【作者】 丁翔；

【导师】 宋治远；

【作者基本信息】 第三军医大学 ， 内科学， 2010， 硕士

【摘要】 背景和目的心房颤动(简称为房颤)是最常见的心律失常之一,其中70岁以上人群发生率高达10%,已经成为威胁人类健康和生活质量的重要疾病之一。房颤能诱导心房发生以心房肌有效不应期(AERP)缩短为特征的电重构及心房肌结构发生适应性与非适应性改变引起的结构重构。“钙超载”是心肌细胞电重构的关键,也可能是房颤发生并持续的重要基础。钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/ calmodulin-dependent protein kinaseⅡ, CaMKⅡ)作为Ca2 +/CaM调节蛋白家族中的一个主要成员,其在细胞的病理生理过程中发挥重要的生物学作用。有研究表明房颤时CaMKⅡ蛋白含量明显增加,其中在左房增加更为明显,而且随着房颤时间的延长CaMKⅡ蛋白含量也逐渐增多。因此,探讨房颤、细胞钙超载和CaMKⅡ三者之间的关系可能有助阐明房颤的确切发病机理。本研究采用钙离子载体伊屋诺霉素(ionomycin)建立新生大鼠心房肌细胞的钙超载模型,在此基础上观察CaMKⅡ抑制剂KN93对新生大鼠心房肌细胞钙负荷的影响,并对细胞CaMKⅡ的变化进行检测,其目的旨在进一步探讨房颤的确切发病机理,并为今后研究CaMKⅡ抑制剂KN93防治房颤发生的可行性奠定实验基础。方法1.用酶消化法分离新生大鼠心房肌细胞并行原代培养。纯化培养96小时后随机分为对照组(C组)、实验组(E1,E2,E3组),依次加入不同浓度ionomycin(0、0.5、1.0、2.0μmol/L),以钙离子指示剂Fluo-3/AM负载心房肌细胞,激光扫描共聚焦显微镜检测心房肌细胞游离钙离子浓度([Ca2+]i),建立新生大鼠心房肌细胞不同程度钙超载模型。2.新生大鼠心房肌细胞原代培养96小时,应用钙离子导入剂ionomycin(1.0μmol/L)建立心房肌细胞钙超载模型,并在KN93三种浓度(0.25、0.5、1.0μmo/L)的干预下,以钙离子指示剂Fluo-3 /AM负载心房肌细胞,激光共聚焦显微镜观察心房肌细胞内游离钙的变化;并应用Western blot方法检测CaMKⅡ表达的变化。结果1.原代培养至第4天,细胞生长密度可以达到瓶底70%左右;免疫组织化学染色鉴定:90%以上培养细胞α-肌动蛋白抗体染色阳性。对照组心房肌细胞活性比率为(73.00士2.37)%,与各实验亚组间心房肌细胞活性相比无显著性差异(P>0.05)。经不同浓度ionomycin处理l h后,各实验亚组心房肌细胞[Ca2+]i明显增高,与对照组相比有显著性差异(431.54±20.97、705.87±28.34、1305.05±53.69 vs 257.08±18.63, P<0.05),且各实验亚组间心房肌细胞[Ca2+]i水平随ionomycin浓度的增加而上升,亚组间差异显著(P<0.05)。光镜下形态学观察也表明,随ionomycin浓度的增加,荧光强度逐渐加强,呈浓度依赖性。2.与对照组比较,钙离子导入剂ionomycin明显增加细胞内钙离子荧光值(660.16士108.47 vs 376.12士57.57,p<0.01);而KN93对细胞内钙离子荧光值无明显影响(389.00士64.01 vs 376.12士57.57,p>0.05)。预先加入三种不同浓度KN93(0.25、0.5、1.0μmo/L)可显著降低ionomycin导致的细胞内钙离子荧光强度的增加幅度(vs 660.16士108.47,p<0.01),与对照组相比也有显著差异(vs 376.12士57.57,p<0.01)。钙超载组细胞CaMKⅡ表达明显增加,与对照组比较,p<0.01;而KN93对细胞CaMKⅡ表达影响不明显。不同浓度KN93(0.25、0.5、1.0μmo/L)预处理后,钙超载细胞CaMKⅡ的表达显著降低,与钙超载组比较,p<0.01。结论1.应用钙离子载体ionomycin能够建立可靠的心房肌细胞钙超载模型。2. CaMKⅡ抑制剂KN93可降低ionomycin引发的大鼠心房肌细胞钙超载,并下调钙超载细胞CaMKⅡ表达。更多还原

