对氯苄基四氯小檗碱（CPU—86017）及L—甲状腺素对大鼠主动脉环α1A、α1B亚型的选择性

目的　研究CPU 86 0 17及L 甲状腺素对血管平滑肌收缩的影响及对α1A、α1B亚型的选择性。方法　以有钙、无钙液中由累积浓度去甲肾上腺素 (norepinephrine ,NE)引起的大鼠胸主动脉环的收缩 ,比较CPU 86 0 17(30 μmol·L-1)对正常及慢性甲状腺给药组 (Thy)大鼠血管平滑肌 (VSM)的α1受体的抑制作用。结果　在无钙液中由α1B引起的内钙释放所致VSM收缩占有钙液中VSM最大收缩的百分率 ,正常组和Thy组分别为 4 8 8%± 8 1%和 6 9.4 %± 9 7% (P <0 .0 1) ,复钙后由α1A引起的外钙内流所致VSM收缩百分率 ,分别为正常组 4 7 7%± 5 0 % ,Thy组为 2 2 1%± 9 4 % (P <0 .0 1)。加入CPU 86 0 17(30 μmol·L-1)后 ,对内钙释放所致的收缩 % ,正常对照组为 35 2 %± 10 1% ,Thy组为 35 2 %± 10 2 % ;而对外钙内流引起的收缩百分率则分别为 13 9%± 7 1%及 2 0 7%± 7 5 %。结论　L 甲状腺素使α1B的作用增强 ,而减弱α1A的作用。CPU 86 0 17在正常情况下对VSM的α1A的抑制强于α1B受体亚型     

AngⅡ对人心房肌细胞膜钾通道电流的影响及替米沙坦的拮抗作用

目的: 观察AngⅡ对人心房肌细胞膜钾电流的作用,揭示其参与房性心律失常的电生理机制,为应用AngⅡ受体拮抗剂治疗房性心律失常提供实验基础.方法: 急性分离单个人心房肌细胞,采用全细胞膜片钳方法记录内向整流钾电流(Ik1)、短暂外向钾电流(Ito).实验分4组:对照组,AngⅡ(0.1 μmol/L)组,替米沙坦(0.01 μmol/L)组,AngⅡ 替米沙坦组.结果: 与对照组相比,0.1 μmol/L AngⅡ使人心房肌细胞膜Ito峰值电流密度明显下降(pA/pF, 6.55±0.52 vs 12.65±1.06,P＜0.01), 在-100mV电压下使IK1 峰值电流密度显著升高 (pA/pF, -9.31±1.02 vs -5.23±0.98, P<0.01).0.01 μmol/L替米沙坦对人心房肌细胞膜Ito IK1无明显影响,但可拮抗AngII的作用;AngⅡ 替米沙坦组的Ito峰值电流密度 (pA/pF,11.74±1.28 )和IK1 峰值电流密度(pA/pF, -6.13±1.15) 与AngⅡ组相比有显著差别(P＜0.01).结论: AngⅡ对人心房肌细胞具有明显的电生理学作用,0.1 μmol/L AngⅡ可促进人心房肌细胞膜IK1并抑制Ito,替米沙坦可拮抗AngⅡ对人心房肌细胞膜钾电流的作用.     

急性心脑血管血栓性疾病患者血浆FⅦ：C、FⅧ：C、凝血酶原及纤维蛋白原的变化

目的检测凝血因子Ⅶ、Ⅷ、Ⅱ、Ⅰ的活性,研究其在急性心脑血管血栓性疾病中的变化.方法分别采用凝血活酶时间、部分凝血活酶时间、发色底物法及凝血酶时间法测定了急性心肌梗死(AMI)和急性缺血性脑卒中(AIS)病人血浆FⅦC、FⅧC、凝血酶原及纤维蛋白原相对活性.结果AMI病人血浆FⅦC为98.3%±36.7%,与青年组的104.0%±29.4%和老年组的114.9%±23.6%比较无明显差异(P值均＞0.05),而AIS病人为152.9%±30.3%,较青年组和老年组显著升高(P值均＜0.001).AMI病人血浆FⅧC为234.2%±57.9%,明显高出青年组的101.2%±32.6%和老年组的116.4%±35.7%(P值均＜0.001).但AIS病人FⅧC±为85.5%±32.6%,低于老年组(P＜0.05).AMI和AIS病人血浆凝血酶原活性分别为130.2%±¨.7%和128.0%±29.4%,高于青年组的108.0%±7.7%(P＜0.001和0.01)和老年组的106.1%±19.4%(P值均＜0.001).AMI和AIS病人血浆纤维蛋白原活性分别为207.2%±53.0%和174.3%±28.5%,二者均显著高于正常青年组的103.3%±¨.6%(P值均＜0.001)和老年组的143.8%±16.8%(P＜0.001和0.01).结论AMI病人FⅧC增高,AIS病人FⅧC增高;二者血浆凝血酶原和纤维蛋白原活性均高于正常水平.     

