血清对分离枯否细胞细菌脂多糖摄取过程的影响

本文应用单克隆抗细胞抗细菌多糖抗体，利用免疫荧光及激光共聚焦扫描技术，观察了分离培养的枯否细胞有无血清条件下对二种不内发子结构细菌脂多糖摄取过程的影响，无血清状态下，枯否细胞加入Ｓ型脂多糖（Ｓ－ＬＰＳ）及Ｒ型脂多糖（Ｒｄ－ＬＰＳ）培育５ｍｉｎ后，其胞闪内抗脂多糖荧光强度显著高于空白对照水平。且Ｓ－ＬＰＳ培育后胞浆内荧光强度增高幅度高于Ｒｄ－ＬＰＳ培养后的增高幅度，１０％小牛血清状态下，Ｓ－ＬＰＳ培     

培养肝内胆管上皮细胞摄取大肠杆菌脂多糖并表达肿瘤坏死因子-α-mRNA

目的观察分离培养肝内胆管上皮细胞对大肠杆菌脂多糖的摄取作用及其肿瘤坏死因子－α－ｍＲＮＡ的表达。方法利用免疫荧光、荧光原位杂交及激光共聚焦扫描技术。结果无血清状态下，培养肝内胆管上皮细胞加入Ｓ型脂多糖（Ｓ－ＬＰＳ）培养１５分钟后，其胞浆内抗脂多糖荧光强度显著高于空白对照水平；与Ｓ－ＬＰＳ培养３小时后其胞浆中肿瘤坏死因子－α－ｍＲＮＡ含量增强，６小时达高峰。胞核中亦有肿瘤坏死－α－ｍＲＮＡ增多，但增加幅度低于胞浆。结论结果提示，分离培养的肝内胆管上皮细胞能摄取脂多糖入胞浆，且强烈表达肿瘤坏死因子－α－ｍＲＮＡ。     

酒精刺激对枯否细胞清除LPS功能的影响

目的和方法：应用免疫荧光及免疫电镜技术，对慢性酒精投与之实验大鼠及重症酒精性肝炎患者肝脏枯否细胞对ＬＰＳ的清除功能进行了观察。结果：经门静脉注射ＬＰＳ１０ｍｉｎ后，Ｌｉｅｂｅｒ’ｓ酒精饮食投与６周之大鼠肝脏枯否细胞内抗ＬＰＳ荧光显色低于正常大鼠，电镜下枯否细胞内抗ＬＰＳ阳性颗粒少见，而肝窦壁内皮细胞及肝细胞内抗ＬＰＳ阳性物增多。重症酒精性肝炎患者肝穿组织枯否细胞内抗ＬＰＳ荧光显色亦显著低于正常对照。结论：酒精刺激后肝脏枯否细胞对ＬＰＳ的清除功能降低，且内毒素可能参与酒精致肝损伤的发生机制     

脂多糖对人脐静脉内皮细胞凋亡的影响及意义

目的：探讨脂多糖（ＬＰＳ）对血管内皮细胞（ＶＥＣ）损伤的机制以及在急性肺损伤（ＡＬＩ）发中的作用。方法：通过建立人脐静脉内皮细胞体外培养,采用流式细胞技术观察ＬＰＳ对人脐静脉内皮细胞（ＨＵＶＥＣ）凋亡的影响。结果：ＬＰＳＬ中入细胞 ,可明显导ＨＵＶＥＣ凋亡,并随着作用时间的延长和ＬＰＳ浓度增加,细胞凋亡率也相应增加,一定范围内呈时间、剂量、效应关系。结论：细胞凋亡是这内皮细胞受损的主要方式之一。     

山莨菪碱抗血栓形成的机制研究

观察中药山莨菪碱对内毒素脂多糖所致血管内皮细胞表达纤溶酶原激活物抑制剂１的作用和作用机制。方法用胰蛋白酶消化培养人脐静脉内皮细胞（ＨＵＶＥＸ）;采用酶联免疫吸附试验法和Ｎｏｒｔｈｅｒｎ印迹方法观察ＨＵＶＥＧＣ条件培养液ＰＡＩ－１蛋白量及ｍＲＮＡ表达用免疫细胞学检测ＨＵＶＥＣ核因子ＮＦ－ｋＢ的核内转移情况,结晶显著啬培养ＨＵＶＥＣ的ＰＡＩ－１蛋白和ｍＲＮＡ表达;然而当山莨菪碱和ＬＰＳ同时作用于ＨＵＶ     

