Phospholipase C-evoked glycerol release in energy depleted rat myocardial cells

Summary Preincubation of rat myocardial cells in hypoxic substrate-free Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) resulted in a substantial decline in high energy phosphates (ATP and CP). Thus, 20 and 60 min preincubation produced a 18 and 72% decline in ATP content, whereas the parallel decline in CP content was 51 and 73%. This energy depletion was accompanied by a change in cell morphology from the initial rod-shaped form to rounded up (hyper-contracted) myocytes. In cells preincubated in substrate-free normoxic buffer, both normal morphology and energy homeostasis were maintained. When energy depleted myocytes later were incubated in the presence of phospholipase C (PLC), this resulted in a substantial release of glycerol, amounting to 92 and 137 nmol/10⁶ cells – 2 h in 20 and 60 min energy depleted myocytes, respectively. In addition, PLC caused an increased leakage of lactate dehydrogenase in energy depleted myocytes. Normal cells, on the other hand, were apparently not affected by PLC. These data suggest that PLC selectively attacks energy depleted and/or structurally damaged myocytes. This could well enhance the breakdown of the natural barrier between the extra- and intracellular compartments and thus augment the cellular damage during ischemia. Moreover, energy depleted myocytes appeared exceptionally sensitive to this enzyme, since the levels required to cause glycerol or lactate dehydrogenase release were several orders of magnitude lower than that required to cause membrane permeation in other cell types.     

Reduction of cell injury in hypoxic cultures of rat myocardial cells by methylprednisolone

Summary An in vitro model to study myocardial cell injury was developed with primary monolayer cultures of rat myocardial cells. Two important conditions associated with myocardial ischemia were simulated by depriving the cultures of oxygen and glucose for a specified period of time. Cellular injury caused by hypoxia and glucose deprivation resulted in significant leakage of lactate dehydrogenase (LDH) from the cells into the culture medium. The cells were not lethally injured by treatments as reflected by a lack of change in cell viability and protein content when compared to controls. Pretreatment of cultures with methylprednisolone for 24 hr provided protection to the cells when challenged by hypoxia and glucose deprivation. Methylprednisolone exhibited a dose-response effect in reducing LDH leakage in cultures, which were subsequently deprived of oxygen and glucose for 4 hr. Similar pretreatment with hydrocortisone had no effect in limiting cellular injury in hypoxic and glucose-deprived cultures. The research was supported by Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute.     

藏红花素抗心肌细胞低氧损伤作用及其机制研究

目的:探讨藏红花素(Crocin)对低氧心肌细胞的保护作用以及低氧诱导因子-1(HIF-1)和脯氨酰羟化酶(PHDs)的调控机制。方法:采用氯化钴(CoCl2)方法建立心肌细胞低氧损伤实验模型。应用Western blot方法检测心肌细胞HIF-1α、血管内皮生长因子(VEGF)、诱导型NO合酶(iNOS)以及PHD1、2、3蛋白表达的变化。结果:与CoCl2低氧组比较,Crocin可显著提高低氧心肌细胞活力。Crocin可进一步促使心肌细胞HIF-1α、及其下游靶基因VEGF、iNOS蛋白表达增强。Crocin可促使心肌细胞PHD2蛋白表达明显增加,PHD3蛋白表达则明显减少。结论:Crocin对低氧心肌细胞具有明显的保护作用,其作用机制与Crocin激活HIF-1介导的低氧反应通路有关,PHDs参与了该反应的病理生理调控过程。     

低氧对胚胎干细胞增殖的影响

目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%～10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%～10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%～10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究.     

低氧对大鼠隔肌放电的影响

目的:探讨睡眠中间歇低氧对大鼠肠肌放电的影响.方法:大鼠分别给予吸入空气,持续低氧和间歇低氧气体,在1 d,3d,7 d,28 d后测定大鼠膈肌放电的情况及采动脉血行血气分析,分析大鼠膈肌放电在不同时期有何不同及其与酸碱平衡之间的关系.结果:与对照组比较,大鼠膈肌放电面积及膈肌放电频率振幅仅在间歇低氧28 d组无统计学...     

Effects of short-term hypoxia on [3H]glutamate release from preloaded hippocampal and cortical synaptosomes

The effect of short-term hypoxia on the release of [³H]glutamate from preloaded hippocampal and cortical synaptosomes was studied in a rapid superfusion system. The technique minimised the loss of released glutamate by reuptake. The results indicated that the effects of short term hypoxia were qualitatively similar to those reported in previous studies using more long-term hypoxia, but were significantly smaller. The non-Ca²⁺-dependent efflux of glutamate from cortical synaptosomes was increased by hypoxia as was the Ca²⁺-dependent release from hippocampal tissue. Possible mechanisms for these findings were discussed. The small amplitude of these changes in comparison to the effects seen in slowly perfused tissue in vitro and in vivo indicated that the contribution made by changes in neuronal efflux to the overall increase in extracellular glutamate seen in hypoxia is relatively minor.     

常氧和急性低氧下大鼠传导性肺动脉平滑肌细胞的电生理特性

本文旨在研究大鼠传导性肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）的电生理特征及对急性低氧的反应。用酶解法急性分离出1-2级分支的PASMCs,通过全细胞膜片钳方法研究常氧及急性低氧状况下细胞钾电流的差异,并在常氧下先后使用iBTX和4-AP阻断大电导钙激活钾离子（large conductance Ca-activated K^＋,BKCa）通道及延迟整流性钾离子（delayed rectifier K^＋,KDR）通道后,观察细胞钾电流特征。根据细胞的大小、形态及电生理特征可将PASMCs分为Ⅰ、Ⅱ、Ⅲ类。iBTX对Ⅰ类细胞几乎无作用,而4-AP几乎完全阻断它的钾电流;Ⅱ类细胞的钾电流在加入iBTX后大部分被抑制,其余的对4．AP敏感;Ⅲ类细胞的钾电流对iBTX及4-AP均敏感。急性低氧对三类细胞的钾电流均有不同程度的抑制,并使Ⅰ类细胞的膜电位显著升高,而Ⅱ、Ⅲ类细胞膜电位升高的程度不如Ⅰ类显著。结果表明,传导性肺动脉有3种形态及电生理特性不同的PASMCs,在急性低氧时其钾电流不同程度地受到抑制,同时静息膜电位也有不同程度去极化,这些可能参与急性低氧时传导性肺动脉舒缩反应的调节。KDR及BKCa通道在3种细胞中的比例不同可能是急性低氧对3种PASMCs影响不同的离子基础。     

Effects of prolonged exposure to hypoxia on morphological changes of endothelial cells plated on fibrin gel

Since tissue oxygenation has a profound effect on capillary growth, the effect of pO2 on endothelial cell functions was studied. Under normoxic conditions, EA.hy926 endothelial cells and HUVEC plated onto fibrin gels in low-serum culture medium underwent rapid and profound morphological changes within 12 to 48 hours depending on the cell line used. Their characteristic cobblestone organisation was transformed into a network of cord-like or tube-like structures. We showed that when exposed to low oxygen concentrations for 3 days, HUVEC and EA.hy926 have their ability to rearrange reduced to around 50 %. With EA.hy926 this effect was amplified by 79% after 9 days of hypoxia. The altered behaviour of hypoxia-adapted cells was not caused by a loss in their fibrinolytic activity. In fact, the fibrin degradation rate and the generated fibrin fragments appeared identical in normoxia and hypoxia. Confocal microscopy and gel densitometry showed that in normoxia the remaining undegraded fibrin gel underwent a dynamic remodeling whereas in hypoxia it remained undisturbed. It is likely that hypoxia induces modification in the factors that integrate matrix information and cytoskeletal organisation in order to contract fibrin.     

间断性低氧对大鼠腓肠肌介电性能的影响

目的：在40Hz～110MHz频率范围观察间断性低氧暴露4周大鼠离体腓肠肌细胞介电性能的改变。方法：采用低压氧舱建立模拟低氧模型,雄性SD大鼠随机分为间断低氧组和正常对照组。利用Agilent 4294A阻抗分析仪测量了离体大鼠腓肠肌的交流阻抗,通过频域介电谱、Cole—Cole图、介电损耗因子频谱、电导率虚部频谱和介电损耗角正切频谱的数据分析,观察间断性低氧暴露对大鼠离体腓肠肌细胞介电性能的影响。结果：间断性低氧暴露4周大鼠腓肠肌的介电常数（εL,εh）降低,介电增量△ε减小,绝缘性降低;低频电导率κL升高,高频电导率κh降低,电导率增量△κ降低;特征频率（f1,f2）增加;介电损失峰值ε”peak、电导率虚部峰值κ”peak和损耗角正切峰值婶谳均降低。结论：间断性低氧暴露致骨骼肌细胞介电性能降低,但其特征频率增加。     

低氧对CD34~ 造血干/祖细胞增殖分化及其对细胞因子依赖性的影响

采用免疫磁珠法分离脐血CD34 造血干 /祖细胞 ,进行低氧和常氧条件下单个核细胞 (MNC)及CD34 细胞的半固体及液体培养 ,计细胞总数和集落产率 ,并通过流式细胞仪检测细胞表型和细胞周期 ,以探讨造血干/祖细胞在低氧环境下增殖分化性能的改变及其对细胞因子反应性的变化。结果显示 :CD34 细胞在低氧条件下生成的BFU E集落数 ( 32 4 8± 41 4/10 4 细胞 )明显增多 (对照为 191 2± 34 5 /10 4 细胞 ,P <0 0 1) ;在无细胞因子存在的液体培养体系中 ,低氧组的BFU E产率 ( 15 2 4± 2 2 6 /10 4 细胞 )明显高于常氧组 ( 74 2± 9 3/10 4 细胞 ,P <0 0 1) ;低氧培养细胞中CD34 细胞的比例高于对照 2 5± 1 2倍 (P <0 0 5 )。但MNC生成的BFU E在常氧和低氧条件下无显著差异。这些结果表明 :体外低氧环境能显著增加CD34 造血干 /祖细胞形成红系祖细胞的产率 ,且使其对细胞因子的依赖性降低 ,并对早期红系祖细胞的维持有增强作用 ,但对粒系祖细胞的增殖则有抑制作用     

间歇性低氧对大鼠心室肌细胞短暂外向电流的影响

利用全细胞膜片箝方法研究间歇性低氧后左、右心室肌细胞短暂外向电流（Ｉｔｏ）的变化,以探讨间歇性低氧增强心肌电稳定性的离子机制。大鼠间歇性暴露于低氧环境２８ｄ（Ｈ２８,６ｈ／ｄ）后,右心室肌细胞的Ｉｔｏ密度较常氧对照组明显增加（１６１８±４６１比６３２±１３５ｐＡ／ｐＦ,Ｐ＜００５）,而左心室肌细胞Ｉｔｏ密度与对照组无明显差异。间歇性低氧暴露４２ｄ（Ｈ４２）动物,其左、右心室肌细胞Ｉｔｏ密度与对照组无明显差异。Ｉｔｏ激活、失活和恢复动力学变化主要表现为Ｈ４２组左、右心室肌细胞的稳态失活曲线明显向负电压方向移位。左心室细胞的半数失活电压（－３８９±２３）ｍＶ与对照组（－３２８±５９）ｍＶ比较,具有显著性差异（Ｐ＜００１）;右心室细胞的半数失活电压（－４１９±４５）ｍＶ与对照组（－３３５±３５）ｍＶ比较,具有显著性差异（Ｐ＜０００１）。据此可推断,Ｉｔｏ密度的改变可反映心室在低氧早期阶段的不同动力学反应。失活动力学改变参与间歇性低氧心脏保护机制     

低氧对培养的不同内径的肺动脉平滑肌细胞增殖的影响

目的和方法：分离培养三种不同内径的肺动脉平滑肌细胞（PASMCs）,用^3H－TdR掺入速率和细胞计数作为细胞增殖的指标,观察低氧对其增殖作用的影响。结果：低氧对三种不同内径的PASMCs（内径分别为＞1000μm、500－800μm、300-400μm）增殖促进作用显著不同,其^3H－TdR掺入速率和细胞计数分别增加23.5％和11.1％、60.0％和33.8％、141.4％和52.0％,选择对低氧最敏感的PASMCs（内径为300－400μm）,进一步探讨低氧促PASMCs增殖作用的细胞机制：钙拮抗剂verapail、蛋白激酶C抑制剂staurosporine(Stau)和细胞Na-H交换抑制剂amiloride可显著降低低氧情况下PASMCs^3H－TdR掺入速率和细胞计数。结论：低氧对三种不同内径的PASMCs增殖促进作用显著不同; Ca^2 、蛋白激酶C和Na^2 －H^ 交换的激活,可能是低氧促PASMCs增殖的重要胞内信息转导机制。     

低氧对胎盘基蜕膜间充质干细胞增殖、凋亡和血管内皮细胞生长因子表达的影响

本文旨在探讨生理低氧和无血清培养条件下人胎盘基蜕膜间充质干细胞（placental decidua basalis—mesenchymal stemcells,PDB-MSCs）的生物学特征和细胞因子的表达情况。采用密度梯度离心法培养获得PDB—MSCs,利用MTT法、流式细胞术检测PDB—MSCs在不同氧浓度（20％02和1％O2）、有无血清（10％FBS和0％FBS）条件下各个时间点（6h、12h、24h、48h、72h、96h）的增殖和凋亡情况,并采用酶联免疫吸附实验（enzyme linked immunosorbent assay,ELISA）检测无血清条件下各个时间点细胞上清液中血管内皮细胞生长因子（vascularendothelial growth factor,VEGF）含量。结果显示,在特定的时间内低氧可以促进PDB—MSCs增殖（P〈0．01,n=3）,而血清对PDB—MSCs增殖的影响与氧浓度关系密切;同时,在本实验条件下,低氧、无血清分别或联合培养不会导致PDB—MSCs凋亡（P〉0．05,n=3）;在无血清条件下,24h时低氧组PDB-MSCs表达较高水平的VEGF。以上结果提示,PDB—MSCs可能成为缺血相关组织工程产品种子细胞的一个新来源。     

3,6-(二甲氨基)-二苯并碘杂六环葡萄糖酸盐对损伤心肌细胞的保护作用

目的：观察3,6-（二甲氨基）-二苯并碘杂六环葡萄糖酸盐（IHC-93）对培养心肌细胞损伤的影响。方法：在培养的心肌细胞建立低氧／复氧损伤模型和过氧化氢损伤模型,观察心肌细胞存活率、乳酸脱氢酶（LDH活性、超氧化物歧化酶（SOD）活性、丙二醛（MDA含量,以研究IHC-93对培养心肌细胞损伤的影响。结果：10、20、40μmol／LIHC-93呈剂量依赖地提高损伤心肌细胞存活率和SOD的活性,减少LDH的释放,降低MDA含量。结论：IHC-93对损伤心肌细胞具有直接保护作用。     

急性低氧暴露小鼠外周血代谢组变化分析

目的：探讨急性低氧对小鼠外周血代谢组的影响。方法：将14只小鼠随机分为正常组和低氧组。用基础饲料喂养2周后,将低氧组减压至6000m模拟高度停留8h,实验结束后,采集静脉血制备血浆待测。在核磁共振波谱仪进行^1H NMR检测,采用模式识别分析方法处理数据。结果：与正常组相比,低氧组乳酸含量明显增加,肉碱水平明显降低;脂类、丙氨酸、丙酮酸、谷氨酰胺、胆碱、牛磺酸和葡萄糖含量升高,缬氨酸、肛羟丁酸、谷氨酸、甘油、甘氨酸和丝氨酸含量下降。结论：急性低氧暴露使小鼠血浆碳水化合物、脂肪代谢和氨基酸代谢谱发生变化,表明低氧后能量代谢以及相关物质含量发生改变。