8-甲氧补骨脂素和长波紫外线在细胞光老化中对端粒缩短机制的影响

Objective To investigate the mechanism of telomere shortening through 8-methoxypsoralen(8-MOP)and subsequent ultraviolet A(UVA)irradiation-induced photoaging model in human dermal fibroblasts(HDFs).Methotis Photoaging model was established by 8-MOP+UVA in skin HDFs.Flow cytometer.enzyme eytochemistry,immunofluorescence,Westem blot and Real-time PCR were employed.Results The percentage of G1 blockage of 8-MOP+UVA group were higher than that of control group at 24、48、72 h and 7 d(61.4%±1.5% vs 32.8%±1.5%.69.5%±2.2% vs 44.9% ±2.3%.88.2%±1.6% vs 59.8%±1.4%,90.7%±2.5% vs 68.5%±2.6%.all P＜0.01).The expression of SA-β-Gal of 8-MOP+UVA group were higher than that of control group at 24、48、72 h and 7 d(34.87%±0.59% vs 7.11%±0.78%,59.38%±0.46% vs 10.57%±0.47%.72.46%±0.98% vs 11.67%±0.87%,94.33%±0.13% vs 12.04%±0.12%,all P＜0.01).8-MOP+UVA treatment could significantly aggravate the oxidative DNA damages,the percentage of 8-oxo-dG positive cell of 8-MOP+UVA group(95.78%±0.14%)were significantly higher than that of control group(7.69%±0.09%,P＜0.01),8-MOP group(9.76%±0.11%,P＜0.01)and UVA group(35.29%±0.14%,P＜0.05).8-MOP+UVA treatment could accelerate the telomere shortening.the relative length of telomere of 8-MOP +UVA group were 2.57±0.05 lower than that of control group(6.63±0.12.P＜0.01).The levels of P53,P21WAF-1 and P16INK-4a of 8-MOP+UVA group were higher than that of control group(3.00±0.88 vs 0.54±0.10,2.50±0.51 vs 0.42±0.06,2.21±0.34 vs 0.38±0.05,all P＜0.01).Conclusion 8-MOP+UVA-induced photoaging of HDFs can be mediated though the regulation of telomere and subsequent P53-dependent signaling pathways.     

8-甲氧补骨脂素和长波紫外线在细胞光老化中对端粒缩短机制的影响

Objective To investigate the mechanism of telomere shortening through 8-methoxypsoralen(8-MOP)and subsequent ultraviolet A(UVA)irradiation-induced photoaging model in human dermal fibroblasts(HDFs).Methotis Photoaging model was established by 8-MOP+UVA in skin HDFs.Flow cytometer.enzyme eytochemistry,immunofluorescence,Westem blot and Real-time PCR were employed.Results The percentage of G1 blockage of 8-MOP+UVA group were higher than that of control group at 24、48、72 h and 7 d(61.4%±1.5% vs 32.8%±1.5%.69.5%±2.2% vs 44.9% ±2.3%.88.2%±1.6% vs 59.8%±1.4%,90.7%±2.5% vs 68.5%±2.6%.all P＜0.01).The expression of SA-β-Gal of 8-MOP+UVA group were higher than that of control group at 24、48、72 h and 7 d(34.87%±0.59% vs 7.11%±0.78%,59.38%±0.46% vs 10.57%±0.47%.72.46%±0.98% vs 11.67%±0.87%,94.33%±0.13% vs 12.04%±0.12%,all P＜0.01).8-MOP+UVA treatment could significantly aggravate the oxidative DNA damages,the percentage of 8-oxo-dG positive cell of 8-MOP+UVA group(95.78%±0.14%)were significantly higher than that of control group(7.69%±0.09%,P＜0.01),8-MOP group(9.76%±0.11%,P＜0.01)and UVA group(35.29%±0.14%,P＜0.05).8-MOP+UVA treatment could accelerate the telomere shortening.the relative length of telomere of 8-MOP +UVA group were 2.57±0.05 lower than that of control group(6.63±0.12.P＜0.01).The levels of P53,P21WAF-1 and P16INK-4a of 8-MOP+UVA group were higher than that of control group(3.00±0.88 vs 0.54±0.10,2.50±0.51 vs 0.42±0.06,2.21±0.34 vs 0.38±0.05,all P＜0.01).Conclusion 8-MOP+UVA-induced photoaging of HDFs can be mediated though the regulation of telomere and subsequent P53-dependent signaling pathways.     

