制备神经细胞实验性缺氧缺糖模型的简易方法

背景: 在神经细胞培养中实验性缺氧缺糖在一定程度上模拟缺血性卒中,对于研究缺血性神经元损伤的进程和病理生理学机制有非常重要的用处。 目的：在神经元培养时制作实验性缺氧缺糖模型。 设计、时间及地点：分组对照观察,实验于2007-01/2008-03在北京大学第三医院中心实验室完成。 材料：17~19 d胎龄的Wistar大鼠。 方法：细胞培养取17~19 d胎龄的Wistar大鼠的皮质神经元做原代细胞培养,并且去掉污染的非神经原细胞。缺氧缺糖的诱导分为3组：实验组将第7天的皮质神经元置于无糖平衡盐溶液和2%去氧酶中,在37 ℃的潮湿保温箱中培育。空白对照组培养基为含20 mmol/L葡萄糖的无去氧酶平衡盐溶液。假性实验组培养基为含20 mmol/L葡萄糖和失活的去氧酶平衡盐溶液。 主要观察指标：以血气分析进行氧浓度的测定;以相差显微镜观察实验组培养细胞神经元死亡状况;以用乳酸脱氢酶检测盒检测乳酸脱氢酶活性;以锥虫蓝染色观察缺氧缺糖对神经元存活力的影响。 结果：氧浓度测定显示在加入去氧酶后培养基迅速产生缺氧状态;乳酸脱氢酶检测显示在用去氧酶和无糖平衡盐处理后,培养基中乳酸脱氢酶释放显著增加;锥虫蓝染色和相差显微镜检查显示经去氧酶和无糖平衡盐处理后实验组的细胞活力明显下降,大部分神经元在6 h死亡。 结论：实验结果显示去氧酶与无糖平衡盐液可联合用于神经元培养时产生缺氧缺糖状态, 其在体外模拟脑缺血的相关研究中有重要作用。     

联合检测CEA、ADA、LDH在鉴别胸腔积液性质上的临床价值

目的探讨结核性和癌性胸腔积液的实验室鉴别诊断。方法将住院患者新鲜抽取的胸腔积液离心沉淀后取上清液分别采用磁酶免法、酶速率法、速率法测定其CEA、ADA、LDH的含量。同法检测其血清CEA、ADA、LDH含量。结果254例胸腔积液中,188例结核性胸腔积液胸水CEA、LDH、ADA与66例癌性胸腔积液比较差异有显著性。结核性胸腔积液患者胸水CEA含量显著低于癌性胸腔积液（P〈0．001）,ADA、LDH含量显著高于癌性胸腔积液（P〈0．001,P〈0．01）;结核性胸腔积液患者胸水LDH、ADA与血清LDH、ADA的比值显著高于癌性胸腔积液患者（P〈0．01）,而胸水CEA与血清CEA的比值则显著低于癌性胸腔积液患者（P〈0．01）。结论癌性胸腔积液的CEA、ADA、LDH的结果与结核性胸腔积液CEA、ADA、LDH的结果差异显著,可通过联合检测胸腔积液中CEA、ADA、LDH辅助判断胸腔积液的性质。     

Direct intestinal cholesterol secretion contributes significantly to total fecal neutral sterol excretion in mice

BACKGROUND & AIMS: Hepatobiliary secretion is generally believed to be an integral step in the pathway of cholesterol excretion from the body. Here we have investigated the validity of this paradigm in mice. METHODS: Cholesterol balance was assessed by measuring intake, excretion, and biliary output in different mouse models. Direct secretion of cholesterol from the luminal side of enterocytes was studied by perfusion of isolated segments of the small intestine in mice. RESULTS: Cholesterol input and output measurements in different mouse models revealed that fecal neutral sterol excretion was higher than the sum of dietary cholesterol intake and biliary cholesterol secretion indicating the existence of an alternative pathway. Here we show that substantial amounts of cholesterol can be secreted directly by enterocytes. Transintestinal cholesterol secretion is a specific process observed throughout the small intestine (proximal > medial > distal). Secretion depended on the presence of a cholesterol acceptor and was strongly stimulated by bile salts and phospholipids. The capacity of the pathway was sufficient to account for the missing cholesterol in the balance studies. The contribution of this pathway to cholesterol excretion in mice is approximately twice that of the biliary pathway. CONCLUSIONS: In mice, the intestine plays a significant role in removal of cholesterol from the body.     

The dynamic changes of LDH isoenzyme 3 and D-dimer following pulmonary thromboembolism in canine

INTRODUCTION: To investigate the time course of changes of lactic acid dehydrogenase (LDH), LDH isoenzymes and D-dimer levels following acute pulmonary thromboembolism (PTE). MATERIALS AND METHODS: Eighteen dogs were randomly divided into three groups. Acute PTE was induced by injection of preformed blood clots into pulmonary artery through femoral vein. Thrombin and human fibrinogen were delivered into blood clots in embolism group I. Only thrombin was delivered into blood clots in embolism group II. The control group received normal saline and human fibrinogen in the same manner. Series of blood samples were collected pre-embolism and post-embolism. LDH isoenzymes proportion and D-dimer levels were measured. RESULTS: At 30 min, 1 h, 2 h, 4 h, 24 h post-embolism, the plasma D-dimer levels from embolism group I were significantly higher than pre-embolism and those from control group at the same intervals (p<0.05). The peak appeared at 2 h post-embolism (2.336+/-0.326 vs. 0.016+/-0.013, p<0.05). At 4 h, 24 h and 48 h post-embolism, total serum LDH activity and LDH-3 proportion from two embolism groups were significantly higher than pre-embolism (p<0.05). The peak of LDH-3 proportion in two embolism groups both appeared at 24 h post-embolism (0.225+/-0.021 vs. 0.108+/-0.030, 0.214+/-0.011 vs. 0.096+/-0.031, respectively. p<0.05). CONCLUSIONS: The LDH-3 and D-dimer levels were changed dynamically with a relative specificity manner during the course of acute massive PTE. Combination the D-dimer assay with LDH-3 may have a potential value in diagnosing acute massive PTE.     

