一过性离体大鼠肝脏灌流模型的建立

目的利用UP-100通用灌流仪建立了一过性离体大鼠肝脏灌流模型。方法用Krebs-Henseleit碳酸氢盐缓冲液进行一过性离体大鼠肝脏灌流,在灌流时长90 min内通过检测AST、ALT和LDH的浓度,观察肝脏外观及胆汁流量,测定肝重变化,进行组织切片观察组织病理变化来评估离体灌流肝脏的损伤程度。结果 90 min灌流时长中,肝脏颜色质地正常,胆汁流量均匀,相应转氨酶释放量少,病理切片结果正常。结论离体大鼠肝脏在UP-100通用灌流仪90 min灌流过程中能保持较好的组织结构和肝脏功能,可用于离体肝脏的药物代谢研究。     

纳洛酮对离体大鼠心脏缺血再灌流损伤的保护作用

本实验采用离体大鼠心脏Langendorff灌流装置.使心脏停灌(旷置)40 min后再恢复灌流30min.夏制心肌缺血再灌流损伤.观察了纳洛酮对再灌流后冠脉流出液中乳酸脱氢酶(LOH)活性,心肌细胞内钙含量.心肌脂质过氧化产物——丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力变化的影响。结果表明.再灌流后应用纳洛酮能显著减少心肌LDH的释放量.心肌细胞内钙聚集、心肌MDA的生成量及SOD活力降低的程度.提示纳洛酮对缺血再灌流心肌具有保护作用。     

丹酚酸B对兔离体心脏缺氧再复氧损伤的保护作用

目的观察丹酚酸B对兔离体心脏缺氧-复氧损伤的保护作用。方法离体兔心脏Langendorff灌流,通氮气饱和的灌流液60min后再恢复含氧灌流液灌流60min,造成缺氧-复氧损伤;自动生化分析仪测定冠脉流出液中肌酸激酶(CK)和乳酸脱氢酶(LDH)活性;光学显微镜观察心肌组织结构改变。结果在缺氧的同时给予丹酚酸B0.3,1和3mg.L-l灌流60min,可降低心率,增加冠脉流量,降低冠脉流出液中CK和LDH的水平,心肌组织形态学损伤明显减轻。结论丹酚酸B对兔离体心脏缺氧-复氧损伤有明显的保护作用。     

γ-亚麻酸甲酯乳剂代谢动力学

本文应用动物全血、组织匀浆体外保温实验和大鼠离体肝脏灌流技术研究了γ-亚麻酸甲酯乳剂的代谢动力学。γ-亚麻酸甲酯在全血中迅速水解;在肝、小肠和肾中也存在代谢;离体肝灌流液中γ-亚麻酸的浓度下降与时间关系经拟合符合单指数方程,消除半衰期为13.2min,肝固有消除率为2.46ml/min.     

脂质过氧化在再灌流性肝损伤中的作用

采用低流率-再灌流模型研究脂质过氧化在再灌流性肝损伤中的作用,肝脏经过90min低速率灌流未见明显损伤,当灌流速率恢复到正常后中心静脉周围区(PC区)细胞发生迅速不可逆损害,并伴有丙二醛生成率大幅度上升,低速率灌流期间,灌流液中黄嘌呤与次黄嘌吟浓度由原来的1.5和3.6μmol·L~(-1)逐步升高至5.5和11.5μmol·L~(-1),自由基清除剂儿茶酸能使再灌流期丙二醛生成率由295 nm01·g~(-1)·h~(-1)下降至109 nmol·g~(-1)·h~(-1),并使LDH释放率减少约50％,PC区细胞死亡率减少89％,再灌流初期用氮饱和灌流液冲洗3 min,使再灌流所致的LDH释放下降约50％,PC区细胞死亡率减少84％,别嘌吟醇(2～6mmol·L~(-1))对防止再灌流性肝损伤表现出明显的剂量效应关系。 与预计结果相反低浓度别嘌呤醇(0.5～1mmol·~(-1))能增加再灌流性肝损伤,400μmol·L~(-1)黄嘌呤则使再灌流性肝损伤明显减轻,其代谢产物尿酸对降低再灌流时丙二醛生成率以及细胞损害均表现出明显的剂量反应关系。     