牛磺酸对小鼠肠推进及大鼠离体回肠平滑肌运动的影响

目的:观察不同浓度的牛磺酸(Tau)对小鼠在体小肠推进运动及大鼠离体回肠平滑肌收缩运动的影响并初步探讨其作用机制.方法:小鼠灌胃给药后测定小肠中阿拉伯胶活性炭粉混合物的推进距离并计算小肠炭末推进比;采用离体器官实验法,以回肠平滑肌的收缩幅度及收缩频率为指标,观察在含钙及无钙营养液中牛磺酸对大鼠离体回肠平滑肌的影响,同时观察硝苯地平对牛磺酸的拮抗作用.结果:低、中剂量牛磺酸可增强小鼠小肠运动功能(P＜0.05和P＜0.01),而高剂量则抑制小鼠小肠运动功能(P＜0.05).低、中剂量牛磺酸均可使正常及无钙K-H营养液中肠管的收缩幅度增加(P＜0.01),且中剂量组作用更明显(P＜0.05),钙通道阻滞剂硝苯地平可完全阻断其作用(P＜0.01).高剂量组则产生相反的作用,而且蠕动频率下降(P＜0.01).结论:牛磺酸具有增强肠平滑肌收缩的作用并具有剂量依赖性,高剂量牛磺酸则产生相反的作用.     

利多卡因对大鼠离体气管平滑肌的作用及机制

目的:研究利多卡因对气管平滑肌的作用及机制.方法:观察利多卡因对乙酰胆碱(ACh)和高钾预收缩大鼠离体气管的作用,对ACh、高钾和CaCl2诱发气管平滑肌收缩作用剂量反应曲线和ACh致两种收缩组分的影响并与维拉帕米进行比较.结果:利多卡因对ACh和高钾诱发的大鼠离体气管平滑肌标本收缩有舒张作用,并不被L-NAME和普萘洛尔阻断.利多卡因浓度依赖性地使ACh、KCl和CaCl2对大鼠离体气管条标本的量效反应曲线右移,最大反应降低.利多卡因0.69和3.45 mmol/L对ACh所致依赖细胞内钙收缩的抑制率及依赖细胞外钙收缩的抑制率差别均十分显著(P＜0.01).结论:利多卡因对离体大鼠气管平滑肌的舒张作用可能是通过非特异性地抑制细胞外钙经电压调节和受体调节钙通道向内流动及抑制细胞内钙释放来实现的.     

花椒毒酚对大鼠离体子宫平滑肌的作用

目的 :研究花椒毒酚 (XT)对大鼠离体子宫平滑肌的作用及其与Ca2 + 的关系。方法 :采用常规离体子宫平滑肌标本运动的实验方法。结果 :XT和Ver明显抑制缩宫素、高钾去极化后钙离子诱导的大鼠离体子宫平滑肌收缩 ,非竞争性拮抗CaCl2 累积量效曲线 ,PD2 值分别为 4 .59±0 .0 8、6 .4 9± 0 .4 1。和Ver不同的是XT不仅明显抑制缩宫素诱导的依赖细胞内钙离子收缩反应 ,而且明显抑制缩宫素诱导的依赖细胞外钙离子收缩反应 ,结论 :XT通过抑制大鼠子宫平滑肌细胞膜上PDC、ROC及内钙释放而发挥其钙拮抗作用。     

