黄芩苷对离体心脏缺血再灌注损伤的保护效应

目的:观察中药黄芩苷对离体状态下缺血再灌注损伤心脏的保护效应。方法:实验于2005-09/12在锦州医学院药理学教研室完成。选用雄性成年SD大鼠60只,头部击昏后迅速取出心脏,采用离体大鼠心脏Lan-gendorff灌流模型制备离体心脏缺血再灌注损伤模型。随机数字表法分为5组,每组12只。①对照组:以含氧K-H液灌注100min。②再灌注组:在对照组基础上灌注含氧K-H液10min后,停止灌流30min,然后再灌注60min。③黄芩苷60μmol/L再灌注组:灌注方法同再灌注组,再灌注时加入60μmol/L黄芩苷溶液。④黄芩苷120μmol/L再灌注组:再灌注时加入120μmol/L黄芩苷溶液。⑤黄芩苷240μmol/L再灌注组:再灌注时加入240μmol/L黄芩苷溶液。应用黄嘌呤测定法测定超氧化物歧化酶活性P应用TAB比色原理测定心肌组织中脂质过氧化产物丙二醛含量P收集各组冠脉流出液测定肌酸激酶、乳酸脱氢酶活性,用以评价心肌细胞受损程度及黄芩苷的保护作用。原位末端标记法标记后应用流式细胞仪进行细胞凋亡率测定,进一步从DNA水平观察细胞受损程度及黄芩苷的保护作用。计量资料差异比较采用t检验,组间数据处理采用方差齐性分析,采用非配对t检验。结果:在实验过程中无动物死亡,全部进入结果分析。①大鼠心肌组织超氧化物歧化酶活性:再灌注组较对照组明显下降[(7301±350),(3400±600)nkat/g,P<0.05];黄芩苷60,120,240μmol/L再灌注组较再灌注组有不同程度增加且随浓度增加作用增强[(5251±717),(5917±172),(6818±633),(3400±600)nkat/g,P<0.05]。②丙二醛含量:再灌注组较正常组增加[(85±17),(275±20)nmol/g,P<0.05],黄芩苷60,120,240μmol/L再灌注组较再灌注组有不同程度下降且呈剂量依赖性[(189±32),(126±27),(97±19),(275±20)nmol/g,P<0.05]。③冠脉流出液中肌酸激酶活性活性变化:再灌注组较正常组增加[(80.28±17.12),(105.43±3.95)mkat/L,P<0.05],黄芩苷60,120,240μmol/L再灌注组较再灌注组降低且有剂量依赖性[(90.86±9.13),(86.90±5.68),(84.40±19.15),(105.43±3.95)mkat/L,P<0.05]。④冠脉流出液中乳酸脱氢酶活性:再灌注组较正常组增加[(47.24±4.56),(68.59±11.74)mkat/L,P<0.05],黄芩苷60,120,240μmol/L再灌注组较再灌注组降低且有剂量依赖性[(50.45±5.37),(49.94±4.86),(47.57±4.58),(68.59±11.74)mkat/L,P<0.05]。⑤大鼠心肌细胞凋亡率:再灌注组较对照组凋亡率明显增加(10.58%,23.73%,P<0.05),黄芩苷60,120,240μmol/L再灌注组比再灌注组降低且呈剂量依赖性(9.35%,15.69%,10.41%,23.73%,P<0.05)。结论:黄芩苷可改善心肌缺血再灌注损伤所致乳酸脱氢酶、肌酸激酶释放量增加及心肌超氧化物歧化酶生成量的降低,降低乳酸脱氢酶、肌酸激酶、丙二醛释放量,增高超氧化物歧化酶活性;减少心肌细胞凋亡率。黄芩苷对离体心脏缺血再灌注所致损伤及凋亡有保护作用,并呈现剂量依赖性。     

不同剂量氯胺酮对大鼠离体缺血再灌注损伤心肌8-异前列腺素水平的影响

目的比较不同剂量氯胺酮对大鼠离体缺血再灌注损伤心肌8-异前列腺素的影响。方法成年Wistar大鼠24只,随机均分为心肌缺血再灌注(IR)组、5μmol/L氯胺酮(KL)组、10μmol/L氯胺酮(KM)组和50μmol/L氯胺酮(KH)组。四组首先采用Langendorff逆灌装置建立离体心脏缺血再灌注模型,以K-H液平衡灌注10 min后,再分别应用不含氯胺酮、含5μmol/L、10μmol/L和50μmol/L氯胺酮的K-H液灌注10 min,之后全心停灌25 min,复灌30 min。测定冠状动脉流出液中总乳酸脱氢酶(LDH)、心尖部心肌组织8-异前列腺素和超氧化物歧化酶(SOD)含量,并取左心室心肌组织观察超微结构变化。结果与IR组比较,KL和KM组8-异前列腺素、SOD及LDH含量均无统计学差异(P>0.05),超微结构变化也未见损伤减轻;KH组8-异前列腺素和LDH含量明显升高(P<0.05),超微结构损伤加重。结论 5μmol/L和10μmol/L氯胺酮对大鼠离体心肌缺血再灌注损伤和8-异前列腺素含量无明显影响,而50μmol/L氯胺酮则增加8-异前列腺素含量、加重心肌损伤。     