林蛙油酶解前后抗疲劳作用对比研究

目的:考察林蛙油酶解前后抗疲劳作用变化情况,为林蛙油酶解工艺的合理性提供理论和实验依据.方法:采用小鼠负重游泳法,观察林蛙油及其酶解后粉末对小鼠运动耐力的影响;对小鼠肝糖原、肌糖原储备的影响;对剧烈运动后小鼠血乳酸(LAC)、血尿素氮(BUN)含量及乳酸脱氢酶(LDH)活力的影响.结果:每天经口服给予小鼠林蛙油及其酶解液437mg/kg,560mg/kg,连续灌胃30d,各用药组均能延长小鼠负重游泳时间,减少小鼠游泳时血尿素氮的产生,增加肝糖原、肌糖原含量,减少血乳酸的产生,LDH活力增加,且各试验组与空白对照组差异显著(P<0.05).在各用药组中,以酶解后组最高,阳性对照组次之,林蛙油组最低,但是各组之间差异不显著.其中游泳后20min血乳酸含量、LDH活力酶解后组与林蛙油组相比具有显著性差异.结论:林蛙油及其酶解液在提高机体对运动负荷的适应能力,延缓疲劳的产生和加速疲劳的消除方面都有很明显的作用,证明林蛙油及其酶解液均有明显的抗疲劳保健功效,且酶解后作用进一步增强.     

门冬氨酸钾镁对离体灌流心脏缺氧的保护作用

目的探讨门冬氨酸钾镁对离体心脏缺氧的保护作用,及2种不同制剂组成方式的门冬氨酸钾镁注射液的效能比较。方法将60只SD大鼠取心脏行Langendorff离体心脏灌流,依照灌流液中钾、镁组成的不同分为A制剂组(门冬氨酸钾 门冬氨酸镁)、B制剂组(天门冬氨酸 氧化镁 氢氧化钾)及对照组(KCl MgSO4)3组,每组各20只动物,3组的K 、Mg2 等剂量。记录离体大鼠心脏在缺氧条件下的HR、左室压力变化及心脏跳动持续时间。结果A制剂组中离体心脏在缺氧后HR及左室压力降低速度缓慢,心脏跳动持续时间达(273.9±65.2)min,与B制剂组(174.7±43.6)min及对照组(121.8±32.9)min相比明显延长(P<0.01)。结论门冬氨酸钾镁液对离体缺氧心脏有保护作用,明显延长缺氧状态下心脏的跳动时间,但制剂B比制剂A的抗缺氧效能差。     

Na+/K+泵参与缺氧诱发的大鼠脑血管收缩的体外研究

目的 探讨Na+/K+泵是否参与缺氧诱发的脑血管收缩.方法 以PSS、K-PSS、oua-PSS、oua-K-PSS或K-free-PSS液灌流大鼠离体基底动脉后5、10、15、30和 60 min时,以压力肌动描记系统记录缺氧前后血管直径的变化.结果 缺氧、K+和哇巴因灌流均可使大鼠基底动脉产生明显收缩,缺氧可增强K+、10-8和10-7 mol·L-1哇巴因对正常基底动脉的收缩(P<0.01),但对K+与10-8或10-7 mol·L-1哇巴因共同预收缩的基底动脉以及5×10-7 mol·L-1哇巴因收缩的基底动脉并无影响.而且用可取消Na+/K+泵活性的无K+-PSS液,不管在正常或缺氧情况下,均能使基底动脉在5 min内达到最大收缩.结论 Na+/K+泵可能参与了缺氧诱发的脑血管收缩,其机制可能与缺氧抑制Na+/K+泵有关.     