应用脂多糖激活人血B淋巴细胞的研究

人血在体外培养时用脂多糖（ＬＰＳ）激活Ｂ淋巴细胞，＾３Ｈ－ＴｄＲ、，＾１４Ｃ－ＵＲ和＾１４Ｃ－缬氨酸作掺入试验反映淋转，测定了正常和各类病人１０６５人次，结果能够反映病情。用单克隆抗体（ＭｃＡｂ）分纯ＣＤ４，ＣＤ８和Ｂ细胞，发现ＬＰＳ只对Ｂ淋巴细胞有激活作用。实验还反映ＣＤ４和ＣＤ８细胞分别对ＬＰＳ激活的Ｂ细胞有辅助和抑制作用。     

α-黑素细胞刺激素体外对星形胶质细胞产生NO和前炎性细胞因子的影响

目的：研究α－黑素细胞刺激素（α－ＭＳＨ）对ＬＰＳ诱导星形胶质细胞产生ＮＯ和前炎性细胞因子的影响。探讨α－ＭＳＨ的抗炎作用机制。方法：分别用ＬＰＳ或α－ＭＳＨ＋ＬＰＳ处理体外培养的大鼠脑星形胶质细胞,用Ｇｒｉｅｓｓ试剂测定ＮＯ,以ＭＴＴ显色法检测ＩＬ－１、ＩＬ－６和ＴＮＦ－α,采用半定量ＲＴ－ＰＣＲ检测ＭＩＦｍＲＮＡ表达。结果：体外培养的星形胶质细胞在ＬＰＳ刺激下产生ＮＯ、ＩＬ－１、ＩＬ－６、ＴＮＦ－α和表达ＭＩＦｍＲＮＡ表达。结果：体外的星形胶质细胞在ＬＰＳ刺激下产生ＮＯ、ＩＬ－１、ＩＬ－６、ＴＮＦ－α和表达ＭＩＦｍＲＮＡ显著增高;若同时给予ＬＰＳ和α－ＭＳＨ,可明显降低ＮＯ、ＩＬ－１、ＩＬ－６和ＴＮＦ－α的产生以及ＭＩＦｍＲＮＡ表达。结论：Ｒ昧α－ＭＳＨ抑制星形胶质细胞产生ＮＯ和前炎性细胞因子与其抑制中枢神     

52 例病毒性肝炎患者肝组织中丙型肝炎病毒 RNA 和 HCAg-NS3 的测定

应用地高辛标记探针原位杂交法和单克隆抗ＨＣＶ－ＮＳ３－ＨＲＰ建立直接酶标免疫组化法分别测定５２例肝炎患者肝组织ＨＣＶＲＮＡ和ＨＣＡｇ－ＮＳ３。结果抗ＨＣＶ阳性组ＨＣＶＲＮＡ检出率５７．１％（１６／２８），ＨＣＡｇ－ＮＳ３检出率５３．６％（１５／２８）；抗ＨＣＶ阴性组其两项检出率均为１２．５％（３／２４）。肝组织中ＨＣＶＲＮＡ阳性物呈蓝紫色细小颗粒存在于肝细胞核或胞浆内，其在肝小叶中的分布可分为３型，即弥漫型、局灶型、散在型。肝组织中ＨＣＡｇ－ＮＳ３阳性物呈棕黄色细小颗粒分布于肝细胞核或胞浆内，以单个或数个阳性细胞散布于肝小叶中。２３例ＨＣＶＲＮＡ或／和ＨＣＡｇ－ＮＳ３阳性病例以肝炎后肝硬化（ＬＣ）病例占多数（１４／２３），其次为慢性重型肝炎（ＣＳＨ）和中度慢性肝炎（ＣＡＨ）。此两种检测方法具有较高符合率（９０．４％，４７／５２），表明病毒核酸及其表达产物均存在于肝细胞内，与ＨＣＶ感染密切相关。这为ＨＣＶ感染诊断提供了直接依据，有利于研究ＨＣＶ感染中病毒复制、慢性化进程、抗病毒治疗监测及重叠感染时病毒相互关系。     

UV-C诱导的体外凋亡SMC内Bcl-2蛋白表达的研究

目的 观察经ＵＶ－Ｃ照射后的凋亡ＳＭＣ内Ｂｃｌ－２蛋白表达的情况。方法 应用组织贴块法进行体外ＳＭＣ培养，ＳＰ免疫组织化学技术和图像分析技术检测经ＵＶ－Ｃ照射后的ＳＭＣ内Ｂｃｌ－２蛋白的表达。结果 ＵＶ－Ｃ照射体外ＳＭＣ后，细胞出现典型的凋亡形态学和生化指标的改变；凋亡ＳＭＣ胞浆内Ｂｃｌ－２表达下降和出现胞浆内Ｂｃｌ－２的迁移和再分布入核。结论 ＵＶ－Ｃ照射可引起体外ＳＭＣ凋亡，且Ｂｃｌ－２可能参     