异丙酚激活蛋白激酶C对心肌缺血再灌注损伤的保护作用

目的:观察异丙酚对大鼠离体心肌缺血再灌注的影响及蛋白激酶C激活的作用。方法:实验于2004-04/2005-03在河北省医学科学院药研室完成。48只大鼠离体心脏随机分为6组,每组8只。分别为:正常对照组,持续灌注Lock液65min;缺血再灌注模型组,用含脂肪乳对照的灌流液灌注15min后,以钳夹主动脉灌注管造成全心常温缺血25min后,恢复再灌注30min,灌流液与预灌时相同;异丙酚15,30,60μmol/L组,缺血前和再灌期的灌流液中分别含相应浓度的异丙酚,余同缺血再灌注模型组;异丙酚60μmol/L 蛋白激酶抑制剂5μmol/L组,灌流液中含5μmol/L的chelerythrine的灌流液,其余同异丙酚60μmol/L组。实验评估:①Powerlab/8s仪记录各组平衡末、缺血前及再灌30min时心率、左室发展压、左室舒张末压、左室压力变化速率、冠状动脉流量等心功能指标。②测定冠状动脉流出液中乳酸脱氢酶、磷酸肌酸激酶活性。③透射电镜观察心肌细胞超微结构变化。④差速离心提取心肌线粒体,测定线粒体超氧化物岐化酶、谷胱甘肽过氧化物酶、ATP酶活性和丙二醛含量。结果:48只大鼠均进入结果分析。①平衡灌注末、缺血前各组间心功能指标差异无显著性(P>0.05),再灌注30min末,异丙酚30,60μmol/L组左室发展压、左室压力变化速率、冠状动脉流量明显高于缺血再灌注模型组(P<0.05),左室舒张末压明显低于缺血再灌注模型组(P<0.05)。异丙酚60μmol/L 蛋白激酶C抑制剂5μmol/L组左室发展压、左室压力变化速率、冠状动脉流量明显低于单纯异丙酚60μmol/L组(P<0.05),但仍高于与缺血再灌注模型组(P<0.05)。②心肌缺血再灌后,冠状动脉流出液中乳酸脱氢酶和肌酸激酶活性明显高于正常对照组(P<0.05)。异丙酚30,60μmol/L组、异丙酚60μmol/L 蛋白激酶C抑制剂5μmol/L组乳酸脱氢酶、肌酸激酶活性明显低于缺血再灌注模型组(P<0.05),异丙酚60μmol/L 蛋白激酶C抑制剂5μmol/L组乳酸脱氢酶和肌酸激酶活性与异丙酚60μmol/L组无明显差异。③与缺血再灌注模型组相比,异丙酚组心肌损伤明显减轻,尤其是60μmol/L组,心肌纤维排列均匀,线粒体膜结构完整,仅轻度水肿,嵴清晰,糖原可见。异丙酚60μmol/L 蛋白激酶C抑制剂5μmol/L组心肌超微结构显示损伤程度重于异丙酚60μmol/L组。④异丙酚30,60μmol/L、异丙酚60μmol/L 蛋白激酶C抑制剂5μmol/L组丙二醛含量低于缺血再灌注模型组(P<0.05),ATP酶、超氧化物歧化酶、谷胱甘肽过氧化物酶活性明显高于缺血再灌注模型组(P<0.05)。异丙酚60μmol/L组与异丙酚60μmol/L 蛋白激酶C抑制剂5μmol/L组相比,上述指标无明显差异。结论:异丙酚对离体大鼠心肌缺血再灌注损伤有保护作用,可能与其抗脂质过氧化和激活蛋白激酶C有关。     

大鼠局灶性脑缺血再灌注后脑组织的氧化损伤

目的:通过观察大鼠局灶性脑缺血再灌注后脑组织超氧化物歧化酶活性和丙二醛含量的变化,进一步探讨大鼠局灶性脑缺血再灌注后氧化损伤的病理生理改变。方法:实验于2004-09/2005-01在泸州医学院神经生物学教研室进行。将雄性成年Wistar大鼠60只随机分为2组:假手术组30只和缺血再灌注组30只。每组分3个观察时间点,分别为手术后6,12,24h,每个时间点10只。采用线栓法制成大脑中动脉闭塞模型,假手术组手术过程同缺血再灌注组,但未插栓线,不造成脑缺血。测定手术后不同时点脑组织超氧化物歧化酶活性和丙二醛水平。结果:在实验过程中有6只大鼠死亡,缺血再灌注组有8只大鼠手术后模型评价为0级,被排除实验,随机补充14只大鼠,最终进入结果分析仍为60只大鼠。①缺血再灌注组术后6,12,24h超氧化物歧化酶活性均低于假手术组眼(289.72±10.67),(534.77±22.67)μkat/L;(330.57±18.17),(539.11±11.50)μkat/L;(377.58±14.67),(550.78±11.50)μkat/L,P<0.05演。②缺血再灌注组术后6,12,24h丙二醛水平显著高于假手术组眼(15.06±0.59),(6.78±0.25)μmol/L;(13.53±1.11),(6.78±0.26)μmol/L;(11.31±0.97),(6.80±0.26)μmol/L,P<0.05演。结论:脑缺血再灌注后,缺血大鼠脑组织内丙二醛水平升高,超氧化物歧化酶活性降低,说明自由基参与了脑缺血再灌注损伤的病理生理过程。     

