血管内皮生长因子165对心肌细胞膜L型钙电流的作用及其对动作电位的影响

发布时间：2018-03-06 21:04

本文选题：血管内皮生长因子165　切入点：L型钙电流　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的细胞因子VEGF(165)是治疗缺血性心脏病和心力衰竭的潜在靶点。研究已证实VEGF165不仅促进形成缺血区域新生血管,还发挥抑制心肌细胞凋亡、减少纤维组织生成以及提高干细胞移植成功率的作用。这些研究结果表明VEGF165不单能够以内皮细胞为靶点进行血管生物学调节,还会影响心肌细胞本身活动。运用不同浓度VEGF165蛋白干预时,心肌细胞的机械活动和电活动可能会发生变化。L型钙通道是心肌细胞膜上重要的电压依赖性钙离子通道,经L型钙通道流入的钙离子,一方面激活钙介导钙释放引发心肌收缩,另一方面参与心肌细胞膜动作电位平台期的形成。在不同浓度VEGF165蛋白的干预下,通过研究心肌细胞膜L型钙电流变化及动作电位的变化,目的在于探索VEGF165对心肌细胞电活动的影响。研究方法首先分离豚鼠心室肌细胞,运用膜片钳技术分别进行电流钳与电压钳实验。1、电压钳实验首先运用硝苯地平检测电压钳设置,得到单个心室肌细胞膜L型钙电流(全细胞)。将分离的心室肌细胞分为0、10、40、100、300 ng/ml VEGF165干预组。在不同浓度VEGF165蛋白干预下,记录各组L型钙电流的电流-电压曲线关系、标准化最大电流值,L型钙通道稳态激活曲线和稳态失活曲线的斜率因子k和半数激活电压V1/2值并比较。在100 ng/ml、300 ng/ml VEGF165干预下,运用VEGF2型受体抑制剂SU5416,检测并比较运用抑制剂前后L型钙电流的电流-电压曲线关系和标准化最大电流值。2、电流钳实验在300 ng/ml VEGF165干预下,以自身作为对照,观察并比较刺激前后静息电位、动作电位时程50、动作电位时程90、动作电位幅度和最大电压变化率。研究结果1、成功确认L型钙电流的电压钳设置。与对照组相比,硝苯地平组的标准化电流为0.04±0.02(P0.05);2、VEGF165以剂量依赖方式刺激豚鼠心室肌细胞膜L型钙电流增强。与未添加VEGF165的对照组相比,40(-1.070±0.049)、100(-1.170±0.042)和300ng/m L(-1.220±0.060)VEGF165干预组标准化L型钙电流明显增加(P0.05)。与40 ng/m L组比较,100和300 ng/m L VEGF165干预组标准化L型钙电流明显增加(P0.05);3、分别比较100 ng/m L VEGF165干预组和对照组的心室肌细胞L型钙通道的稳态激活和稳态失活曲线,两组间半数激活电压(V1/2)和斜率系数(k)无明显差异;4、VEGFR2抑制剂明显消除100 ng/m L(-1.170±0.019 vs-0.923±0.033)和300ng/m L(-1.220±0.025 vs-0.911±0.030,P0.05)VEGF165介导的L型钙电流增加;5、高浓度的VEGF165干预前后,心室肌细胞膜APD90和动作电位的其他参数没有明显变化。研究结论在10-100 ng/m L浓度范围内,通过与细胞膜上的VEGFR2结合,VEGF165能够以剂量依赖方式增强心室肌细胞膜L型钙电流。VEGF165介导的钙电流增强作用与心室肌细胞膜L型钙通道的电压门控特性无关,且不影响心肌细胞动作电位特性,有助于心脏电活动稳定性。
[Abstract]:Background and objective the cytokine VEGF 165) is a potential target for the treatment of ischemic heart disease and heart failure. It has been demonstrated that VEGF165 not only promotes the formation of neovascularization in ischemic regions, but also inhibits cardiomyocyte apoptosis. These results suggest that VEGF165 can not only target endothelial cells for vascular biological regulation, but also increase the success rate of stem cell transplantation. When treated with different concentrations of VEGF165 protein, the mechanical and electrical activities of cardiomyocytes may change. L-type calcium channel is an important voltage-dependent calcium channel in myocardial cell membrane. Calcium ion influx via L-type calcium channel, on the one hand, activates calcium-mediated calcium release and induces myocardial contraction, on the other hand, participates in the formation of action potential plateau of myocardial cell membrane. Under the intervention of different concentrations of VEGF165 protein, By studying the changes of L-type calcium current and action potential of myocardial cell membrane, the purpose of this study was to explore the effect of VEGF165 on the electrical activity of cardiac myocytes. The patch-clamp technique was used to carry out current clamp and voltage clamp experiments respectively. First, nifedipine was used to detect the setting of voltage clamp. L-type calcium current (L-type calcium current) of single ventricular myocyte membrane was obtained. The isolated ventricular myocytes were divided into two groups: one group was treated with different concentrations of VEGF165 protein, the other was divided into three groups. Under different concentrations of VEGF165 protein, the current-voltage curves of L-type calcium current were recorded. The slope factor k and the half activation voltage V1 / 2 of the normalized maximum current value / L type calcium channel steady-state activation curve and steady-state inactivation curve were compared and compared. VEGF2 receptor inhibitor SU5416 was used to detect and compare the relationship between current and voltage curve of L-type calcium current before and after the use of the inhibitor and the standardized maximum current value of .2.Current-clamp experiment was carried out under the intervention of 300 ng/ml VEGF165 and itself as control. Observe and compare the resting potential, action potential duration 50, action potential duration 90, action potential amplitude and maximum voltage change rate before and after stimulation. The standardized current of nifedipine group was 0.04 卤0.02P0.05P0.05VEGF165 increased the L-type calcium current in guinea pig ventricular myocytes in a dose-dependent manner. Compared with the control group without VEGF165, the standardized L-type calcium current increased significantly between the control group (40 卤1.070 卤0.049) and the control group (300ngr 路m L-1.220 卤0.060 VEGF165) and the control group (300ngmL-1.220 卤0.060VEGF165). The standardized L-type calcium current in 40 ng/m L group was significantly increased than that in 300 ng/m L VEGF165 group. The steady-state activation and steady-state inactivation curves of L-type calcium channel in ventricular myocytes were compared between the 100 ng/m L VEGF165 intervention group and the control group. There was no significant difference between the two groups in the half activation voltage (V 1 / 2) and slope coefficient (k). 4 VEGFR2 inhibitors significantly eliminated 100 ng/m L ~ + -1.170 卤0.019 vs-0.923 卤0.033) and 300 ng / m L ~ (-1) -1.220 卤0.025 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911 卤0.030 vs-0.911, respectively. There was no significant change in APD90 and other parameters of action potential in ventricular myocytes. VEGF165 could enhance the L-type calcium current in ventricular myocytes in a dose-dependent manner. The enhancement of calcium currents mediated by VEGF165 was independent of the voltage-gated characteristics of L-type calcium channels in ventricular myocytes. And it does not affect the action potential characteristics of cardiomyocytes and contributes to the stability of cardiac electrical activity.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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