子水平和NF-κB的活性改变,以及肝脏功能和病理改变情况。结果模型组小鼠生存时间为8～10 h,模型建立后小鼠血清TNF-α、IL-6和MCP-1水平显著升高,在2～4h达到高峰;肝脏外观出现瘀血和出血,肝脏小叶被严重破坏,肝细胞严重坏死和出血;血清ALT/AST水平在模型诱发后持续迅速上升;整体成像显示NF-κB的活性在4～6 h达到高峰。正常对照组小鼠以上指标无显著变化。结论成功建立LPS/D-GalN诱发的NF-κB转基因小鼠的急性致死性肝损伤模型。     

LPS／D—GalN诱发ⅣF—KB转基因小鼠急性致死性肝损伤模型的建立

目的以NF—KB转基因BALB／c小鼠建立一个LPS／D—GaIN诱发的急性致死性肝损伤模型。方法采取腹腔注射高剂量的LPS／D-GalN建立急性致死性肝损伤小鼠模型,观察模型小鼠的促炎症细胞因子水平和NF—KB的活性改变,以及肝脏功能和病理改变情况。结果模型组小鼠生存时间为8—10h,模型建立后小鼠血清TNF—a、IL-6和MCP-1水平显著升高,在2—4h达到高峰;肝脏外观出现瘀血和出血,肝脏小叶被严重破坏,肝细胞严重坏死和出血;血清ALT／AST水平在模型诱发后持续迅速上升;整体成像显示胛-KB的活性在4～6h达到高峰。正常对照组小鼠以上指标无显著变化。结论成功建立LPS／D-GalN诱发的M-船转基因小鼠的急性致死性肝损伤模型。     

薄芝糖肽对D-GalN所致大鼠急性肝衰竭的影响

目的:探讨薄芝糖肽对组织损伤的保护作用.方法:Wister大鼠腹腔注射D-GalN(D-氨基半乳糖)诱发急性肝衰竭(ALF).48h后存活的大鼠随机分成4组,分别尾静脉注射不同剂量薄芝糖肽注射液或等量生理盐水,连续2w.观测动物存活率、血清谷丙转氨酶(ALT)以及肝组织学检查.结果:薄芝糖肽注射液3个剂量治疗组大鼠的存活率分别为42.1%、57.9%和63.2%,生理盐水对照组的存活率为21.1%,实验组与对照组的存活率相差显著(p＜0.05).治疗组大鼠ALT水平在给药第2d即明显下降,第7d基本恢复正常;对照组直到实验结束才恢复正常.4组动物病理切片显示,注射D-GalN后肝细胞大量坏死,呈现ALF状态.第15d高剂量治疗组基本恢复正常,但对照组仍见散在肝细胞坏死灶及,汇管区炎性细胞浸润.结论: 薄芝糖肽注射液可明显提高ALF大鼠存活率,改善肝功能.提示薄芝糖肽注射液可用于临床救治急性肝功能衰竭或重症肝炎.     

D-氨基半乳糖敏化小鼠内毒素休克模型的优化(英文)

目的:建立D-氨基半乳糖敏化小鼠内毒素休克的规范化模型。方法:选择对内毒素休克小鼠死亡率有影响的3个因素:D-氨基半乳糖增敏剂量、内毒素攻击剂量及其给药途径每个因素选取3个水平,按L9(34)正交试验表安排试验。以小鼠48h死亡率为评价指标,并通过验证试验,优化与实验要求相符的造模条件。结果:各因素对动物死亡率影响程度依次为:D-氨基半乳糖增敏剂量>内毒素给药途径>内毒素攻击剂量。采用D-氨基半乳糖600 mg.kg-1敏化小鼠,腹腔注射内毒素0.5 mg.kg-1的给药方案为佳。结论:规范内毒素休克模型的复制条件,将有利于科学评价药物的抗内毒素活性。     