骨骼肌缺血再灌注损伤中血管功能障碍与丹参的干预效应

目的:观察骨骼肌缺血再灌注损伤血清一氧化氮、诱导型一氧化氮合酶、内皮素1含量的变化及中药丹参的干预作用。方法:实验于2005-01/12在福建中医学院骨伤系实验室完成。实验分组:选用雄性SD大鼠84只,按随机数字表法分为丹参组、生理盐水组,每组42只,每组又分缺血再灌注10,20,30,40,50,60,90min7个时间点,每个时间点6只。实验方法:①制备大鼠左侧提睾肌缺血再灌注损伤模型。②丹参组于缺血2h时腹腔注射丹参注射液,生理盐水组给予相应剂量生理盐水;分别于缺血再灌注10,20,30,40,50,60,90min抽取腹主动脉血。实验评估:①采用硝酸还原酶法测定血清一氧化氮浓度。②采用比色法测定诱导型一氧化氮合酶活性。③采用放射免疫法测定内皮素1含量。结果:纳入大鼠84只,均进入结果分析。①一氧化氮、诱导型一氧化氮合酶的表达量:缺血再灌注10,20min两组一氧化氮、诱导型一氧化氮合酶的表达量无明显差异(P>0.05);缺血再灌注30,40,50,60,90min丹参组一氧化氮和诱导型一氧化氮合酶的表达量均高于生理盐水组[一氧化氮分别为(70.95±2.10),(68.21±2.23)μmol/L;(77.05±2.28),(72.20±1.56)μmol/L;(81.12±2.74),(74.60±1.90)μmol/L;(68.81±2.32),(62.03±2.80)μmol/L;(57.08±3.02),(46.77±3.01)μmol/L;诱导型一氧化氮合酶分别为(515.17±47.54),(459.78±37.27)μkat/L;(629.46±44.19),(499.37±29.46)μkat/L;(673.73±29.96),(584.77±58.48)μkat/L;(590.62±31.96),(507.78±31.82)μkat/L;(485.33±38.27),(378.64±38.04)μkat/L],差异有非常显著性意义(t=2.238,4.332,4.783,4.569,5.922;2.246,6.000,3.317,4.499,4.843,P<0.05,0.01)。缺血再灌注50min两组一氧化氮、诱导型一氧化氮合酶的表达量均达到最高峰,此后表达量开始下降。②内皮素1的表达量:缺血再灌注40min两组内皮素1的表达量均达到最高峰,此后表达量开始下降;缺血再灌注10min两组间内皮素1的表达量无明显差异(P>0.05),缺血再灌注20,30,40,50,60,90min丹参组内皮素1的表达量低于生理盐水组[分别为(145.77±26.54),(237.76±14.41)ng/L;(197.32±21.80),(258.50±40.20)ng/L;(124.44±6.00),(189.58±7.24)ng/L;(115.88±10.84),(165.93±10.43)ng/L;(103.96±3.84),(158.05±10.62)ng/L;(84.42±6.16),(113.69±10.41)ng/L],差异有非常显著性意义(t=7.462,3.278,16.959,8.149,11.731,5.926,P<0.01)。结论:骨骼肌缺血再灌注损伤导致血清中一氧化氮、诱导型一氧化氮合酶、内皮素1的表达量发生改变,丹参可能通过调整一氧化氮和内皮素1的平衡,改善缺血再灌注损伤造成的血管内皮功能障碍。     

