【作者】 余薇；

【导师】 林国生；

【作者基本信息】 武汉大学 ， 内科学， 2011， 博士

【摘要】 目的：观察维拉帕米(Ver)对糖尿病大鼠心肌缺血再灌注(I/R)后心功能,细胞内[Ca2+]i以及L-型钙电流(ICa-L)影响,探讨其防治糖尿病心肌I/R损伤的作用和机制。方法：通过链脲佐菌素诱导糖尿病大鼠模型,在糖尿病形成后的6-14周龄给予Ver(8mg/kg/d)灌胃,利用Langendorff离体心脏灌流系统复制大鼠心肌I/R模型,观察正常对照组、糖尿病组、Ver对照组和糖尿病组的心功能变化,随后双酶法急性分离各组心肌细胞,激光扫描共聚焦显微镜加Fluo-3/AM荧光染色技术和全细胞膜片钳技术分别观察心肌细胞内Ca2+荧光强度和ICa-L大小。结果：1与正常对照组相比,糖尿病组大鼠心脏经I/R后,心脏左室发展压(LVDP)和舒张末压(LVEDP)以及收缩压最大上升速率(dp/dtmax),冠状动脉流量(CF)都明显减小(P<0.01),收缩压最大下降速率(dp/dtmin)明显增加(P<0.01)o Ver糖尿病组大鼠离体心脏经I/R作用后,心功能得到明显改善,与糖尿病组相比,LVDP、LVEDP、dp/dtmax、CF各值均明显增加(P<0.01), dp/dtmin明显减少(P<0.01)。2糖尿病组大鼠心肌细胞经I/R作用后,细胞内Ca2+荧光强度明显高于正常对照组(P<0.01); Ica-L的电流密度值在各指令电位下,均明显减小,其电流-电压(I-V)曲线明显上抬到所有I-V曲线最上部,最大峰值在指令电位+30mV。在Ver糖尿病组,心肌细胞经I/R影响后,细胞内Ca2+荧光强度明显弱于糖尿病组(P<0.01); Ica-L值小于正常对照组,明显大于糖尿病组的ICa-L,其I-V曲线高于正常对照组,而显著低于糖尿病组,最大峰值在+20mV。当指令电位为+20mV时,糖尿病组大鼠心肌细胞经I/R损伤后,ICa-L电流密度由正常对照组的(-8.17±2.07)pA/pF减小为(-3.21±0.54)pA/pF(P<0.01); Ver对照组和糖尿病组心肌细胞ICa-L的电流密度分别变为(-7.14±2.17)pA/pF (P>0.05)和(-6.81±0.76)pA/pF(与正常对照组相比,P<0.05,与糖尿病组相比,P<0.01,与Ver对照组相比,P>0.05)。结论：糖尿病大鼠心脏在I/R影响下心功能减弱可能与心肌细胞I/R时细胞内[Ca2+]i增加和ICa-L减小密切相关。Ver长期治疗可以缓解糖尿病慢性作用带给心肌I/R损伤引起的心功能下降,其机制可能是Ver调控糖尿病心肌细胞膜上ICa-L内流大小,优化心肌细胞内[Ca2+]i平衡,避免I/R时糖尿病心肌细胞内Ca2+触发Ca2+释放作用引发的细胞内Ca2+超载。更多还原

【Abstract】 Objectives:To investigate the effects and mechanism of verapamil preventing ischemia/reperfusion (I/R) injury via observing the cardiac performance and intracellular free [Ca2+]i as soon as L-type calcium current (ICa-L) in cardiomyocytes of diabetes mellitus rats.MethodsDiabetes rats by streptozotocin-induced were fed verapamil (8mg/kg/d) from 6 tol4 wk of age.The hearts models of I/R in vitro rats which randomly divided into normal control group, diabetes group, verapamil control and diabetes groups were setup to observe the changes of heart function through using Langendorff-perfusion system. The fluorescence intensity of intracellular Ca2+was detected with Flup-3/AM loading by the laser scanning confocal microscope. The ICa-L was recorded via whole-cell patch clamp technique in enzymatically dissociated single rat ventricular myocytes.Results1 With I/R injury of the vitro rat hearts in diabetes group, the valures of left ventricular developed pressure (LVDP), diastolic end pressure (LVEDP), the maximum rising rates of left ventricular pressure (dp/dtmax) and coronary arterial flow (CF) were all significantly decreased, and the maximum dropping rates of left ventricular pressure (dp/dtmin) were increased (compared with normal control group, P<0.01, respectively). But some parameters mentioned above such as LVDP, LVEDP, dp/dtmax, CF were increased, and dp/dtmin was obviously decreased in verapamil diabetes group (compared with diabetes group, P<0.01, respectively).2 The fluorescence intensities of intracellular free Ca2+ in cardiomyocytes of diabetes rats were markedly stronger after influence of I/R injury (compared with normal control group, P<0.01). Under every command potential, the current density of ICa-L was significantly decreased, and the current-voltage (Ⅰ-Ⅴ) curve was changed up to the top, as soon as the peak clamp potential was-30mV with same I/R condition in diabetes rats. In verapamil diabetes group, The fluorescence intensities of intracellular free Ca2+ were significantly reduced in I/R injury procedure (compared with diabetes group, P<0.01). ICa-L were partly recovered near normal control group, and I-V curve was changed among normal control and diabetes group. The peak clamp potential was -20mV. When clamp voltage was -20mV, the current densities of ICa-L were significantly decreased from (-8.17±2.07) pA/pF in normal control group to (-3.21±0.54) pA/pF in diabetes group (P<0.01), and (-7.14±2.17) pA/pF in verapamil control group (compared with normal control, P>0.05), and (-6.81±0.76) pA/pF in verapamil diabetes group(compared with normal group, P<0.05, and with diabetes group, P<0.01, and with verapamil control group, P>0.05).ConclusionThe poor heart function was tightly correlate to that the [Ca2+]i was increased and ICa-L was decreased with I/R injury in diatetes rat hearts. Verapamil-treated in long time could significantly inhibit I/R injury induced the severely cardiac performance in diabetes rats, which was attributed to that verapamil might adjust ICa-L influx, and normalize the balance of intercellular [Ca2+]i, as soon as blocked the Ca2+ overload trigged by effects of Ca2+induced Ca2+ release in diabetes cardiomyocytes更多还原