束缚应激对D-氨基半乳糖联合脂多糖诱导小鼠肝损伤的保护作用

目的探讨束缚应激对D-氨基半乳糖联合脂多糖（D–galactosamine and lipopolysaccharide combination,D＋L）诱导的小鼠肝损伤的保护作用。方法正常BALB/c小鼠随机分为正常对照组（con）、应激对照组（str）、模型组（D＋L）、束缚应激组（D＋L＋str）。con组小鼠常规饲养;str组小鼠给予定时定量的束缚应激;D＋L组小鼠腹腔注射D-氨基半乳糖和脂多糖的混合溶液,1次/2天;D＋L＋str组小鼠腹腔注射等量D＋L混合液后,给予与str组相同的束缚应激。第8周,各组小鼠取血检测血清AST、ALT,肝脏固定后HE及Masson染色观察小鼠肝脏结构、细胞形态及纤维化程度。结果第8周D＋L＋str组与D＋L组小鼠相比,血清ALT和AST显著降低（P〈0.01）,AST/ALT显著增高（P〈0.01）;HE及Masson染色显示,D＋L组小鼠肝小叶结构紊乱,出现结节性增生及大量上皮细胞核浓缩、溶解、坏死,枯否氏细胞浸润,而D＋L＋str组未见明显病理变化;纤维化程度评分显示,D＋L＋str组与D＋L组小鼠相比,病理评分与纤维显色吸光度值均显著降低（P〈0.05）。结论束缚应激对D＋L诱导的小鼠肝损伤具有一定保护作用。     

BAPN诱导小鼠胸主动脉夹层合并急性肺损伤模型的建立

目的 采用β-氨基丙腈(β-aminopropionitrile monofumarate, BAPN)1 g/(kg·d)饮水给药的方式,构建一种可行性高、稳定的胸主动脉夹层(thoracic aortic dissection, TAD)合并急性肺损伤(acute lung injury, ALI)的小鼠模型,为研究TAD合并ALI提供合理的动物模型。方法 选取45只SPF级3周龄C57BL/6J雄性小鼠,随机分为CON组15只(正常饮食水)和BAPN组30只(与无菌水配置成1 g/(kg·d)的溶液饮水给药),持续4周。实验期间,观察两组小鼠一般情况、成模率,通过测量小鼠胸主动脉最大直径和主动脉组织苏木素-伊红(HE)染色,验证小鼠TAD模型并将BAPN组分为TAD组和Non-TAD组。进一步检测CON组、Non-TAD组和TAD组小鼠肺组织HE病理染色、湿干重比(dry/wet weight ratio, W/D)及肺泡灌洗液(broncho alveolar lavage fluid, BALF)中总蛋白水平和白细胞介素-1β(interleukin-1β,IL-1β)、白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)表达情况验证小鼠TAD合并ALI模型。结果 BAPN干预明显延缓小鼠体重和饮水量的增加。与CON组和Non-TAD组相比,TAD组小鼠胸主动脉最大直径明显增粗(P<0.05);主动脉HE染色显示主动脉中层明显增厚,主动脉壁结构破坏、紊乱;肺组织HE染色显示肺间质明显水肿及炎性渗出,伴肺泡腔扩大,肺泡壁上皮脱落及透明膜形成,肺损伤病理评分显著增加(P<0.05);肺组织W/D、BALF中总蛋白水平及IL-1β、IL-6、TNF-α表达也明显升高(P<0.05),而另外两组上述指标无明显差异。结论 通过BAPN饮水给药的方式,可成功建立胸主动脉夹层合并急性肺损伤的小鼠模型。     

急性肝损伤大鼠模型的血浆氨基酸代谢变化模式

以Ｄ－氨基半乳糖（Ｄ－Ｇａｌａｃｔｏｓａｍｉｎｅ,Ｄ－ＧａｌＮ）造成急性肝损伤（急性肝炎、急性肝坏死）大鼠模型后、对照观察了急性肝损伤大鼠血浆氨基酸的变化,建立了大鼠急性肝损伤时血浆氨基酸的变化模式并对其发生机理进行了探讨。大鼠血浆氨基酸的测定采用聚酰薄层荧光分析技术,其测定结果是：急性肝炎组,酪氨酸（Ｔｙｒ）、天冬氨酸（Ａｓｐ）、谷氨酰胺（Ｇｌｎ）和鸟氨酸（Ｏｒｎ）升高,精氨酸（Ａｒｇ）下降,其余氨基酸无显著变化。急性肝坏死组,除Ａｒｇ显著下降外,其余所有氨基酸都显著升高,而两组支链氨基酸（ＢＣＡＡ）／芳香族氨基酸（ＡＡＡ）克分子比值均显著下降。     

贡菊黄酮抗小鼠急性肝损伤作用的研究

为研究黄山贡菊中黄酮的提取纯化方法及其抗小鼠急性肝损伤作用,采用乙醇回流法从黄山贡菊中提取粗黄酮,聚酰胺柱层析纯化,真空冷冻干燥获得黄酮粉末。对以四氯化碳造模的急性肝损伤小鼠分别给予联苯双酯及不同剂量的贡菊黄酮灌胃,测定血清中GPT/ALT、GOT/AST活性,测定肝组织中SOD活性和丙二醛含量,HE染色观察肝组织病理形态学的改变。结果表明,黄山贡菊黄酮可以降低急性肝损伤小鼠血清中GPT/ALT、GOT/AST活性,提高肝组织的SOD活性,降低丙二醛含量,对减轻肝脏病理组织损伤有积极作用。     