【Abstract】 Background and objectiveAtrial fibrillation (AF for short) is one of the most common arrhythmia, in which the incidence in people over 70 years of age up to 10%, has become a threat to human health and quality of life one of the important diseases. Occurrence of atrial fibrillation can be induced by atrial effective refractory period (AERP) is characterized by reduced atrial electrical remodeling and the structure of adaptive and non adaptive changes caused by remodeling."Calcium overload" is the key to electrical remodeling, atrial fibrillation and may also be an important basis for continuing. Calcium / calmodulin-dependent protein kinaseⅡ(calcium / calmodulin-dependent protein kinaseⅡ, CaMKⅡ) as a Ca2 + / CaM-regulated protein family of one of the main members, the pathophysiological process in the cell play an important biological role. Research has shown that CaMKⅡatrial fibrillation significantly increased protein content, which increased more significantly in the left atrium, and with the AF time CaMKⅡprotein content also increased gradually. Therefore, the study of atrial fibrillation, calcium overload and the relationship between CaMKⅡmay help the prevention and treatment of atrial fibrillation. In this study, calcium ionophore ionomycin Neonatal rat atrial muscle cell calcium overload model, based on the observation of this CaMKⅡinhibitor KN93 on neonatal rat atrial myocytes of load, and cell Detection of changes in CaMKⅡfor CaMKⅡinhibitors on the prevention and treatment of atrial fibrillation feasibility study lays the foundation.Methods1. The atrial myocytes were disassociated from neonate rats with digestion method and cultured for 96 hours. Co-incubated with the ionomycin(0、0.5、1.0、2.0μmol/L, respectively), the cells were divided into control group and experiment group(E1, E2 and E3) and loaded with Ca2+ indicator Fluo-3 /AM. The level of intracellular Ca2+ ([Ca2+]i) were measured with laser confocal scanning microscope.2. The atrial muscle cells were primarily cultured for 96 h and a model of calcium overload for atrial muscle cell in neonate rat was established by using calcium ionophore(ionomycin, 1.0μmol/L). In the present of Fluo-3 /AM(an indicator of calcium), intracellular calcium and the expression of CaMKⅡwere detected under the intervention of KN93(0.25、0.5、1.0μmo/L).Results:1. More than 90% of cultured cells were positive toα-actin antibody. The activity ratio of cells in control group was 73.00士2.37%. Compared with the each subgroup of experiment, there was no significant difference(p>0.05). One hour after treated with different concentrations of ionomycin, the [Ca2+]i of atrial muscle cell was significantly increased in all the experimental groups(431.54±20.97, 705.87±28.34, 1305.05±53.69 vs 257.08±18.63, respectively, p<0.05). There was significant difference between each subset of the experimental group. [Ca2+]i level and the fluorescence intensity of the myocytes increased gradually in a dose-dependent manner with the concentration of ionomycin.2. Compared with the control group, the intracellular Ca2+ was increased significantly by ionomycin (660.16士108.47 vs 376.12士57.57,p<0.01) and KN93 has no effect on it(389.00士64.01 vs 376.12士57.57, p>0.05). Added KN93 (0.25、0.5、1.0μmo/L) in advance, the fluorescence intensity of intracellular Ca2+ induced by ionomycin was significantly decreased (vs 660.16士108.47, p<0.01). the expression of CaMKⅡwas increased in calcium overload group( vs control group, p<0.01) and KN93 has no effect on it. a Added KN93 (0.25、0.5、1.0μmo/L) in advance, the expression of CaMKⅡin cell of calcium overload was significantly reduced (vs calcium overload group,p<0.01).Conclusion1. calcium overload model with primary atrial myocytes of neonate rats can be built by ionomycin.2. KN93, an inhibitor of CaMKⅡ, can reduce the calcium load of atrial muscle cell induced by ionomycin and decrease the expression of CaMKⅡ.更多还原