异丙酚对乙酰胆碱所致豚鼠离体气管平滑肌收缩作用的影响

目的:研究异丙酚对乙酰胆碱(Ach)所致豚鼠离体气管平滑肌收缩的作用及作用机制.方法:采用离体气管环模型,以营养液中加入β2受体阻滞剂和营养液中无Ca2 的方法,通过力-位移换能器记录其张力变化,观察异丙酚对豚鼠离体气管平滑肌β2受体和细胞内Ca2 浓度变化的影响.结果:(1)100,300μmol/L异丙酚的舒张作用分别为(81.4±7.6)%和(33.6±6.6)%,与对照组比较,差异十分显著(P＜0.01);(2)100,300μmol/L异丙酚使Ach所致气管平滑肌收缩的量效曲线右移,但最大收缩无明显变化.10 μmol/L普萘洛尔不影响100μmol/L异丙酚抑制乙酰胆碱收缩气管平滑肌量效曲线的作用.(3)100,300μmol/L异丙酚可剂量依赖地显著抑制Ach所致气管平滑肌依内钙性收缩、依外钙性收缩和总收缩,与对照组比较差异十分显著(P＜0.01).结论:异丙酚能抑制乙酰胆碱对离体豚鼠气管平滑肌的收缩作用,且有剂量相关性.异丙酚抑制离体豚鼠气管平滑肌收缩的机制可能为抑制细胞内Ca2 释放和跨膜细胞外Ca2 内流,与β2受体无关.     

A型肉毒毒素对P物质所致大鼠胃体、胃底离体平滑肌收缩的抑制作用

目的 观察A型肉毒毒素(botulinum toxin type A,BTX-A)对P物质(substance P,SP)所致肌条收缩的影响,探讨BTX-A在SP与NK1受体结合过程中可能存在的作用机制.方法 取大鼠胃体、胃底平滑肌制备肌条并随机分为对照组、SP组、SP+ APTL-SP(NK1受体拮抗剂)组、BTX-A组、BTX-A+ SP组、SP+ BTX-A组,采用Biolap420E生物机能实验系统记录肌条收缩数据.结果 SP增加胃体平滑肌自发性收缩张力及振幅、胃底平滑肌自发性收缩张力(P均＜0.01);APTL-SP降低SP引发的胃体、胃底平滑肌收缩张力(P＜0.01); BTX-A作用后的胃体、胃底平滑肌条振幅降低(P均＜0.01).BTX-A降低SP引发的胃体(P＜0.05,P＜0.01)、胃底(P均＜0.01)平滑肌自发性收缩张力及振幅;SP对BTX-A作用后的胃体、胃底平滑肌收缩能力未产生增强作用.结论 SP可增强胃体、胃底平滑肌收缩能力,而BTX-A可抑制SP对胃体、胃底平滑肌的收缩作用.     

Method for human peripheral blood eosinophil isolation for patch-clamp study]

OBJECTIVE: To establish a rapid and economic method for isolating human peripheral blood eosinophils with high viability for patch-clamp studies and investigate the electrophysiological properties of Ca(2+)-activated K(+) channel of the isolated cells. METHODS: Peripheral blood eosinophils were isolated by modified discontinuous Percoll density gradient centrifugation, and the electric currents in the single Ca(2+)-activated K(+) channels of the cells were recorded using patch-clamp technique with cell-attached configuration. RESULTS: The purity of the eosinophils from healthy donors reached (90.5+/-1.6)%, with a viability rate over 99% and recovery rate of (48.2+/-6.9)%. The isolated cells were morphologically intact, from which Ca(2+)-activated K(+) channel activity could be detected. CONCLUSION: The peripheral blood eosinophils isolated using this rapid, simple and highly efficient method are characterized by high purity and viability without obvious cellular injuries, which are ideal for patch-clamp studies.     

TNF-α的心肌负性肌力作用机制研究

目的 :探讨肿瘤坏死因子α(TNF-α)对心肌的负性肌力作用机制。方法 :采用离体大鼠乳头肌张力测定 ,细胞内双波长钙荧光检测和 ATP酶分析方法。结果 :1TNF-α抑制大鼠右心室乳头肌的收缩力 ,2 0 U/ ml和 2 0 0U/ ml TNF-α灌流 10 min后 ,乳头肌收缩力分别减小到对照的 91%和 76 % (P均 <0 .0 1) ;2 TNF-α处理后对心肌细胞钙瞬态变化幅度无明显影响 (0 .97± 0 .0 5 vs0 .95± 0 .0 7,P>0 .0 5 ) ;3TNF- α明显抑制肌浆网 Ca2 + - ATPase活性 ,平均抑制率达到 2 0 % ;4 TNF- α拮抗肌浆网 Ca2 + - ATPase对底物中不同水平的 Ca2 +和 ATP的正反应性 ;5TNF- α对肌膜 Ca2 + - ATP酶和 Na+ / K+ - ATP酶活性无明显抑制作用。结论 :TNF- α抑制心肌收缩力的负性肌力作用机制可能部分与心室肌浆网 Ca2 + - ATP酶活性下降有关 ,而与膜上的 Ca2 + - ATP酶和 Na+ / K+ - ATP酶活性无关。     