氯氮平对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:观察氯氮平对局灶性脑缺血再灌注损伤的保护作用,并与尼莫地平进行阳性对照。方法:实验于1999年在郑州大学医学院药理教研室实验室进行,取Wistar大鼠240只,单纯随机分成缺血再灌注组,氯氮平24,12,6mg/kg组,尼莫地平组和假手术组6组,每组40只。氯氮平24,12,6mg/kg组腹腔注射相应剂量的氯氮平,尼莫地平组腹腔注射0.2mg/kg尼莫地平,缺血再灌注组和假手术组腹腔注射等体积生理盐水,均为1次/d,连续7d。给药7d后除假手术组外其他5组大鼠栓塞法建立大脑中动脉局灶性缺血再灌注模型。测定再灌注2h缺血侧脑组织含水量、丙二醛含量和超氧化物歧化酶活性;流式细胞仪定量分析细胞凋亡率;Fura-2负载,以荧光分光光度计测定细胞内游离钙的变化。结果:经补充后240只大鼠进入结果分析。①脑组织含水量:缺血再灌注组高于假手术组([81.62±0.15)%,(75.81±0.23)%,P<0.01],其他4组均低于缺血再灌注组(P<0.01)。②丙二醛含量:缺血再灌注组高于假手术组([10.85±0.38),(4.07±0.63)μmol/g,P<0.01],其他4组均低于缺血再灌注组(P<0.01)。③超氧化物歧化酶活性:缺血再灌注组低于假手术组([82.47±10.73),(280.15±10.32)Nu/mg,P<0.01],其他4组均高于缺血再灌注组(P<0.01)。④细胞内游离钙浓度:缺血再灌注组高于假手术组([574.87±14.56),(215.76±10.84)nmol/L,P<0.01],其他4组均低于缺血再灌注组(P<0.01)。⑤细胞凋亡率:假手术组为0,缺血再灌注组为28%,氯氮平6,12,24mg/kg组及尼莫地平组分别为19%,12%,5%,13%。结论:①6～24mg/kg氯氮平可显著降低细胞内游离钙含量,抑制缺血再灌注诱导的神经细胞凋亡,提示氯氮平对缺血再灌注所致神经细胞损伤的保护作用可能与其钙拮抗以及抗脂质过氧化有关。②氯氮平的神经保护作用与尼莫地平相似。     

大鼠实验性肠缺血再灌注损伤过程中组织因子表达的研究

目的 :研究大鼠肠缺血再灌注损伤过程中组织因子(tissue factor,TF)和相关调控分子及凝血系统的变化。方法:将40只雄性Wistar大鼠随机等分为假手术(Sham)组和肠缺血再灌注损伤(ischemia reperfusion injury,I/R)组。夹闭I/R组大鼠的肠系膜上动脉60 min后恢复血供,再灌注15 min;Sham组大鼠只开腹暴露肠系膜上动脉,不予夹闭,持续75 min。分别于缺血前、缺血60 min和再灌注15 min取血液标本,于再灌注结束后取缺血再灌注区的肠黏膜组织进行检测。结果:I/R组大鼠肠缺血60 min和再灌注15 min时,血液中TF、凝血酶-抗凝血酶复合物(thrombin-antithrombin complex,TAT)和丙二醛(malonal dehyde,MDA)含量分别为(33.45±6.12)ng/L和(46.12±8.16)ng/L、(9.64±2.19)μg/L和(20.11±6.29)μg/L、(23.65±4.25)μmol/L和(37.69±6.43)μmol/L,均明显高于Sham组(P均<0.01)。结论:大鼠肠缺血再灌注损伤以及由此诱发的过氧化损伤引起TF合成与释放增多,进而激活凝血系统,导致TAT水平增高。NF-κB和EGR-1作为TF的2个重要调控因子在大鼠肠缺血再灌注损伤过程中上调了TF的基因转录和表达。     

激光共聚焦显微镜观察前胡丙素对新生大鼠缺氧再灌注心肌细胞内游离钙的影响

目的激光共聚焦显微镜观察缺氧再灌注损伤对心肌细胞内Ca2+浓度([Ca2+]i)的影响,以及前胡丙素对模拟心肌缺氧再灌注过程中减轻钙超载的作用。方法采用SD大鼠乳鼠进行心肌细胞培养,建立模拟缺氧再灌注模型。实验分4组:正常组:细胞正常培养;缺氧预适应组:预先缺氧2 h,复氧1 h,再缺氧12 h后,复氧2 h;前胡丙素预处理组:先予前胡丙素单体终浓度为100μmol/L作用1 h,再行缺氧12 h,后复氧2 h;模拟缺氧再灌注组缺氧12 h,后复氧2 h。细胞上清检测LDH值。心肌细胞以Fluo-3/AM荧光指示剂负载,应用激光共聚焦显微镜技术检测心肌细胞([Ca2+]i)变化。结果模拟缺氧再灌注组心肌细胞([Ca2+]i)荧光强度值显著高于前胡丙素预处理组及缺氧预适应组(P<0.01),前胡丙素预处理组与缺氧预适应组细胞内荧光强度无明显组间差异。结论心肌细胞缺氧再灌注损伤导致Ca2+超载,而前胡丙素有明显减轻心肌细胞模拟缺氧再灌注时Ca2+超载的作用。应用激光扫描共聚焦显微镜技术可以直观地进行细胞内钙离子研究。     