光化学诱导小鼠急性脑梗塞模型的建立

目的建立光化学诱导小鼠急性脑梗塞模型.方法采用5因素4水平正交试验建立光化学诱导小鼠急性脑梗塞模型的最佳实验条件.结果最佳实验条件为照射时间5?min、照射强度为1?Lx、玫瑰红剂量100?mg/kg、伊文思蓝剂量100?mg/kg、处死时间4?h.结论通过结果验证,证明该模型是成立的.     

c-Myc抑制剂10058-F4减轻脂多糖/右旋半乳糖胺诱导的小鼠急性肝损伤

本研究探讨c-Myc抑制剂10058-F4对脂多糖(lipopolysaccharide, LPS)和右旋半乳糖胺(D-galactosamine, D-Gal)诱导的急性肝损伤的影响及其可能机制。32只雄性BALB/c小鼠(Mus musculus)分为4组：正常对照组、 10058-F4组、 LPS/D-Gal组和10058-F4+LPS/D-Gal组。通过腹腔注射LPS/D-Gal诱导小鼠急性肝损伤,10058-F4在注射LPS/D-Gal前0.5 h经腹腔注入。注射LPS/D-Gal 1.5 h后处死小鼠并采集样本,分别采用实时荧光定量PCR法和ELISA法检测肝组织中肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的mRNA水平和血清中TNF-α的水平。另取32只小鼠经上述相同处理后,注射LPS/D-Gal 6 h后处死并采集样本,检测小鼠血清中转氨酶活性及白介素-6(interleukin-6, IL-6)的水平、肝组织中IL-6的mRNA水平及肝组织中半胱氨酸天冬氨酸特异性蛋白酶(cysteinyl aspartate specific p...     

粒细胞集落刺激因子对小鼠内毒素性急性肝损伤的保护作用

目的观察粒细胞集落刺激因子对小鼠内毒素性急性肝损伤的作用,并对其机制进行初步探讨。方法昆明（KM）小鼠随机分为模型组、预防组和正常组,模型组小鼠腹腔注射内毒素（LPS）10mg／kg或30mg／kg,预防组于造模前1小时皮下注射重组人粒细胞集落刺激因子（rhG-CSF）500btg／kg,正常组注射等剂量的生理盐水,观察各组小鼠的存活率及造模后6h、24h小鼠肝脏组织病理变化,全自动生化分析仪检测血清丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AsT）的水平,酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子（TNF—a）和白介素10（IL-10）的水平。结果预防组小鼠存活率与模型组相比无明显差异（80％VS66．7％,P〉0．05）;预防组肝组织损伤及肝功能酶学指标ALT和AST均好于模型组（P〈0．05）;预防组小鼠血清IL-10的表达水平在6小时点明显高于模型组（P〈0．05）,24小时点与模型组相比无明显差异（P〉0．05）,血清TNF-a表达水平与模型组相比差异无统计学意义（P〉0．05）。结论粒细胞集落刺激因子对小鼠内毒素性急性肝损伤具有保护作用,对小鼠的存活率无明显影响。     

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.     

Blood-brain barrier in acute liver failure

Brain edema remains a challenging obstacle in the management of acute liver failure (ALF). Cytotoxic mechanisms associated with brain edema have been well recognized, but evidence for vasogenic mechanisms in the pathogenesis of brain edema in ALF has been lacking. Recent reports have not only shown a role of matrix metalloproteinase-9 in the pathogenesis of brain edema in experimental ALF but have also found significant alterations in the tight junction elements including occludin and claudin-5, suggesting a vasogenic injury in the blood-brain barrier (BBB) integrity. This article reviews and explores the role of the paracellular tight junction proteins in the increased selective BBB permeability that leads to brain edema in ALF.     

Neuropathology of acute liver failure

Cerebral edema has been identified in all forms of liver disease and is closely related to the development of hepatic encephalopathy. Cerebral edema is most readily recognized in acute liver failure (ALF), while the main cause of death in patients with ALF is multi-organ failure; brain herniation as a result of intracranial hypertension does remain a major cause of mortality. The mechanisms responsible for cerebral edema in ALF suggest both cytotoxic and vasogenic injury. This article reviews the gross and ultrastructural changes associated with cerebral edema in ALF. The primary cause of cerebral edema is associated with astrocyte swelling, mainly perivascular edema and ammonia still remains the primary neurotoxin involved in its pathogenesis. The astrocytic changes were confined to the gray matter. The other organelles involved in the pathogenesis of ALF include mitochondria, basement membrane, pericytes, microglial cells, blood-brain barrier (BBB) etc. Discrete neuronal changes have recently been reported. Recent studies in animal and humans have demonstrated the microglial changes which have the potential to cause neuronal dysfunction in ALF. The alterations in BBB still remain unclear though few studies have showed disruption of tight junction proteins indicating the involvement of BBB in cellular swelling.     