匹那地尔对大鼠缺血心肌钙调控的作用

目的 :探讨大鼠缺血心肌再灌注 (I- R)损伤的钙调控机制及匹那地尔对心肌 I- R损伤的保护作用机理。方法 :取 SD大鼠 ,心室肌细胞酶解分离 ,心肌细胞静息 1～ 2 h后随机分为 4组 :对照组、高钾停搏液组、匹那地尔强化组、格列苯脲拮抗组。 4组细胞在 2 4℃下保存 2 h后 ,复氧、复灌 2 0 min同时测定 [Ca2 + ]i 瞬态变化、肌浆网内贮钙释放功能。结果 :经匹那地尔强化处理的心肌细胞在 I- R过程中 [Ca2 + ]i 瞬态变化恢复率明显高于高钾停搏液组 (90 .2 7%～ 95 .5 7% vs6 7.0 5 %～ 80 .11% ,P<0 .0 1)。肌浆网内贮钙释放能力 (173.15 %± 2 6 .0 1% )明显高于高钾停搏液组 (112 .0 0 %± 16 .93% ) (P<0 .0 1)。经匹那地尔强化处理的心肌细胞在咖啡因诱导的钙释放后 ,胞内钙离子浓度从高水平回复到舒张末静息水平的时间为 (3.2 0± 0 .71) ms,显著短于高钾停搏液组 (3.93± 0 .4 6 ) ms(P<0 .0 5 )和对照组 (4 .6 8± 0 .77) ms(P<0 .0 1)。结论 :匹那地尔通过肌浆网和细胞膜 Na+ /Ca2 +交换体功能来调节钙瞬态变化 ,减少细胞内钙超载使心肌细胞在 I- R过程中保持较好的钙调控功能。     

性激素对老龄大鼠颏舌肌细胞肌浆网Ca2+-ATP酶活性及其基因表达的影响

目的:观察雌、雄激素对老龄大鼠颏舌肌细胞肌浆网(SR)Ca2 -ATP酶活性及其基因表达的影响,探索性激素影响上气道稳定性的机制.方法:将24只老龄雄性大鼠随机分为3组:老龄对照组、老龄雌激素组(肌注苯甲酸雌二醇,每次0.1 mg/kg,每周2次,共4周)和老龄雄激素组(肌注丙酸睾酮,每次2.5 mg/kg,每周2次,共4周).采用定磷法检测颏舌肌SR Ca2 -ATP酶活性,荧光定量RT-PCR测定SR Ca2 -ATP酶mRNA表达的变化.结果:与对照组相比,老龄雌激素组颏舌肌SR Ca2 -ATP酶活性[前者34.24 ± 6.14 μmol Pi/(mg protein·hour),后者 38.39 ± 8.49 μmol Pi/(mg protein·hour)]及其mRNA表达水平(前者0.029 7 ± 0.014 0,后者0.031 4 ± 0.017 4 )差异无统计学意义(P＞0.05);老龄雄激素组SR Ca2 -ATP酶活性[22.91 ± 4.56 μmol Pi/(mg protein·hour)]下降到对照组的67%(P＜0.01),其mRNA表达水平(0.017 2 ± 0.008 6)亦明显下降(P＜0.05).结论:雄激素可能通过降低颏舌肌SR Ca2 -ATP酶活性、抑制其mRNA的表达而影响老龄大鼠颏舌肌的功能;而雌激素没有此项作用.     

Effects of Atractylodes Macrocephala on the Cytomembrane Ca^2＋-activated K^＋ Currents in Cells of Human Pregnant Myometrial Smooth Muscles