促红细胞生成素预处理对肢体缺血再灌注损伤的影响

目的:观察促红细胞生成素预处理对骨骼肌缺血再灌注损伤的影响。方法:实验于2004-08/2005-01在解放军第二五二医院中心实验室完成。①采用大鼠右后肢缺血再灌注模型,将30只实验大鼠随机分为3组,每组10只。假手术组:仅显露股血管鞘,不做缺血再灌注,经腹腔注射同体积生理盐水;对照组:经腹腔注射同体积生理盐水,持续缺血4h,再灌注1h。促红细胞生成素预处理组(预处理组):经腹腔注射5000u/kg人重组促红细胞生成素,24h后持续缺血4h,再灌注1h。②测定各组血清磷酸激酶,乳酸脱氢酶丙二醛和腓肠肌99Tcm亚甲基二磷酸钠吸收量变化。③电镜观察腓肠肌超微结构变化。结果:30只大鼠全部进入结果分析。①对照组和预处理组血清磷酸激酶[(121.627±18.112),(84.417±14.594)μkat/L]、乳酸脱氢酶[(20.065±4.676),(13.354±5.229)μkat/L]明显高于假手术组[(50.247±16.066),(7.732±1.256)μkat/L](P<0.05);对照组和预处理组丙二醛[(10.36±2.65),(6.55.±2.19)nmol/L]及99Tcm亚甲基二磷酸钠吸收量[(16.69±3.14),(11.66±3.87)%/(g·min)]均明显高于假手术组[(3.54±1.89)nmol/L,(9.12±1.96)%/(g·min)(P<0.05)]。②预处理组各指标与对照组相比显著降低(P<0.05)。结论:促红细胞生成素对肢体骨骼肌缺血再灌注损伤具有保护作用。     

瑞舒伐他汀对兔心肌缺血-再灌注损伤内皮功能的影响

目的 研究兔心肌缺血-再灌注损伤中内皮功能改变及瑞舒伐他汀对其影响.方法 将16只新西兰大白兔随机分为两组:心肌缺血-再灌注损伤组(对照组),瑞舒伐他汀干预组(药物组).建立心肌缺血-再灌注模型,心电图显示ST段明显弓背向上抬高(≥0.2 mV)表示结扎左前降支成功;40 min后剪断结扎线,心电图ST段回落1/2以上,标志再灌注成功.分别在缺血前,缺血40 min,再灌注60 min和再灌注180 min四个时相点留取血标本,检测一氧化氮(nitric oxide,NO)、内皮素-1(endothe-lin-1,ET-1)含量.运用SPSS 11.5软件,采用重复测量资料的方差分析,以P<0.05为差异具有统计学意义.结果 对照组与药物组血清NO含量随缺血及再灌注时间的延长而降低,血浆ET-1含量随缺血及再灌注时间的延长而增高;缺血前对照组与药物组血清N0含量[(109.875±32.255)μmol/Lvs.(114.500±37.405)μmol/L,P>0.05]及血浆ET-1含量[(221.111±28.125)pg/mL vs.(204.594±31.790)pg/mL,P>0.05]差异无统计学意义;缺血40 min、再灌注60 min、再灌注180 min时,药物组血清NO含量[分别为(63.125±18.962)、(43.500±16.518)、(29.625±14.162)μmol/L vs.(82.000±13.825)、(63.375±17.541)、(50.250±18.987)μmol/L,P<0.05]均显著高于对照组,药物组血浆ET-1含量[分别为(331.785±35.341)、(375.914±45.204)、(459.829±70.110)pg/mL vs.(282.541±38.928)、(315.152±55.263)、(377.795±60.427)pg/mL,P<0.05]均显著低于对照组.结论 瑞舒伐他汀通过升高血清中NO,降低血浆ET-1,从而改善兔心肌缺血-再灌注损伤时内皮功能.     

Na+/Ca2+交换抑制剂对慢性阻塞性肺疾病患者肺泡巨噬细胞功能的影响

目的观察Na /Ca2 交换抑制剂Benzamil对慢性阻塞性肺疾病(COPD)患者肺泡巨噬细胞(AM)胞浆中游离钙离子(〔Ca2 〕i)浓度及其对生成肿瘤坏死因子-α(TNF-α)、丙二醛(MDA)的影响。方法采用支气管肺泡灌洗、细胞培养和荧光指示剂的方法,测定36例COPD稳定期患者及36例健康体检者AM内〔Ca2 〕i浓度及其生成的TNF-α和MDA含量。结果1COPD组患者AM内〔Ca2 〕i〔(68.26±7.24)nmol/L〕、TNF-α〔(5.74±0.42)ng/L〕和MDA〔(3.77±0.61)μg/L〕水平均较健康对照组〔分别为(60.61±6.26)nmol/L、(2.06±0.20)ng/L、(1.91±0.19)μg/L〕明显增高(P均<0.01);2给予缺氧后,COPD组AM内〔Ca2 〕i浓度〔(168.34±17.58)nmol/L〕、TNF-α〔(9.67±1.01)ng/L〕和MDA〔(11.21±1.01)μg/L〕水平均较刺激前增高(P均<0.01);3先加Benzamil孵育AM再缺氧,可使胞浆内〔Ca2 〕i浓度〔(129.21±14.33)nmol/L〕、TNF-α〔(6.78±0.52)ng/L〕和MDA〔(8.47±0.79)μg/L〕水平均较单纯缺氧时明显减少(P均<0.01)。结论Benzamil可抑制由缺氧引起AM内〔Ca2 〕i浓度增高及其产生的TNF-α、MDA含量;提示通过调节AM激活可抑制TNF-α、MDA的分泌。     