水飞蓟宾在大鼠小肠中的吸收特性

目的考察水飞蓟宾在大鼠各肠段的吸收。方法以一定浓度的水飞蓟宾溶液作为灌流液，以0.1 mL·min^-1进行大鼠不同肠段的单向灌流,于不同时间收集肠灌流液并肝门静脉取血，分别用HPLC测定灌流液和血中药物浓度。结果实验结果表明190 μg·mL^-1水飞蓟宾在不同肠段的吸收速率常数(k_a)和有效透过系数(P_eff)是十二指肠>空肠>回肠>结肠。质量浓度为80 μg·mL^-1的水飞蓟宾灌流液在十二指肠的吸收与190和300 μg·mL^-1的吸收情况均有显著性差异(P<0.05)，但质量浓度为190 μg·mL^-1与300 μg·mL^-1的吸收之间无显著性差异(P>0.05)。肝门静脉血中药物分析也显示十二指肠>空肠>回肠>结肠。结论水飞蓟宾在小肠全肠道均有吸收且有高浓度饱和现象。     

白藜芦醇在缺糖缺氧/再灌注损伤不同时间窗对大鼠皮质神经元的保护作用

目的 研究白藜芦醇在缺糖缺氧/再灌注损伤不同时间窗内对大鼠皮质神经元的保护作用及机制.方法 大鼠皮质神经元缺糖缺氧处理150 min,随即恢复正常培养24 h.实验分为正常组,模型组,白藜芦醇预处理组、造模时处理组、造模后处理组和全程处理组.倒置显微镜下观察细胞形态.化学比色法测定LDH活性和GSH含量.MTT法检测细胞活力.TUNEL检测细胞凋亡.免疫印迹法检测Bcl-2、Caspase-3 蛋白表达.结果在白藜芦醇全程处理组中,各浓度白藜芦醇均可降低培养上清液LDH活性,升高细胞内GSH含量(P<0.05),其中以40 μmol·L-1白藜芦醇作用最佳.在缺糖缺氧再灌注损伤的不同时间内给予白藜芦醇,均较模型组增强神经元活力,减少神经元凋亡,上调Bcl-2蛋白、下调Caspase-3蛋白的表达(P<0.05),其中全程处理组作用最佳,其次为预处理组.结论 白藜芦醇对缺糖缺氧/再灌注损伤的神经元有剂量依赖性保护作用,而且全程处理组作用最佳,预处理组次之.其保护作用机制可能至少部分是通过调节凋亡相关蛋白而实现.     

原钒酸钠的细胞毒理学实验研究

目的　探讨原钒酸钠 (SOV)对心肌细胞的毒性作用及其机制。方法　采用循环灌流的方法急性分离成年豚鼠心肌细胞 ,使其在原钒酸钠浓度为 1 ,1 0 ,1 0 0 μmol·L- 1 ,1mmol·L- 1 作用后 ,分别检测 30 ,60 ,1 2 0 ,1 80min 4个时相心肌细胞保存液中LDH、CK含量以及心肌细胞蛋白含量和Na+ ,K+ ATP酶活性 ,检测心肌细胞凋亡率和细胞活力 ;电镜下观察死亡和凋亡细胞的细胞器和细胞核形态学变化。结果　在原钒酸钠 1 0 0 μmol·L- 1 和 1mmol·L- 1 两个组的各时相保存液中LDH、CK以及心肌细胞中蛋白含量增加 ;心肌细胞中Na+ ,K+ ATP酶活性和细胞活力降低 ,与对照组相比差异有显著性 (P <0 0 1 )。流式细胞仪显示 :在 1 80min时 1 0 0 μmol·L- 1 和 1mmol·L- 1 两组的细胞凋亡率与对照组相比增加 (P <0 0 5)。结论　在原钒酸钠浓度高于 1 0 0μmol·L- 1 且作用时间在 30min以上时 ,心肌细胞出现不可逆性损害     

Effect of Trolox C on hypoxia/reoxygenation-induced injury in isolated perfused rat liver

Livers isolated from 18 hours fasted rats were subjected to N(2) hypoxia (for 45 min) followed by reoxygenation (for 45 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). Lactate and alanine were added as gluconeogenic and ureagenic substrates and Trolox C was also added to perfusate. Oxygen consumption, lactate dehydrogenase (LDH), alanine transaminase (ALT), total glutathione, oxidized glutathione, bile flow, glucose and urea were measured. After hypoxia oxygen consumption significantly dropped but Trolox C had no influence on this decrease. ALT and LDH were significantly increased by hypoxia/reoxygenation. This increase was markedly attenuated in the presence of Trolox C. The total glutathione and oxidized glutathione efflux increased following hypoxia, which were prevented by the treatment of Trolox C. Bile flow rate decreased following hypoxia/reoxygenation but did not continue to decrease in the reoxygenation phase by Trolox C. Following hypoxia/reoxygenation glucose and urea releases decreased. Trolox C had no influence on inhibition of glucose and urea production. These results suggest that Trolox C protected the liver cells against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model.     