The study examined the inhibitory effect of Atractylodes macrocephala(AM) on the uterine contraction during premature delivery and explored its electrophysiological mechanism by studying the effects of AM on the Ca2 -activated K currents of pregnant human myometrial smooth muscle cells with or without the treatment with interleukin-6.Single cells were acutely isolated from pregnant human myometrial smooth muscles.Whole-cell Ca2 -activated K currents were recorded by using an Axopatch1-D amplifier.The cells were divided into three groups: group A in which AM was added into perfusate,group B,in which interleukin-6 was added into perfusate) and group C in which AM was added into perfusate after addition of interleukin-6.IL-6 10 ng/mL inhibited BKca by 36.9%±13.7% as compared with control(P<0.01).AM at 2 mg/mL raised BKca by 36.7%±22.6% or 45.2%±13.7% with or without the treatment of IL-6,respectively(P<0.01).It is concluded that AM was able to enhance the BKca of pregnant human myometrial smooth muscle cells treated or un-treated with interleukin-6 and its effect on the BKca IL-treated cells was stronger that its effect on BKca of untreated cells.Our results suggested that AM can help to maintain the membrane potentials and the resting status of pregnant human myometrial smooth muscle cells.     

华蟾素对大鼠胸主动脉的缩血管作用及其机制

目的:研究中成药华蟾素对大鼠离体胸主动脉环的作用及其机制。方法:采用大鼠离体胸主动脉灌流模型,观察华蟾素对血管张力的影响。结果:华蟾素(2.5、5.0、7.5、10.0 g/L)浓度依赖性地收缩去内皮的大鼠胸主动脉环,收缩幅度分别为(16.3±3.39)%、(52.5±7.70)%、(60.9±8.84)%、(69.2±11.34)%,显著高于内皮完整的大鼠胸主动脉环收缩幅度[(6.2±2.07)%、(14.7±4.91)%、(17.6±5.86)%、(20.3±6.78)%](P<0.01);在无Ca2 液中,10.0 g/L华蟾素对去内皮主动脉环的收缩幅度[(7.1±0.79)%]显著低于有Ca2 液中的收缩幅度[(78.1±4.32)%](P<0.01);维拉帕米(1-0 7m o l/L,V er)预处理可消除10.0 g/L华蟾素的缩血管作用(P<0.01);一氧化氮合酶抑制剂L-NAM E(1-0 4m o l/L)预处理内皮完整的主动脉环,可增强10.0 g/L华蟾素的缩血管作用(P<0.01)。结论:华蟾素对大鼠离体胸主动脉有收缩作用,其缩血管机制与其促使细胞外Ca2 经电压依赖性钙通道内流进入血管平滑肌细胞有关;同时,华蟾素作用于血管内皮细胞,引起血管舒张因子一氧化氮的释放,部分抵消其缩血管效应。     

问号钩端螺旋体磷脂酶C的活性及其内化时细胞内游离Ca2+水平的变化

目的:了解不同毒力的钩端螺旋体(简称钩体)对细胞内游离Ca2 水平的影响及其磷脂酶C(PLC)活性与细胞内游离Ca2 水平的关系.方法:建立问号钩体Vero和J774A.1细胞感染模型.采用fluo-3/AM胞内Ca2 特异荧光标记激光共聚焦技术,检测强毒力的问号钩体黄疸出血群赖型56601株和弱毒力的波摩那群波摩那型56608株及无毒力的双曲钩体Patoc Ⅰ株作用的Vero和J774A.1细胞胞内游离Ca2 水平.以[3H]PIP2为底物,采用同位素法检测上述3株钩体培养物上清、胞浆和胞膜中PLC的活性.结果:正常Vero和J774A.1细胞胞内游离Ca2 基础值分别为(102.3±8.2)%和(105.9±7.3)%,在观察时间内Patoc Ⅰ株钩体作用细胞的荧光强度变化百分数一直波动于(102.3±8.2)%～(102.2±8.3)%.问号钩体56601株感染的Vero和J774A.1细胞胞内游离Ca2 浓度呈双峰型增高,第一峰荧光强度变化百分数分别为(430.5±35.7)%和(747.5±18.5)%,第二峰荧光强度变化百分数分别为(380.6±17.4)%和(804.6±22.4)%.问号钩体56608株感染Vero和J774A.1细胞,其胞内游离Ca2 浓度均呈缓慢的单一坡型升高,其最大荧光强度变化百分数分别为(235.0±19.3)%和(402.4±17.4)%,明显小于56601株问号钩体(P<0.01).问号钩体56601株和56608株及双曲钩体Patoc Ⅰ株的培养物上清、胞浆蛋白和胞膜蛋白成分中均显示PLC活性(P<0.05).结论:不同毒力的问号钩体所感染细胞的胞内游离Ca2 水平及其峰型有明显差异,而且与被感染细胞的种类有关,与PLC活性无关.     

胆囊收缩素对豚鼠结肠平滑肌及其细胞膜L-型钙电流和膜电位的影响

目的 探讨胆囊收缩素8肽(CCK-8S)对豚鼠近端结肠平滑肌及细胞膜L-型钙电流和膜电位的电生理作用及其机制.方法 (1)采用RM6240多道生理信号采集处理系统记录CCK-8S对豚鼠近端结肠平滑肌肌条收缩的影响;(2)检测Fluo-2/AM标记的近端结肠平滑肌细胞膜内钙离子浓度([Ca2+]i)变化;(3)用EPC-10膜片钳放大器测量全细胞模式下CCK-8S对近端结肠平滑肌细胞膜静息电位(RP)、动作电位(AP)和L-型钙通道电流(ICa-L)的影响.结果 CCK-8S(10-1mol/L)作用后:(1)豚鼠近端结肠平滑肌肌条收缩幅值和频率分别为对照组的(149±12)%和(132±13)%(均P＜0.05);(2)近端结肠平滑肌细胞膜的[Ca2+]i为对照组的(738±24)%(P＜0.05);(3)RP、AP峰值及AP快速复极时间分别为对照组的(52±9)%、(140±4)%和(61±13)%(均P＜0.05),该效应可被预先加入的CCK1受体拮抗剂地伐西匹和(或)L-型钙通道阻断剂尼非地平阻断(n=8,均P＜0.05),而预先向灌流液中加入CCK2受体拮抗剂CI 988后,CCK-8S对RP、AP峰值及AP快速复极时间的作用仍存在;(4)从-60 mV去极化至+10 mV,ICa-L为对照组的(138±7)%(P＜0.05),但分别可被相应拮抗剂抑制;当向电极内液加入肝素(10-6 mol/L)或向细胞外液加入staurosporine(10-6 mol/L)后,CCK-8S对Ica-L均没有影响(2.9%±2.6%和1.9%±2.3%,均P＞0.05).结论 CCK-8S通过CCK1受体促进近端结肠平滑肌自发性收缩频率和强度;其机制与激活1,4,5-三磷酸肌醇介导的蛋白激酶C途径进而促进L-型钙通道开放有关.

Abstract：

Objective To investigate the effects and mechanism of cholecystokinin octapeptide (CCK-8S) on the contractile activity of smooth muscles,L-type calcium current and membrane potentials of proximal colon myocytes in guinea pig.Methods ( 1 ) Strips of proximal colon were obtained from adult guinea pigs.The contraction of these stripes was measured by a RM6240 multi-channel physiological signal system.(2) Suspension of single smooth muscle cells (SMCs) were obtained from proximal colon and isolated by enzymatic digestion.The effect of CCK-8S on intracellular calcium concentration ( [Ca2+] i) of SMCs was examined by fura-2-1oaded miscrofluorimetric measurement.(3) Resting potential ( RP),action potential (AP) and L-type calcium current (ICa-L ) were recorded by patch-clamp technique.Results ( 1 )The contractile amplitude and frequency of muscle stripes enhanced by CCK-8S ( 10 -7 mol/L) were ( 149 ±12)% and (132 ± 13 )% respectively of those of control group (all P ＜ 0.05 ).They were significantly attenuated by pretreating strips with CCK1 receptor antagonist devazepide ( 10-7 mol/L),L-type calcium channel blocker nifedipine ( 10 -5 mol/L),Ca2+ -ATPase inhibitor TG (thapsigargin) ( 10-5 mol/L) and BA (boric acid) (10-5 mol/L) respectively.(2) [Ca2+]i of SMCs intensified by CCK-8S was (738 ±24)% of that of control group.And it was inhibited by pretreating SMCs with devazepide(all P ＜0.05).(3) After the superfusion of CCK-8S,RP depolarized to (52 ±9)%,the exogenously stimulated peak values of AP rose to (140±4)% and fast repolarization time of AP decreased to (61 ± 13)% (all P ＜0.05).They were significantly inhibited when these cells were pretreated with devazepide and/or nifedipine (n = 8,P ＜0.05 for each group) whereas CI 988 had little effect.(4) The CCK-8S-evoked ICa-L of SMCs at the voltage of + 10 mV was boosted to ( 138 ± 7 )%.Such an effect was suppressed by a pretreatment with nifedipine,devazepide,TG and BA respectively.In the presence of an inhibitor of inositol 1 ,4,5-trisphosphate (IP3 ) receptors,heparin (10-6 mol/L) and an protein kinase C (PKC) inhibitor,saurosporine (10-6 mol/L),CCK-8S did not significantly intensify ICa-L (all P ＞ 0.05 ).Conclusion CCK-8S promotes proximal colon contraction by CCK1 receptors through the activation of IP3-mediated PKC pathway.     