粉防己碱对脂多糖致大鼠胰腺腺泡细胞损伤的保护作用及其机制研究

目的探讨粉防己碱（Tet）对脂多糖（LPS）诱发大鼠胰腺腺泡细胞损伤的保护作用及其机制。方法胶原酶法分离雄性SD大鼠胰腺腺泡细胞，预先经Tet（50μmol／L、100μmol／L）处理15min后，再经LPS（10mg／L）或正常培养液处理，在0、1、4和10h采集上清液，检测其中丙二醛（MDA）古量及超氧化物歧化酶（SOD）、磷脂酶A2（PLA2）活性；采用四甲基偶氮唑蓝比色法（MTT）检测胰腺腺泡细胞存活情况；部分胰腺腺泡细胞经Fluo-3／AM荧光探针负载后，于相应时间点采用灌流方式给予Tet或LPS，激光共聚焦显微镜观察单个胰腺腺泡细胞内钙离子浓度（[Ca^2＋]i）。结果Tet可减轻LPS所致的细胞损伤（P均〈0.05），抑制LPS诱发的胰腺腺泡细胞[Ca^2＋]i升高（P均〈0．05），降低细胞培养上清液中MDA含量和PLA2活性、增加SOD活性。结论Tet可能通过抑制钙超载、增强抗氧化能力以及减少胰酶活化，减少LPS所致胰腺腺泡细胞损伤，从而发挥对胰腺腺泡细胞的保护作用。     

Allantoin as a marker of oxidative stress in human erythrocytes

Abstract Background: Uric acid is the final product of purine metabolism in humans. It was determined that this compound has important antioxidative properties and it may be oxidized to allantoin by various reactive oxygen species. Therefore, the measurement of allantoin may be useful for the determination of oxidative stress in humans. Methods: We measured allantoin and uric acid in human plasma and erythrocytes obtained from patients with chronic renal failure before hemodialysis (n=30) and blood donors (n=30). We used a method based on selective isolation of allantoin from deproteinized plasma and erythrocyte lysate samples on AG 1-X8 resin and its derivatization to glyoxylate-2, 4-dinitrophenylhydrazone. Separation of glyoxylate-2, 4-dinitrophenylhydrazone from interfering substances was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. Uric acid was determined by reversed phase HPLC with UV/VIS detection at 292 nm. Results: We found significant differences in allantoin and uric acid concentration between the patients with chronic renal failure and the control group both in plasma (20.5+/-6.5 mumol/L and 323.9+/-62.9 mumol/L vs. 2.1+/-1.1 mumol/L and 270.1+/-62.3 mumol/L, p<0.05) and erythrocytes [82.8+/-39.1 nmol/g hemoglobin (Hb) and 110.7+/-28.8 nmol/g Hb vs. 20.1+/-6.1 nmol/g Hb and 82.1+/-23.7 nmol/g Hb, p<0.05]. Conclusions: Significant higher levels of allantoin in both plasma and erythrocytes of patients with chronic renal failure indicate that allantoin may be used as a good marker of oxidative stress. Clin Chem Lab Med 2008;46:1270-4.     

Effects of lysophosphatidylcholine on cultured heart cells: correlation of rate of uptake and extent of accumulation with cell injury

In this study we evaluated the effects of lysophosphatidylcholine, a possible mediator of ischemic damage, on cultured neonatal rat heart cells. The rate and duration of lysophosphatidylcholine accumulation was correlated with Ca++ uptake and cell injury. The rate of carbon 14-labeled lysophosphatidylcholine accumulation during superfusion of the cells by 10 to 100 mumol/L 14C-labeled lysophosphatidylcholine was proportional to the concentration of lysophosphatidylcholine in the perfusate. Rapid accumulation of lysophosphatidylcholine (0.235 nmol/mg protein per minute), which occurred during 10 minutes of exposure to 100 mumol/L lysophosphatidylcholine, resulted in Ca++ overload and cell lysis. In contrast, slow accumulation of lysophosphatidylcholine by myocytes, which occurred during prolonged (1 hour) exposure to a sublethal micellar concentration (80 mumol/L) or very prolonged exposure (6 hours) to a submicellar concentration of lysophosphatidylcholine (10 mumol/L) did not result in Ca++ overload or irreversible injury despite more total lysophosphatidylcholine accumulation than during a single 10-minute exposure to 100 mumol/L lysophosphatidylcholine (p less than 0.005). Repeated brief exposures (5 minutes) to 100 mumol/L lysophosphatidylcholine separated by 20-minute recovery intervals also resulted in more lysophosphatidylcholine accumulation than during the lethal 10-minute exposure to 100 mumol/L lysophosphatidylcholine but did not result in irreversible injury. We therefore conclude that cardiac myocytes can tolerate slow accumulation of lysophosphatidylcholine and that factors other than the quantity of lysophosphatidylcholine accumulating in cells are determinants of the degree of injury sustained from exposure to lysophosphatides.     

