文献类型：学位论文

中文题名：当归挥发油对血管紧张素Ⅱ所致心肌细胞肥大CaN通路及T通道的影响

英文题名：Effects of Angelicanaphtha on CaN Signal Pat-hways and T-type Calcium Channel of Angio-tensinⅡ-induced Hypertrophy Cardiomyocy-te

作者：王雨薇[1];

第一作者：王雨薇

机构：[1]甘肃中医学院;

第一机构：甘肃中医药大学

导师：李应东;甘肃中医学院

授予学位：硕士

语种：中文

中文关键词：当归挥发油;血管紧张素Ⅱ;心肌细胞肥大;钙超载;T型钙离子通道

外文关键词：Angelicanaphtha;AngiotensinII;cardiomyocyte hypertrophy;Ca2+ overloading;T-type calcium channel

年份：2014

摘要：目的： 观察当归挥发油/(Angelicanaphtha，AN/)对血管紧张素Ⅱ/(AngiotensinⅡ,AngⅡ/)诱导的肥大心肌细胞CaN通路及T型钙离子通道的影响，探讨当归挥发油抑制AngⅡ诱导心肌细胞肥大的作用及其机制。 方法： 选择H9c2心肌细胞株，将细胞浓度调整到1×106cells//mL，37℃、5/%CO2培养箱中培养72h。用10-4mg·L-1血管紧张素Ⅱ作用于心肌细胞24h成功建立心肌细胞肥大模型。应用超临界萃取装置提取当归挥发油。实验分为5组：空白对照组；模型组；低剂量/(5mg·L-1/)组、中剂量/(10mg·L-1/)组、高剂量/(20mg·L-1/)组。MTT法检测心肌细胞活力的改变；激光共聚焦显微镜检测线粒体膜电位和细胞内钙离子浓度，用试剂盒检测的活性及表达变化、用western blot检测CaN蛋白、T型钙离子通道相关基因蛋白Cav3.1/(CACNA1G/)、Cav3.2/(CACNA1H/)以及凋亡蛋白caspase-3、caspase-12的表达情况。 结果： 1.MTT法检测心肌细胞存活率：血管紧张素Ⅱ造模后心肌细胞存活率明显增加，与对照组比较，差异有显著性/(P＜0.05/)。与血管紧张素Ⅱ组相比，当归挥发油干预组心肌细胞的细胞存活率均有所下降，差异有统计学意义/(P＜0.05/)。 2.激光共聚焦检测细胞内钙离子浓度：与空白组相比，模型组Ca2+平均荧光强度明显增强，差异有统计学意义/(P＜0.05/)；与模型组比较，当归挥发油干预组Ca2+平均荧光强度明显减弱，差异有统计学意义/(P＜0.05/)。 3.激光共聚焦显微镜检测线粒体膜电位，与空白组相比，模型组AngⅡ/(浓度10-4mg·L-1/)细胞体积增大，平均荧光强度明显增强，差异有统计学意义/(P＜0.05/)；与模型组比较，当归挥发油干预组细胞体积缩小，线粒体数目减少，荧光强度明显减弱，差异有统计学意义/(P＜0.05/)。 4.流式细胞术检测细胞周期，与空白组相比，模型组G2期细胞增多，差异具有显著性/(P＜0.05/)；与模型组相比，当归挥发油干预组的G2期细胞有所减少，差异统计学意义/(P＜0.05/)。 5.Western Blot检测CaN蛋白、Cav3.1/(CACNA1G/)、Cav3.2/(CACNA1H/)蛋白的表达情况：AngⅡ组与空白组比较，CaN、Cav3.1、Cav3.2表达增高，caspase-3和caspase-12凋亡相关蛋白表达增高，差异具有统计学意义/(P＜0.05/)，与模型组比较，当归挥发油干预组CaN、Cav3.1、Cav3.2及caspase-3、caspase-12表达水平降低，差异具有显著性/(P＜0.05/)。 结论： 1．当归挥发油可通过钙离子阻滞作用和抑制心肌细胞凋亡来治疗心肌细胞肥大。 2．本研究从微观上证实了当归挥发油为“血中气药”和中医的气血理论。
Objective: To explore the mechanism of Angelicanaphtha /(AN/) on AngiotensinII/(AngII/)-induced cardiac myocyte hypertrophy. By observing impact on calciumconcentration, cell Cycle, mitochondrial membrane potential changes and the proteinexpression of CaN, Cav3.1, Cav3.2, Caspase-12and Caspase-3induced by AngII inthe myocardial cell. Methods: Cells with concentration of1x106//mL were cultured in closed incubator with37℃and5/%CO2for72h. Cardiomyocyte hypertrophy model was established by10-4mg·L-1AngII treatment. AN was made by CO2supercritical fluid extraction.Subjects were divided into5groups: normal control group, model group /(myocardialcell with AngII, final concentration10-4mg·L-1/), AN low-dose intervention group/(treatment myocardial cells with AN of5mg·L-1for24h/); AN middle-doseintervention group /(treatment myocardial cells with AN of10mg·L-1for24h/); ANhigh-dose intervention group/(pretreatment myocardial cells with AN of20mg·L-1for24h/); the cell viability of cardiomyocytes was detected by MTT assay; themitochondrial membrane potential and calcium concentration were detected with aconfocal microscopy; the cell cycle was detected by flow cytometry; the proteinexpression of CaN, Cav3.1, Cav3.2, caspase-12and caspase-3were detected bywestern blotting assay. Results: 1. MTT assay for myocardial cell viability detection: Compared with the normalcontrol group, cell viability of myocardial cells was significantly increased in modelgroup /(P＜0.05/). Compared with the model group, the cell viability was significantlydecreased in all AN groups /(P＜0.05/). 2. Calcium concentration of myocardial cells: Compared with the normal controlgroup, the calcium concentration of myocardial cells was significantly increased in model group /(P＜0.05/). Compared with the model group, the calcium concentrationof myocardial cells was significantly decreased in all AN groups /(P＜0.05/). 3. Confocal laser scanning results: Compared with normal control group theRh123fluorescence intensity of myocardial cells in the model group was very high,but in AN interventionn group was significantly reduced./(P＜0.05/). 4. Flow cytometry results: Compared with normal control group the ration of G2phase cells in the model group was significantly increased, but in AN interventionngroup was significantly reduced./(P＜0.05/). 5. Western blot results: There was a certain amount of expression of CaN, Cav3.1,Cav3.2, caspase-12and caspase-3protein in normal control group. These proteinsexpression in model group significantly was increased/(P＜0.05/). In AN interventiongroup, CaN, Cav3.1, Cav3.2protein expression was significantly decreased/(P＜0.05/),caspase-12and caspase-3protein expression was significantly lower/(P＜0.05/). Conclusions: 1. Angelicanaphtha can treat myocardial cell hypertrophy by blocking ofCa2+influx and inhibiting myocardial apoptosis. 2. This study confirmed the angelica naphtha from the micro for blood gasmedicine and traditional Chinese medicine theory of qi and blood.