EFFECTS OF GLIBENCLAMIDE, GLIMEPIRIDE, AND GLICLAZIDE ON ISCHEMIC PRECONDITIONING IN RAT HEART

Objective To compare the influence of different sulfonylureas on the myocardial protection effect of ischemic preconditioning (IPC) in isolated rat hearts, and ATP-sensitive potassium channel current (IKATP) of rat ventricular myocytes. Methods Isolated Langendorff perfused rat hearts were randomly assigned to five groups: (1) control group, (2) IPC group, (3) IPC glibenclamide (GLB, 10 μmol/L) group, (4) IPC glimepiride (GLM, 10 μmol/L) group, (5) IPC gliclazide (GLC, 50 μmol/L) group. IPC was defined as 3 cycles of 5-minute zero-flow global ischemia followed by 5-minute reperfusion. The haemodynamic parameters and the infarct size of each isolated heart were recorded. And the sarcolemmal IKATP of dissociated ventricular myocytes reperfused with 10 μmol/L GLB, 1 μmol/L GLM, and 1 μmol/L GLC was recorded with single-pipette whole-cell voltage clamp under simulated ischemic condition. Results The infarct sizes of rat hearts in IPC (23.7%±1.3%), IPC GLM (24.6%±1.0%), and IPC GLC (33.1%±1.3%) groups were all significantly smaller than that in control group (43.3%±1.8%; P<0.01, n=6). The infarct size of rat hearts in IPC GLB group (40.4%±1.4%) was significantly larger than that in IPC group (P<0.01, n=6). Under simulated ischemic condition, GLB (10 μmol/L) decreased IKATP from 20.65±7.80 to 9.09±0.10 pA/pF (P<0.01, n=6), GLM (1 μmol/L) did not significantly inhibit IKATP (n=6), and GLC (1 μmol/L) decreased IKATP from 16.73±0.97 to 11.18±3.56 pA/pF(P<0.05, n=6). Conclusions GLM has less effect on myocardial protection of IPC than GLB and GLC. Blockage of sarcolemmal ATP-sensitive potassium channels in myocardium might play an important role in diminishing IPC-induced protection of GLM, GLB, and GLC.     

铁对白细胞介素-2舒血管效应的调制作用

目的 :研究微量元素铁对白细胞介素 - 2 (IL- 2 )舒血管效应的调制作用及其机理。方法 :采用离体主动脉环灌流模型 ,在苯肾上腺素 (1μmol/ L)预收缩基础上 ,测定经柠檬酸铁胺 (FAC)处理后 IL- 2对血管张力的变化 ;用分光光度法测定血管一氧化氮合酶 (NOS)活性。结果 :FAC(0 .1～ 10μmol/ L )孵育 30 min后对血管的张力无影响 ,却能浓度依赖性的抑制 IL - 2 (1～ 10 0 0 U/ ml)的舒血管效应 (P<0 .0 5～ 0 .0 1)。 IL - 2 (1、10、10 0、10 0 0 U/ ml)单独作用后的血管张力分别为加药前的 (78.4 7± 4 .31) %、(6 6 .86± 5 .5 5 ) %、(5 2 .6 2± 4 .5 1) %和 (4 2 .39± 4 .2 7) %。10 μmol/ L FAC预处理后再加 IL- 2 ,血管张力为 (89.81± 1.94 ) %、(86 .13± 3.11) %、(77.16± 5 .6 6 ) %和 (6 8.76± 5 .6 9) %。L-精氨酸 (1mmol/ L)孵育取消了 FAC对 IL- 2舒血管效应的抑制作用。IL- 2 (10 0 0 U/ ml)使 NOS的活性从 (9.86± 0 .5 4) U/ ml prot显著增高为 (2 2 .10± 1.87) U/ ml prot。FAC(10 μmol/ L)单独孵育 30 min NOS的活性为 (10 .5 9± 0 .5 9) U/ ml prot,但是 FAC预处理再加 IL - 2后 ,NOS活性显著降低为 (15 .71± 0 .89) U/ ml prot;高钙 (2 .5 mmol/ L )预处理 ,不改变 FAC对