细胞内钙与K562/A02细胞多药耐药相关性的研究

本研究旨在探讨白血病耐药细胞的发生及其逆转机制与细胞内钙离子浓度的关系。用甲基四唑蓝法(MTT)测定柔红霉素 (daunomycin ,DNR)的细胞毒性 ,用Fura 2 /AM方法测定耐药细胞株K5 6 2 /A0 2及其敏感株K5 6 2的静息 [Ca2 ]i水平 ,并观察了柔红霉素及耐药调节剂汉防己甲素 (Tetrandrine,Tet)、屈洛昔芬 (droloxifene,DRL)单独或联合应用后细胞内游离钙离子浓度的变化。结果表明 :1μmol/LTet,5 μmol/LDRL均能增加DNR对耐药细胞系K5 6 2 /A0 2的细胞毒作用 ,IC50 (半数抑制量 )分别为 ( 7.2 8± 2 .0 6 ) μg/ml,( 7.5 8± 3.4 4 ) μg/ml,逆转倍数为 2 .94和 2 .82倍。两药联合作用明显增强 ,IC50 为 ( 1.6 6± 0 .4 1) μg/ml,逆转倍数达 12 .9倍。静息状态下K5 6 2 /A0 2细胞的游离钙离子浓度显著高于K5 6 2细胞。 1μmol/LTet,5 μmol/LDRL单独作用于K5 6 2 /A0 2细胞引起 [Ca2 ]i的明显升高 ,两者联合应用有拮抗作用。结论 :K5 6 2 /A0 2细胞内Ca2 浓度的增高可能是导致其耐药的原因之一 ,但耐药调节剂Tet,DRL对耐药细胞 [Ca2 ]i的影响在其逆转耐药中的作用有待进一步研究     

Glutathione, glutathione S-transferase, and transmembrane transport of glutathione conjugate in human neutrophil leukocytes

Glutathione (GSH) conjugation is important in the detoxification of carcinogens and other exogenous electrophilic drugs and chemicals. The conjugation is catalyzed by GSH S-transferase. Neutrophil GSH content was 12.3 +/- 2.5 (mean +/- SD) nmol/10(7) cells, and transferase activity of cytosol preparations was 0.187 +/- 0.035 nmol/min/mg protein with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Maximal activity was found at a pH of 7.0 and a temperature of 40 degrees C. Apparent Km of transferase was 1.25 +/- 0.18 mmol/L CDNB, and apparent Vmax was 0.621 +/- 0.22 nmol/min/mg. GSH-CDNB conjugate was quantitated by HPLC in cells and in medium after CDNB exposure. Transport of conjugate from cells to medium increased with CDNB concentration to 50 mumol/L, and kinetic data showed two saturable transport mechanisms with apparent Km of 5.90 mumol/L and 0.265 mumol/L, respectively. Cellular GSH content fell rapidly with CDNB concentration greater than 2.5 mumol/L, and was depleted in a 10-minute incubation at a concentration of greater than 50 mumol/L CDNB. Neutrophils have a significant content of GSH and a significant amount of transferase activity, and transport of GSH conjugate involves two distinct saturable pathways. GSH depletion can be accomplished with a relatively low concentration of an exogenous chemical and could impair the ability of the cells to carry out their phagocytic functions.     

Red blood cell calcium level in chronic renal failure: effect of continuous ambulatory peritoneal dialysis

Red blood cell (RBC) calcium, calcium 45 influx, and calcium extrusion as indicated by Ca-stimulated, Mg-dependent adenosine triphosphatase (CaATPase) was determined in patients with chronic renal failure (CRF), patients with CRF receiving continuous ambulatory peritoneal dialysis (CAPD) treatment, and controls. Cell calcium, which in the controls was 5.5 mumol/L of cells, was elevated in patients with CRF--30.6 +/- 6.8 mumol/L of cells (p less than 0.002)--and in patients receiving CAPD-23.6 +/- 6.7 mumol/L of cells (p less than 0.02). Basal CaATPase activity in controls was 850.7 +/- 66.7 nmol inorganic phosphate per milligram of protein per hour. It was suppressed in patients with CRF and patients receiving CAPD: 504.9 +/- 34.4 nmol inorganic phosphate per milligram of protein per hour and 618.2 +/- 47.3 nmol inorganic phosphate per milligram of protein per hour, respectively (p less than 0.01). Calmodulin-stimulated CaATPase revealed a pattern similar to that of CaATPase basal activity. RBC calcium showed an inverse correlation with CaATPase activity (r = -0.935, p less than 0.005) in patients with CRF. Calcium influx was increased in patients with CRF and in patients receiving CAPD: 12.00 +/- 1.34 mumol/L of cells per hour and 13.60 +/- 1.70, respectively, compared with 4.61 +/- 0.39 mumol/L of cells per hour in controls (p less than 0.001). Patients with CRF have elevated RBC calcium levels mainly related to decreased extrusion and to increased influx. CAPD fails to improve substantially these abnormalities. Plasma vanadium levels were markedly elevated in patients undergoing hemodialysis and marginally in patients receiving CAPD.(ABSTRACT TRUNCATED AT 250 WORDS)     