补体膜攻击复合物刺激血管内皮细胞胞浆游离钙离子浓度的变化

目的 在单个细胞水平，观测亚溶破补体膜攻击复合物（MAC）诱导的血管内皮细胞[Ca^2 ]i的动态变化。方法 将原代培养的人脐静脉内皮细胞，以Fluo-4/AM(1-6μmol/L）和0.02%PluronicF-127进行细胞钙荧光指示剂负载后，于0℃组装亚溶破剂量的MAC，在37℃条件下，以LSCM实时监测MAC沉积引起的胞浆钙荧光强度的变化。结果 MAC沉积后，多数细胞的钙荧光强度迅速增强，以后随时间的推移，升高的钙荧光逐渐下降，直至恢复至静息水平，定量分析选定的胞浆区域钙荧光强度的变化发现，不同细胞在[Ca^2 ]i的变化高度，持续时间和胞内钙振荡 的特征等方面存在异质性。结论 MAC沉积到内皮细胞表面后，可诱导胞浆游离钙离子的浓度增高，且不同细胞[Ca^2 ]i的变化存在明显的异质性。     

培养肝内胆管上皮细胞摄取大肠杆菌脂多糖并表达肿瘤坏死因子…

观察分离培养肝内胆管上皮细胞对大肠杆菌脂多糖的摄取作用及其肿瘤坏死因子－α－ｍＲＮＡ的表达。     

Effects of Culture Substrates and Normal Hepatic Sinusoidal Cells on in Vitro Hepatocyte Synthesis of Apo-SAA

Primary hepatocyte cultures synthesize apo-SAA upon stimulation with supernatant from lipopolysaccharide (LPS)-treated macrophages. The matrices on which the hepatocytes were grown influence their basal apo-SAA synthetic capability. Fibronectin was superior. Coculturing hepatocytes with hepatic sinusoidal cells did not adversely affect the ability of hepatocytes to synthesize and secrete apo-SAA into the culture medium. In 72 h, clear islands of endothelial cells nestled in layers of hepatocytes. Both apo-SAA, and apo-SAA, were made in considerable quantities but no evidence could be obtained that the apo-SAA were free of apo-A-1. The coculturing of hepatocytes with liver sinusoidal cells, the site of ultimate AA deposition, is a first step in establishing an in vitro system for AA amyloidogenesis.     

Binding of 125I-endothelin-1 to fat-storing cells in rat liver revealed by electron microscopic radioautography.

Summary Localization of intravenously injected [¹²⁵I]-endothelin-1 was examined in rat liver by light and electron microscopic radioautography. At 10 min after injection, silver grains were localized along the sinusoidal wall, i.e., mostly on the thin processes of fat-storing cells and sinusoidal endothelial cells, and also on the Kupffer cells and the microvilli of hepatocytes. About 35% of the total silver grains were located on the processes of fat-storing cells at 10 min. The grain density (number of silver grains/cell area) of fat-storing cells was three-fold that of Kupffer cells, and 18-fold that of hepatocytes. At 60 min, 60% of the total grains were observed on the fat-storing cells, though the value of grain density was not changed. Silver grains were internalized into the cytoplasm of fat-storing cells and often associated with multivesicular bodies. In contrast, the grain density of endothelial cells and Kupffer cells decreased with time. These results indicate that hepatic fat-storing cells have a considerable number of endothelin-binding sites, and incorporate bound endothelin into cytoplasm.     

Ultrastructurural picture of cyclophosphamide-induced damage to the liver

We studied the ultrastructural reorganization of hepatocytes and other populations of liver cells after cyclophosphamide treatment. Single administration of cyclophosphamide was followed by signifi cant ultrastructural changes in two major populations of liver cells (hepatocytes and sinusoidal endothelial cells). This treatment was also accompanied by reactive changes in Kupffer cells, lymphocytes, and plasma cells migrating into the spaces of Disse. Cyclophosphamide-induced spatial reorganization of hepatocytes was associated with a progressive decrease in the structural density of mitochondria, signifi cant reduction of the smooth endoplasmic reticulum, and increase in autophagocytosis (e.g., sequestration of glycogen). Intracellular reorganization of endothelial cells was related to the following two factors: damage (necrobiosis) of some cells; and increase in metabolic processes, phagocytosis, and regenerative reactions in other cells.     

Deacylation of bacterial lipopolysaccharide in rat hepatocytes in vitro

The possible role of liver parenchymal cells in the uptake and degradation of bacterial lipopolysaccharide (LPS) was investigated in vitro by employing radiolabelled LPS as substrate. Hepatocytes obtained from Wistar rats by collagenase treatment were found to take up LPS only when it was not linked to the polysaccharide of O-antigen. The amount of LPS taken up increased with time and after 48 h incubation it increased in a dose-dependent manner up to at least 30 micrograms. When incubated with LPS radiolabelled exclusively in the fatty-acid moiety, cultured hepatocytes released lipophilic materials into the culture medium. These were identified as beta-hydroxytetradecanoic acid and triglyceride, in the ratio of 7:I. These results indicate that the R-form of LPS which lacks the O-antigen polysaccharide is taken up and deacylated in hepatocytes, and the derived fatty acids are released into the culture medium either in the free form or after conversion to triglyceride.     

Deacylation of bacterial lipopolysaccharide in rat hepatocytes in vitro.

The possible role of liver parenchymal cells in the uptake and degradation of bacterial lipopolysaccharide (LPS) was investigated in vitro by employing radiolabelled LPS as substrate. Hepatocytes obtained from Wistar rats by collagenase treatment were found to take up LPS only when it was not linked to the polysaccharide of O-antigen. The amount of LPS taken up increased with time and after 48 h incubation it increased in a dose-dependent manner up to at least 30 micrograms. When incubated with LPS radiolabelled exclusively in the fatty-acid moiety, cultured hepatocytes released lipophilic materials into the culture medium. These were identified as beta-hydroxytetradecanoic acid and triglyceride, in the ratio of 7:I. These results indicate that the R-form of LPS which lacks the O-antigen polysaccharide is taken up and deacylated in hepatocytes, and the derived fatty acids are released into the culture medium either in the free form or after conversion to triglyceride.     

Connective tissue components in cultured parenchymal and nonparenchymal cells of rat liver. Immunohistochemical studies

Connective tissue components were detected immunohistochemically in cultured rat hepatocytes and sinusoidal lining cells. In the Ito and endothelial cells, the staining reactions to prolyl hydroxylase were strong. The reaction in the hepatocytes, however, was weaker, and there was no staining reaction to prolyl hydroxylase in the Kupffer cells. In the Ito cells, types III and IV collagen were clearly positive, whereas reactions to type I collagen were very weak. In the endothelial cells, type IV collagen stained clearly, whereas the reaction for type I collagen was very weak, and there was no reaction for III. Laminin was clearly positive in the Ito and endothelial cells. Connective tissue components were not detectable in the Kupffer cells. The hepatocytes were positive for types I, III, and IV collagen, but the staining reactions to type IV collagen were weak. Laminin staining was almost negative. These results suggest that type IV collagen is produced mainly by nonparenchymal cells and that types I and III collagen are produced by both parenchymal and nonparenchymal liver cells.     

Ultrastructural localization of xanthine oxidoreductase activity in isolated rat liver cells

Xanthine oxidoreductase (XOR) can exist in a dehydrogenase form (XD) and an oxidase form (XO). The D-form uses NAD as cofactor and the O-form uses oxygen as second substrate and produces oxygen radicals. Both enzymes have a high affinity for hypoxanthine and xanthine as substrate and produce uric acid, a potent antioxidant. In the present study, XOR activity was demonstrated with the ferricyanide method in permeabilized isolated rat liver cells at the electron microscopical level. Moreover, ultrastructural localization of XO activity in these cells was studied with the cerium salt method. Activity of both XOR and XO was found in matrix and core of peroxisomes of rat liver parenchymal cells. Only XOR activity was present as well in the cytoplasm of rat liver parenchymal cells. In Kupffer cells and sinusoidal endothelial cells, XOR activity was demonstrated in vesicles and occasionally on granular endoplasmic reticulum. XO activity was not found in Kupffer cells and sinusoidal endothelial cells. The presence of uric acid oxidase activity in matrix and core of peroxisomes as was found previously suggests further breakdown of purines to allantoin in peroxisomes. It is suggested that the major function of XOR activity in the cytoplasm of rat liver parenchymal cells and in sinusoidal cells is not the production of oxygen radicals, but rather the production of uric acid which can act as a potent antioxidant.     

A novel immunocytochemical staining method that preserves cell membranes: Application for demonstrating Ca2+ pump ATPase in the liver

We have devised a method for immunogold staining of unosmicated, plastic-embedded cells that gives high levels of specific staining without sacrificing cell ultrastucture. Important conditions include PLP (periodate-lysine-paraformaldehyde) fixation, postfixation with uranyl acetate to preserve membrane phospholipids, dehydration with acetone, low-temperature embedding in LR gold resin, and use of osmium tetroxide to stain thin sections after immunogold labeling. We developed this method to localize plasma membrane calcium pump ATPase in rat hepatocytes and hepatic sinusoidal endothelial cells. Most gold particles of Ca²⁺ pump ATPase were easily assigned to bile canalicular membranes in rat hepatocytes, and the gold particles of Ca^2? pump ATPase were located on the labyrinthlike structures of the endothelial sinusoidal fenestrae in rat hepatic sinusoidal endothelial cells. In these studies, it was useful to preserve the cell membrane for postembedding methods.