Effects of chlorpromazine on plasma membrane permeability and fluidity in the rat brain: a dynamic positron autoradiography and fluorescence polarization study

Antipsychotic drugs have been widely used in psychiatry for the treatment of various mental disorders, but the underlying biochemical mechanisms of their actions still remain unclear. Although phenothiazine antipsychotic drugs have been reported to directly interact with the peripheral plasma membrane, it is not known whether these drugs actually affect plasma membrane integrity in the central nervous system. To clarify these issues, we investigated the effect of chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, on plasma membrane permeability in fresh rat brain slices using a dynamic positron autoradiography technique and [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG) as a tracer. Treatment with CPZ (> or =100 microM) resulted in the leakage of [(18)F]FDG-6-phosphate, but not [(18)F]FDG, suggesting that the [(18)F]FDG-6-phosphate efflux was not mediated by glucose transporters, but rather by plasma membrane permeabilization. The leakage of [(18)F]FDG-6-phosphate was followed by slower leakage of cytoplasmic lactate dehydrogenase, suggesting that CPZ could initially induce small membrane holes that enlarged with time. Furthermore, the addition of CPZ (> or =100 microM) caused a decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, which implies an increase in membrane fluidity. CPZ loading dose-dependently increased both membrane permeability and membrane fluidity, which suggested the involvement of a perturbation of membrane order in the mechanisms of membrane destabilization induced by antipsychotic drugs.     

灯盏花素对大鼠脑微血管内皮细胞损伤的保护作用

目的 观察灯盏花素对谷氨酸致原代培养的大鼠脑微血管内皮细胞（rBMECs）损伤的保护作用。方法 灯盏花素（200、100、50 μmol/L）作用于rBMECs 24 h后，加入谷氨酸（终浓度为1 mmol/L）培养18 h，MTT法检测rBMECs细胞活性，并按试剂盒方法检测细胞上清液中乳酸脱氢酶（LDH）水平、细胞中丙二醛（MDA）水平和超氧化物歧化酶（SOD）活性。结果 谷氨酸（l mmol/L）使原代培养的rBMECs明显受到损伤，灯盏花素高、中浓度（200、100 μmol/L）可显著对抗谷氨酸造成的rBMECs损伤，抑制LDH释放，降低MDA水平，增强SOD活性。结论 灯盏花素对谷氨酸所致原代培养的rBMECs损伤具有明显的保护作用，其机制与增强细胞抗氧化能力有关。     

LDH及sTfR在成人急性溶血性贫血中的意义

目的 探讨血清乳酸脱氢酶(lactic dehydrogenase,LDH)及可溶性血清转铁蛋白受体(soluable transferritin receptor,sTfR)在无慢性贫血史成人急性溶血性贫血(acute hemolutic anemia,AHA)诊断中的意义.方法 无慢性溶血性疾病史的32例成人急性溶贫患者采用酶标比色法测定血清LDH水平,ELISA方法检测血清sTfR水平并与正常体检者进行比较分析.结果 急性溶贫患者血清sTfR水平为49.3±13.1 nmol/L,对照组为15.5 ±2.1 nmol/L,两组比较有统计学差异(P＜0.05);急性溶贫患者血清LDH水平为531.1 ±111.2 IU/L,对照组为105.5 ±42.1 IU/L,两组比较有统计学差异(P＜0.05);不同程度AHA患者血清LDH及sTfR不同.结论 血清LDH、sTfR活性可作为无慢性溶血性疾病成人AHA诊断筛选.     

Myeloperoxidase-mediated bioactivation of 5-hydroxythiabendazole: A possible mechanism of thiabendazole toxicity

Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity. Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been proposed to yield a reactive intermediate. Here we show that this reactive intermediate can be catalyzed by myeloperoxidase (MPO), a neutrophil-bourne peroxidase. Using a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPO-generated metabolites in cell-based models including primary rat proximal tubule epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells. Timecourse experiments with MPO showed complete turnover of 5OH-TBZ within 15 min and a dramatic leftward shift in dose-response curves after 12 h. After a 24 h exposure in vitro, the LC₅₀ of this reactive intermediate was 23.3 ± 0.2 μM reduced from greater than 200 μM from 5OH-TBZ alone, an approximately 10-fold decrease. LC₅₀ values were equal in all cell types used. Comparison of lactate dehydrogenase leakage and caspase 3/7 activity revealed that cell death caused by the reactive intermediate is primarily associated with necrosis rather than apoptosis. This toxicity can be completely rescued via incubation with rutin, an inhibitor of MPO. These results suggest that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate which may play a role in TBZ-induced toxicity.     

Preventive effect of Piracetam and Vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture

The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N₂, 5% CO₂) for 3 h and followed by 1 h of reoxygenation (21% O₂ and 5% CO₂) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia + Piracetam, Hypoxia + Vinpocetine, Normoxia + Piracetam and Normoxia + Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1 mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture.