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目的研究中药大黄素对模拟冷缺血再灌注后肝细胞内钙离子浓度及细胞凋亡的影响。方法体外培养肝细胞株HL-7702，随机分为对照组和大黄素处理组。对照组未予大黄素处理，大黄素处理组按100、10、1μmol／L分为高、中、低3个浓度组，建立模拟冷缺血再灌注模型，流式细胞技术检测各组细胞内钙离子浓度及细胞凋亡水平，分别检测各组细胞培养上清液乳酸脱氢酶水平。结果冷缺血8h再灌注6h后，高、中、低浓度大黄素处理组钙离子荧光强度分别为（24．12±0．51）、（26．35±1.34）、（39．12±1．94），均显著低于对照组的105．29±1．01（P〈0．01）。高、中、低浓度大黄素处理组细胞凋亡率分别为（5．46±0．41）％、（10．64±0．64）%、（11．90±0．50）％，均显著低于对照组的（25．40±1．41）％（P〈0．01）。高、中浓度大黄素处理组上清液LDH含量分别为（179．67±18．57）u／L、（198．83±14．22）u／L，显著低于对照组的（351．33±34．16）u／L（P〈0．01）。结论大黄素可降低模拟冷缺血再灌注后的肝细胞内钙离子浓度，抑制细胞凋亡，减轻肝细胞损伤。

细胞内钙超载在急性胰腺炎中发挥关键性作用，而白藜芦醇可以减轻胰腺炎的严重程度。有关白藜芦醇治疗胰腺炎的机制尚未明确。本研究旨在阐明细胞内钙离子调节失衡和白藜芦醇对大鼠急性胰腺炎的治疗效应间的关系。将雄性SD大鼠随机分成3组：假手术组（OS）、重症急性胰腺炎组（SAP）和白藜芦醇治疗组（RES）。通过胰胆管逆行注射牛磺胆酸钠建立重症急性胰腺炎模型，而白藜芦醇通过静脉给药。

目的 探讨肾综合征出血热（HFRS）患者血小板内游离钙离子浓度（[Ca^2＋]i）与疾病的相关性。方法 用流式细胞仪检测细胞中Fluo-3的荧光强度，对42例确诊的HFRS患者血小板内[Ca^2＋]i进行测定，并以30例正常人作为对照。结果 检测显示，多尿期HFRS患者的[Ca^2＋]i显著高于正常对照组（P〈0．01），而发热期、少尿期及恢复期的[Ca^2＋]i与对照组之间无统计学差异（P〉0．05）。结论 HFRS发病过程中有多种因素可以活化血小板，多尿期患者的血小板处于活化状态，而发热期和少尿期患者的血小板可能由于各种原因的功能缺陷导致其[Ca^2＋]i并不升高。

P-selectin engagement of P-selectin glycoprotein Iigand-1 （PSGL-1） causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector （Fluo-4 AM）, the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demon- strated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellu- lar calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neu- trophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase （Syk）. These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectininduced calcium signaling of neutrophils in flow.

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity （MIA）, secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins a4131 and 05131, facilitating cell detachment and promoting formation of me- tastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I （COPI）- and coat protein complex II （COPII）-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca2＋ ions. Interestingly, the Ca2＋-activated K＋-channel, subfamily N, member 4 （KCa3.1）, known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant- negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling.As a newly discovered universal and fundamental mitochondrial phenomenon,superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria.Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown.Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence,and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response.Quantitatively,the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0？F/F0 of Rhod-2 signal.In contrast,large mitochondrial calcium transients（e.g.,peak△F/F0-2.8,duration-2 min）in the absence of steady-state elevations failed to alter the flash activity.These results indicate that physiological levels of sustained,but not transient,mitochondrial calcium elevation acts as a potent regulator of superoxide flashes,but its mechanism of action likely involves a multi-step,slow-onset process.

The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1-7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Phe- amide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels （at 120 minutes of exposure） and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 μmol/L gonadotropin-inhibitory hormone or 1 μmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.

目的研究藏红花素通过影响Ca^2＋内流对谷氨酸盐诱导的视网膜神经节细胞（RGCs）凋亡的影响及可能机制。方法分离大鼠RGCs,以0.1、1mmol/L的谷氨酸盐刺激RGCs 24、48h,建立RGCs凋亡模型,并用0.1、1.0、3.0μmol/L浓度梯度藏红花素分别处理。Annexin V-FITC/PI双标检测细胞凋亡率,Fluo-3/AM荧光标记Ca^2＋检测胞内钙离子浓度,Western blot检测藏红花素对胞内钙离子介导的凋亡信号分子calpain和CaMKⅡ表达的影响。JC-1荧光染色和Western blot分别检测藏红花素对线粒体膜电位和线粒体凋亡相关信号分子Caspase-3、Caspase-9、Bcl-2/Bax表达的影响。结果 0.1mmol/L谷氨酸盐刺激24h,RGCs细胞凋亡率与对照组差异无统计学意义（P〉0.05）;而当刺激48h时,RGCs的凋亡率达到（43.050±2.616）%,差异有统计学意义（P〈0.01）。高剂量谷氨酸盐（1mmol/L）刺激24、48h的RGCs凋亡率为（46.450±1.061）%和（45.500±3.253）%,较对照组均显著增加,差异有统计学意义（P〈0.01）。用1mmol/L谷氨酸盐刺激RGCs 12h后加入0.1、1.0、3.0μmol/L藏红花素再处理12h,不同浓度藏红花素均可显著抑制细胞凋亡（P〈0.01）,且抑制效率具有剂量依赖性。另外,1.0μmol/L藏红花素组的谷氨酸盐诱导的胞外Ca^2＋内流减少及钙依赖蛋白Calpain1和CaMKⅡ的表达减弱,线粒体膜电位增高,Caspase-3和Caspase-9的表达减少,Bcl-2/Bax表达上调。结论藏红花素抑制谷氨酸盐诱导的RGCs凋亡,其机制可能与阻止胞外Ca^2＋内流,抑制钙依赖的凋亡信号通路和线粒体凋亡信号通路有关。

目的观察AMP依赖的蛋白激酶（AMPK）对高糖刺激内皮细胞凋亡的抑制作用,并初步探讨其机制。方法体外培养MS-1内皮细胞株,分别用AMPK激动剂、AMPK抑制剂、钙库依赖性钙离子通道（SOCC）抑制剂2-APB和（或）高糖处理,另设对照组（未经任何方式干预）。采用TUNEL法检测细胞凋亡情况,激光共聚焦显微镜检测细胞内钙离子（Ca2＋）内流,Western blotting检测SOCC蛋白Stim1和Orai1的表达。结果与对照组比较,高糖能够明显诱导内皮细胞凋亡,增加Stim1和Orai1蛋白表达（P〈0.05）。与高糖组比较,AMPK抑制剂＋高糖能够明显增强高糖诱导的内皮细胞的凋亡（P〈0.05）,而AMPK激动剂＋高糖能够明显抑制高糖诱导的内皮细胞凋亡,并降低Stim1和Orai1蛋白表达（P〈0.05）。与对照组比较,高糖能够明显诱导内皮细胞Ca2＋内流;与高糖组比较,2-APB＋高糖能够明显抑制高糖诱导的内皮细胞Ca2＋内流,并阻断高糖对内皮细胞凋亡的诱导作用,而AMPK激动剂能够明显抑制高糖诱导的内皮细胞Ca2＋内流。结论 AMPK能够通过降低Stim1和Orai1蛋白的表达,抑制SOCC介导的Ca2＋内流,进而阻断高糖刺激的内皮细胞凋亡,对内皮细胞功能起重要的保护作用。

目的 了解内皮素-1（ET-1）对小梁细胞收缩状态调节的作用方式，以求探索新的有效而不良反应小的抗青光眼药物。方法 通过组织块培养的方法获得三代牛眼小梁细胞，Fulo-3／AM负载后于激光扫描共聚焦显微镜（LSCM）下动态扫描不同浓度ET-1作用后胞内荧光强度变化，得出[Ca^2＋]i值。结果 10^-9、10^-8、10^-7mol／LET-1可使小梁细胞[Ca^2＋]i呈剂量依赖性升高，其中10^-8mol／L的ET-1可使小梁细胞[Ca^2＋]i由327．5±24．57升高至373．60±40．18（n=8，P〈0．01）。结论 一定浓度的ET-1可通过收缩小梁网而致眼内压升高；其受体阻滞剂或转换酶抑制剂可能为原发性开角型青光眼的治疗开辟新途径。