适于研究药物相互作用的肝脏灌流模型

目的:改进和建立大鼠离体肝脏灌流模型.方法:采用Krebs-Henseleit 缓冲液(pH7.4)为港灌流液基液,含2%透析48h的牛血清白蛋白组分V、20%(V:V)洗过的人红细胞和0.3%葡萄糖.灌流速度1.5ml·min~(-1)·g~(-1),温度(37±0.5)℃,灌流压力1.7～1.33pKa.测定大鼠灌流过程中胆汁分泌量、耗氧量及灌流液pH、Na~ 、K~ 水平,并观察肝脏外观变化和组织切片的细胞形态学,评定肝脏功能.结果:本系统灌流中大鼠的各项考察指标正常,离体肝脏的存活力可达3h.结论:该模型适用于研究药物在肝脏代谢中的相互作用及其发生机制,也可用于研究某些药物特殊的代谢动力学特征.     

Determination of the extent of ischemic damage and the effect of the calcium antagonist, verapamil following coronary artery ligation in the rat

Apart from pharmacological interventions, four methods can be used to induce myocardial damage in the isolated, perfused heart. These are (i) total global ischemia, where perfusion is stopped completely; (ii) partial ischemia where perfusion is restricted; (iii) regional ischemia, produced by occlusion of the coronary circulation, and (v) hypoxia where the oxygenated buffer is replaced with a buffer bubbled with nitrogen. Using rat hearts, coronary artery occlusion was found to have potential as a screening device for antiischemic compounds. In these studies 45Ca uptake and enzyme release were found to increase with ligation time. The inclusion of the Ca2+ antagonist verapamil (0.01 to 1 microM) resulted in a concentration-dependent inhibition of 45Ca uptake (IC50 = 68 nM); however the proportion of tissue damaged remained unchanged. Similar findings were obtained in the presence of the dihydropyridine Ca2+ antagonist nicardipine (0.1 or 1 microM). Measurement of enzyme release during the reperfusion period confirmed significant correlations between levels of either lactate dehydrogenase (LDH) or creatine kinase (CK) and 45Ca uptake. Studies involving LDH show that cation uptake precedes enzyme release (r = 0.93; p = less than 0.001).     

MARKERS OF PHYSICAL INTEGRITY AND METABOLIC VIABILITY OF THE PERFUSED HUMAN PLACENTAL LOBULE

1. Peripheral lobules of term placentae obtained from healthy females at Caesarian section were perfused using separate maternal and fetal circulations for 6 h periods under either oxygenated or anoxic conditions. 2. Markers of physical integrity during setting-up and initial perfusion were establishment of dual perfusion within 25 min of placental delivery, pressure in the fetal capillary network less than 40 mmHg, leakage of perfusate from fetal to maternal compartments less than or equal to 2 ml/h, and overlap of maternal with fetal perfusion as indicated visually by appropriate blanching and verified by a fetal artery to vein oxygen gradient of greater than or equal to 90 mmHg. 3. Post-perfusion markers of metabolic viability were most reliably indicated by glucose consumption (oxygenated 7.8 +/- 1.5, anoxic 17.7 +/- 1.2 mmol/kg per h), lactate production (oxygenated 8.5 +/- 1.4, anoxic 33.9 +/- 2.5 mmol/kg per h) and human placental lactogen production (oxygenated 41.2 +/- 9.8, anoxic 12.2 +/- 3.4 mg/kg per h).     

