【作者】 刘琴；

【导师】 王琛；

【作者基本信息】 苏州大学 ， 麻醉学， 2011， 硕士

【摘要】 目的观察七氟烷预处理延迟性保护(SWOP)对大鼠心肌细胞缺氧复氧(H/R)损伤的影响,探讨自噬在七氟烷SWOP对H/R损伤心肌细胞延迟性保护中的机制。方法H9c2大鼠心肌细胞随机分为5组,CON组、H/R组(缺氧2 h,复氧1 h)、七氟烷预处理延迟性保护组(SWOP组,给予2.5%七氟烷SWOP 1 h,间隔24 h后进行H/R)、自噬抑制剂组(3-MA组,在培养基内加入终浓度为10 mM 3-MA培养1 h,间隔24 h后进行H/R)、3-MA+SWOP组(2.5%七氟烷SWOP前15 min给予10 mM 3-MA,间隔24 h后进行H/R)。实验结束后取心肌细胞,MTT法检测细胞存活率,流式细胞仪检测凋亡率,采用透射电镜观察细胞内自噬小体和凋亡的变化,Western Blot法检测LC3-Ⅱ、Beclin 1和Bcl-2的蛋白表达。结果细胞存活率和凋亡率:与H/R组相比,SWOP组、3-MA组和3-MA+SWOP组心肌细胞存活率增加(P<0.05),凋亡率减少(P<0.05)。电镜结果:H/R组心肌细胞内溶酶体被激活,自噬和凋亡同时存在,SWOP组自噬小体和溶酶体减少。Western blot结果:与CON组相比,H/R组和SWOP组LC3-Ⅱ、Beclin 1表达上调(P<0.05);与H/R组相比,SWOP组LC3-Ⅱ、Beclin 1蛋白上调幅度降低(P<0.05),SWOP组、3-MA组和3-MA+SWOP组Bcl-2表达上调(P<0.05)。结论七氟烷SWOP可通过下调LC3-Ⅱ、Beclin 1,上调Bcl-2的表达,以减少24 h后H/R损伤心肌细胞的自噬性死亡和凋亡,增加细胞存活率,从而发挥其延迟性心肌保护作用。更多还原

【Abstract】 ObjectiveTo investigate the delagyed protection of Sevoflurane preconditioning (SWOP) against myocardial hypoxia/reoxgenation (H/R) injury and the mechanism of autophagy during the cardioprotection on rat cardiomyocytes.MethodsIn this study, H9c2 rat cardiomyocytes were randomly divided into five groups: Control (CON) group; H/R group, rat cardiomyocytes was exposed in the airtight container for 2 h followed by 1 h of reoxgenation; SWOP group, rat cardiomyocytes was exposed to 1 h of 2.5% sevoflurane 24 h before H/R; The inhibitor of autophagy 3-methyladenine (3-MA) was added to culture medium 15 min before sevoflurane exposure (3-MA+SWOP group) or cells were treated by 3-MA alone (3-MA group). Rat myocardiocytes were collected after different treatments. Cell proliferation was detected by MTT assay. Apoptosis rate was analyzed by flow cytometry. Autophagosomes and apoptosis were observed with transmission electron microscopy (TEM). The expression of autophagy protein microtubule associated protein light chain3-Ⅱ(LC3-Ⅱ), Beclin 1 and anti-apoptosis factor B-cell lymphoma-2 (Bcl-2) was determined by western blot.ResultsThe cell proliferation of SWOP group, 3-MA group and 3-MA+SWOP group were significantly increased compared with the H/R group (P<0.05), and the apoptosis rate was decreased (P<0.05). TEM releaved that the formation of autophagosome and apoptosis was decreased in the sevoflurance preconditioning groups compared with the H/R groups. The expression of LC3-Ⅱand Beclin 1 were upregulated in H/R and SWOP groups compared with CON group (P<0.05), which were lower in SWOP group, 3-MA group and 3-MA+SWOP group than in H/R group, but Bcl-2 expression was upregulated.ConclusionThe results of our study indicated that Sevoflurane preconditioning (SWOP) 24 h before H/R produced delayed myocardial protection against H/R injury by increasing cell proliferation and decreasing apoptosis rate in cardiomyocytes. SWOP may inhibit autophagic cell death and apoptosis by down-regulation of autophagy protein LC3-Ⅱand Beclin 1 expression and up-regulation of Bcl-2 expression during reoxgenation.更多还原