2型糖尿病大鼠血管平滑肌细胞大电导钙激活钾通道电流的变化

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1、目录12型糖尿病大鼠血管平滑肌细胞大电导钙激活钾通道电流的变化„„„„„„„„„„„„„„„„„„„„„„„11.1中文摘要„„„„„„„„„„„„„„„„„„„„„11.2英文摘要„„„„„„„„„„„„„„„„„„„„„31.3前言„„„„„„„„„„„„„„„„„„„„„„„61.4材料与方法„„„„„„„„„„„„„„„„„„„„91.5结果„„„„„„„„„„„„„„„„„„„„„„„131.6讨论„„„„„„„„„„„„„„„„„„„„„„„211.7结论„„„„„„„„„„„„„„„„„„„„„„„271.8参考文献„„„„„„„„„

2、„„„„„„„„„„„„281.9英汉缩略词对照表„„„„„„„„„„„„„„„„„312致谢„„„„„„„„„„„„„„„„„„„„„„„3232型糖尿病血管病变相关基因研究（综述）„„„„„„332型糖尿病大鼠血管平滑肌细胞大电导钙激活钾通道电流的变化摘要目的：本实验通过运用全细胞穿孔膜片钳技术观察2型糖尿病大鼠胸主动脉和肠系膜动脉血管平滑肌细胞（vascularsmoothmusclecells,VSMCs）大电导钙激活钾通道（bigconductancecalciumactivatedpotassiumchannel,BKca）的电流变化，

3、探讨BKca与2型糖尿病血管病变之间的关系。方法：1.2型糖尿病大鼠模型的建立：雄性SD大鼠适应性喂养一周后称体重，随机分为两组，对照组以普通饲料喂养，高脂组喂以高脂高糖饲料。4周后高脂组SD大鼠腹腔一次性注射STZ25mg/kg（pH4.2的0.1mol/L枸橼酸缓冲液配成0.25％浓度)；对照组腹腔注射等体积的0.1mol/L枸橼酸缓冲液。2.2型糖尿病大鼠模型的鉴定：于STZ注射一周后心脏采血测空腹血糖、血脂、血胰岛素含量，成模鼠继续高脂喂养,于8、12周末分批用3％戊巴比妥钠30mg/kg腹腔注射麻醉后，取胸主动脉及肠系膜小动脉供试验用。3

4、.膜片钳实验：（1）细胞悬液制作：将分离的动脉剪切成碎片，置于细胞分离液中，37℃恒温水浴振荡，经酶液两步消化使细胞间质及周围的连接组织被酶解，然后将被消化后的动脉组织放于无钙台式液中，选取具有较强立体感表面光滑的平滑肌细胞，采用全细胞穿孔膜片钳技术记录电流。（2）全细胞穿孔膜片钳步骤：在室温下进行实验，使微管电极尖端与周围细胞间形成电化学绝缘，阻抗约10～100GΩ，形成全细胞构型。用Clampex10.1软件采集电流，Clampfit10.1软件系统进行数据分析，统计电流幅值和制作I-V曲线。（3）单通道膜片钳步骤：在形成全细胞构型后，将电极尖

5、端提出液面，再放回浴液中形成内面向外式膜片；用pClamp9.0软件记录通道的电流信2+号。结果：1.通道的开放概率随着电压和单通道浴液中的Ca浓度的增加而增大。2.急性酶分离的大鼠胸主动脉VSMCsBKca电流的基本特性：（1）BKca宏观电流具有明显的电压、钙依赖性及外向整流的性质；（2）比较对照组、高脂组八周、高脂组十二周BKca电流：对照组（n=8）、高脂八周组（n=6）、高脂十二周组（n=6）的电流幅值分别为352.31±82.56pA（+60mV）、719.25±45.03pA（+60mV）、911.58±77.82pA（+60mV）。

6、经t检验，对照组与高脂八周组，t=9.785，P<0.05；正常组与高脂十二周组，t=12.845，P<0.05；高脂八周与高脂十二周组，t=5.240，P<0.05。3.急性酶分离的大鼠肠系膜动脉VSMCsBKca电流的基本特性：（1）BKca宏观电流具有明显的电压、钙依赖性及外向整流的性质；（2）比较对照组、高脂组八周、高脂组十二周BKca电流：对照组（n=5）、高脂八周组（n=5）、高脂十二周组（n=5）的电流分别为169.88±67.13pA（+60mV）、330.41±55.04pA（+60mV）、568.77±62.97pA（+60mV

7、）。经t检验，对照组与高脂八周组，t=4.135，P<0.05；对照组与高脂十二周组，t=9.690，P<0.05；高脂八周与高脂十二周组，t=6.373，P<0.05。结论：1.本实验成功复制了T2DM大鼠模型。2.急性酶分离的大鼠胸主动脉和肠系膜动脉平滑肌细胞BKca具有电压依赖性和钙依赖性，其通道电流具有外向整流特征。3.高脂组的BKca电流幅值比对照组高，同时高脂十二周组的BKca电流幅值比高脂八周组高。关键词：大电导钙激活钾通道；胸主动脉；肠系膜动脉；血管平滑肌细胞；2型糖尿病ThechangesofBKcaintype2diabetes

8、ratvascularsmoothmusclecellsAbstract:bjective:Thisexperimentb