Beneficial actions of two novel calcium entry blockers in the isolated perfused hypoxic cat liver

The effects of two new calcium entry blockers, anipamil and ronipamil, were studied during 150 min of normoxic or hypoxic perfusion in isolated perfused cat livers. Hypoxic livers in which the vehicle for these inhibitors (i.e., ethanol) was injected intravenously prior to isolation of the liver, exhibited significantly higher increases in perfusion pressure, perfusate lactate dehydrogenase and cathepsin D activities, compared to control normoxic perfused livers. In contrast, the livers isolated from cats pretreated with calcium entry blocker anipamil and subsequently perfused under hypoxic conditions showed no significant difference in any of these variables from the control normoxic perfused livers. Ronipamil given intravenously 30 minutes prior to isolation also significantly protected the liver during hypoxia. The protection afforded by anipamil and ronipamil appears to be related to their inhibition of Ca++ influx which has been linked to cell death in hepatocytes.     

Protective effects of the calcium antagonists diltiazem and TA3090 against hepatic injury due to hypoxia.

Recent work has shown that dihydropyridine-type calcium channel blockers such as nitrendipine protect against ischemic liver damage in the rat in vivo (Thurman RG, Apel E and Lemasters JJ, J Cardiovasc Pharmacol 12: S113-S116, 1988), suggesting that calcium antagonists may have clinical value in preventing ischemic and hypoxic hepatic injury. This study was designed to examine the effects of two benzothiazepine-type calcium channel blockers, diltiazem and TA3090, in the hypoxic perfused rat liver. Livers were isolated and perfused briefly with oxygen-saturated buffer, followed by perfusion for 80 min with nitrogen-saturated buffer with diltiazem or TA3090 (20-200 microM), and concluding with 20 min of perfusion with oxygen-saturated buffer. In control preparations, maximal lactate dehydrogenase (LDH) release into effluent perfusate following hypoxia averaged about 1100 U/L. Diltiazem and TA3090 decreased LDH release at all concentrations studied; both drugs were most effective at the 100 microM concentration (71 and 73% inhibition, respectively). Oxygen uptake by control livers decreased 78% following hypoxia; diltiazem and TA3090 reduced this effect markedly, with maximal effectiveness again observed with 100 microM (O2 uptake was decreased by 22% with 100 microM diltiazem and by only 9% with 100 microM TA3090). Histological examination for nuclear uptake of the vital dye trypan blue revealed necrosis of parenchymal cells along with cell shrinking and consequent expansion of the sinusoids in control livers. Perfusion with diltiazem markedly reduced parenchymal cell death but did not alter the pattern of cell damage observed. In contrast, livers perfused with TA3090 during hypoxia had virtually no parenchymal cell damage, although moderate damage to nonparenchymal cells in the sinusoids occurred. The difference in mechanisms responsible for the phenomena which occur with diltiazem and TA3090 is not completely understood; however, these and other calcium antagonists clearly have powerful hepatoprotective effects against ischemia and hypoxia.     

Effect of perfused rat mandibular-gland pHI on the ratio of procainamide concentration in saliva to that in venous effluent

The saliva to venous-effluent concentration ratio (S/E ratio) for procainamide (PA) was determined and compared with the ratio calculated by using the intracellular pH value of glandular cells. Exposed mandibular gland was perfused in situ with Krebs-Ringer bicarbonate buffer containing PA (10-100 micrograms/ml) and acetylcholine (ACh, 0.1 to 10 microM) or pilocarpine (10 microM). These perfusion conditions maintained almost normal physiological function of the mandibular gland throughout the perfusion period of 60 min, since the salivary Na+ and K+ concentrations were kept at almost constant levels, comparable with those reported in vivo, and the salivary flow, pH and protein level were also stabilized. Under fixed stimulation conditions with 1 microM ACh or 10 microM pilocarpine, the perfusate PA concentration ranging from 20 to 100 micrograms/ml did not affect the S/E ratio (approximately 0.3). There was a negative correlation between the S/E ratio and salivary pH when stimulated with 0.1 to 10 microM ACh. However, Matin's equation [S. B. Matin et al., Clin. Pharmac. Ther. 16, 1052 (1974)] employing venous effluent and salivary pH values did not explain fully these observed ratios. In contrast, Borzelleca's model [J. F. Borzelleca and J. W. Putney, J. Pharmac. exp. Ther. 174, 527 (1970)] for salivary drug transport using intracellular pH of the mandibular gland cells predicted S/E ratios relatively close to the observed values when the gland was perfused at pH 7.4 or 8.0.