酪氨酸激酶和磷酸酶及蛋白激酶C在As2O3调控NB4细胞和皮层神经元凋亡中的作用

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶及蛋白激酶C(PKC)在三氧化二砷 (As2O3 )调控白血病细胞和人脑皮层神经元凋亡中的作用 ,观察As2 O3 对人脑皮层神经元和白血病细胞的胞浆游离钙 ([Ca2 ]i)浓度的影响。方法　用Fluo 3/AM荧光探针标记人白血病细胞和人脑皮层神经元[Ca2 ]i,激光共聚焦显微镜实时测定不同浓度As2 O3 干预后 [Ca2 ]i的变化并观察蛋白酪氨酸激酶和磷酸酶抑制剂对 [Ca2 ]i变化的影响。磷基转移法测定细胞膜和胞浆的PKC活性。琼脂糖凝胶电泳法观察细胞DNA的片段化。结果　 1μmol /L的As2 O3 使NB4细胞的 [Ca2 ]i明显增高 ,而对人脑皮层神经元 [Ca2 ]i影响不明显。 2 μmol /L以上的As2 O3 引起两种细胞 [Ca2 ]i增高 ,此作用被磷酸酶抑制剂钒酸盐呈浓度依赖性促进 ,被蛋白酪氨酸激酶抑制剂金雀异黄素呈浓度依赖性抑制 ,2 ,5 ,10μmol/L钒酸盐作用 2 4 0s时NB4细胞的 [Ca2 ]i总增加率分别为 (6 .5± 2 .3) % ,(2 1.7± 2 .1) %和 (4 9.2± 2 .5 ) % ;人脑皮层神经元为 (6 .7± 2 .1) % ,(19.4± 2 .5 ) %和 (5 2 .3± 2 .7) % ;2 ,5 ,10 μmol/L金雀异黄素作用 2 4 0s时NB4细胞的总抑制率分别为 (6 .7± 2 .9) % ,(2 5 .6± 2 .5 ) %和 (5 2 .2± 3.5 ) % ;人脑皮层     

异丙酚对急性呼吸窘迫综合征时肾Gq/11蛋白的干预作用

目的探讨异丙酚是否通过影响Gq/11蛋白的表达对肾脏起保护作用。方法采用尾静脉注射油酸(OA)法复制急性呼吸窘迫综合征(ARDS)大鼠模型。将健康雄性Wistar大鼠随机分为对照组(C组)、OA组和异丙酚预处理组(P组),并观察血压及相关酶的变化。用蛋白质免疫印迹法检测肾脏中Gq/11蛋白含量。结果P组Gq/11蛋白含量(124.68±19.38)%较C组(100.00±0)%增加了(24.68±19.38)%(P<0.01),但明显低于OA组(149.34±20.04)%(P<0.01)。P组肾组织血管紧张素转换酶(16.52±1.37)μmol·min-·g-1、乳酸脱氢酶(1.20±0.16)kU/g活性和丙二醛(1.51±0.35)μmol/g含量也均明显低于OA组分别为(17.56±1.02)μmol·min-1·g-1,(1.41±0.16)kU/g,(1.94±0.16)μmol/g,P均<0.01。结论预先注射异丙酚能降低Gq/11蛋白在肾脏中的含量,并减轻ARDS时肾损害的程度。     

Characterization of riluzole-induced stimulation of large-conductance calcium-activated potassium channels in rat pituitary GH3 cells.

BACKGROUND: Riluzole is known to be an inhibitor of glutamatergic neurotransmission. Transmitter release from nerve terminals can be regulated by the activity of large-conductance Ca(2+)-activated K+(BKCa) channels. METHODS: The ionic mechanism of actions of riluzole was investigated in neuroendocrine (GH3 and PC12 cells), using the whole-cell patch-clamp and inside-out excised patch configurations. RESULTS: In GH3 cells, riluzole at 0.3-100 mumol/L increased the amplitude of Ca(2+)-activated K+ current (IK(Ca)) in a concentration-dependent manner with a half maximal concentration of 5 mumol/L. The riluzole-induced increase in outward current was not be suppressed by glibenclamide (10 mumol/L) or apamin (200 nmol/L). However, iberiotoxin (200 nmol/L) or tetrandrine (10 mumol/L) can effectively suppress riluzole-induced IK(Ca). Under inside-out patch recording mode, riluzole (10 mumol/L) applied intracellularly can increase the opening probability of large-conductance Ca(2+)-activated K+(BKCa) channels, but did not affect their single-channel conductance. The riluzole-induced change in the kinetic behavior of BKCa channels is due to an increase in mean open time and a decrease in mean closed time. Riluzole caused a left shift in the midpoint for voltage-dependent opening. Riluzole-stimulated activity of BKCa is independent on internal Ca2+. Riluzole (30 mumol/L) did not affect the amplitude of voltage-dependent K+ current, but it produced a slight reduction of L-type voltage-dependent Ca2+ current. Under current clamp mode, riluzole (10 mumol/L) decreased the firing rate of action potentials induced by thyrotropin releasing hormone (10 mumol/L) in GH3 cells. In rat pheochromocytoma PC12 cells, riluzole also increased the activity of BKCa channels without altering their channel conductance. CONCLUSION: This study shows that riluzole can stimulate the activity of BKCa channel in neuroendocrine cells.