The neurological manifestations of acute liver failure

Acute liver failure is a disorder which impacts on multiple organ systems and results from hepatocellular necrosis in a patient with no previous history of chronic liver disease. It typically culminates in the development of liver dysfunction, coagulopathy and encephalopathy, and is associated with high mortality in poor prognostic groups. In acute liver failure, some patients may develop cerebral edema and increased intracranial pressure although recent data suggest that intracranial hypertension is less frequent than previously described, complicating 29% of acute cases who have proceeded to grade 3/4 coma. Neurological manifestations are primarily underpinned by the development of brain edema. The onset of encephalopathy can be rapid and dramatic with the development of asterixis, delirium, hyperreflexia, clonus, seizures, extensor posturing and coma. Ammonia plays a definitive role in the development of cytotoxic brain edema. Patients with acute liver failure have a marked propensity to develop renal insufficiency and hence impaired ammonia excretion. The incidence of both bacterial and fungal infection occurs in approximately one third of patients. The relationship between inflammation, as opposed to infection, and progression of encephalopathy is similar to that observed in chronic liver disease. Intracranial pressure monitoring is valuable in identifying surges in intracranial hypertension requiring intervention. Insertion of an intracranial bolt should be considered only in the subgroup of patients who have progressed to grade 4 coma. Risk factors for developing intracranial hypertension are those with hyperacute and acute etiologies, progression to grade 3/4 hepatic encephalopathy, those who develop pupillary abnormalities (dilated pupils, sluggishly responsive to light) or seizures, have systemic inflammation, an arterial ammonia >150 μmol/L, hyponatremia, and those in receipt of vasopressor support. Strategies employed in patients with established encephalopathy (grade 3/4) aim to maintain freedom from infection/inflammatory milieu, provide adequate sedation, and correct hypo-osmolality.     

Therapeutic hypothermia in the management of acute liver failure

A large body of experimental data and preliminary clinical studies point to the induction of mild hypothermia (32-35 °C) as a valuable approach to control the development of brain edema and intracranial hypertension in acute liver failure (ALF). The ability of hypothermia to affect multiple processes probably explains its efficacy to prevent these cerebral complications. Remarkably, mild hypothermia has been shown to prevent or attenuate most of the major alterations involved in the pathogenesis of the cerebral complications of ALF, including the accumulation of ammonia in the brain and the circulation, the alterations of brain glucose metabolism, the brain osmotic disturbances, the accumulation of glutamate and lactate in brain extracellular space, the development of inflammation and oxidative/nitrosative stress, and others. Limited information suggests that the systemic effects of hypothermia may also be beneficial for some peripheral complications of ALF. Translation of the beneficial effects of therapeutic hypothermia into standard clinical practice, however, needs to be confirmed in adequately designed clinical trials. Such trials will be important to determine the safety of therapeutic hypothermia, to identify which patients might benefit from it, and to provide the optimal guidelines for its use in patients with ALF.     

气雾攻击法感染结核病小鼠模型的建立及其综合评价

目的 模拟自然感染方式建立结核病小鼠模型,并对其病理变化进行综合评价.方法 通过气雾攻击方式将结核分枝杆菌H37Rv接种至C57BL/6J小鼠体内.在感染后的4周、6周、8周对小鼠进行micro-CT活体动态扫描,无菌分离肺脏和脾脏,肉眼观察病变情况,活菌菌落计数,组织病理检测(HE和抗酸染色).结果 肉眼观察和micro-CT扫描发现,不同时间小鼠肺部感染情况逐渐加重,至感染后第8周时病变弥漫至整个肺部;HE染色肺组织出现弥漫性肉芽肿样实变;抗酸染色可见结核分枝杆菌.结论 通过大体病变、病理、影像、菌落计数几个方面对建立的小鼠模型进行综合分析,证明利用气雾攻击法感染的结核病小鼠模型建立成功;该模型在形成病变时与结核患者的情况存在一定差异,对其完善的综合评价有助于在相关研究中对该小鼠模型的合理应用.     

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.