【作者】 陈和平；

【导师】 徐亚利；

【作者基本信息】 江西医学院 ， 药理学， 2004， 硕士

【摘要】 目的：探讨14-3-3蛋白参与心肌细胞缺氧预处理延迟保护作用的可能性。方法：实验用SD新生大鼠（1～3 d），雌雄不拘，无菌取出乳鼠心脏，经分离附着组织，胰蛋白酶消化，纯化制成心肌细胞，在培养的第4 d随机分为6组：正常对照（Control）组、PD98059+Control组、缺氧/复氧（A/R）组、缺氧预处理(APC)组、DMSO+APC组、PD98059+APC组。各组分别进行以下指标观察：①心肌细胞搏动频率；②细胞存活率（MTT法）；③培养液中乳酸脱氢酶（LDH）活性；④透射电镜观察细胞超微结构；⑤Western Blotting法检测14-3-3蛋白的表达变化。结果：①细胞搏动频率：PD98059+Control组(95±13 beat/min)与Control组(98±16 beat/min)比较无显著性差异（p>0.05）； A/R组(13±3 beat/min)与Control组相比，有显著性差异（p<0.01）；APC组(92±10 beat/min)、DMSO+APC组(90±8 beat/min)对正常心肌细胞搏动频率无明显影响（p>0.05），与A/R组比较均有显著性差异（均p<0.01）；APC组、DMSO+APC组之间无显著性差异（p>0.05）；PD98059+APC组(18±6 beat/min)细胞搏动频率显著弱于APC组（p<0.01），与A/R组比较无显著性差异（p>0.05）。②细胞存活率：PD98059+Control组(91.2±12.5%)与Control组(89.6±10.8%)比较无显著差异（p>0.05）；A/R(38.9±7.2%)组与Control组比较，有显著差异（p<0.01）；APC组(78.2±10.6%)、DMSO+APC组(80.6±12.4%)分别与A/R组比较，有显著性差异（均p<0.01），APC组、DMSO+APC组之间无显著性差异（p>0.05）；PD98059+APC组(57.9±6.8%)与APC组比较，有显著性差异（p<0.01）。③LDH活性：PD98059+Control组(3.45±0.42 IU/L)与Control组(3.06±0.51 IU/L)比较无显著性差异（p>0.05）；A/R组(30.26±4.88 IU/L)与Control组比较，有显著性差异（p<0.01）；APC组（14.12±2.06 IU/L）、DMSO+APC组(12.65±2.81 IU/L)分别与A/R组比较，有显著性差异（p<0.01），APC组、DMSO+APC组之间无显著性差异（p>0.05）；PD98059+APC组(28.52±4.62 IU/L)与 APC组<WP=6>比较，有显著性差异（p<0.01），与A/R组比较LDH活性无显著性差异（p>0.05）。④.14-3-3蛋白的表达：Control组心肌细胞有少量14-3-3蛋白表达，PD98059+Control组为Control组的0.98±0.12倍，两组之间无显著性差异（p>0.05）；A/R组为Control组的3.52±0.46倍，（p<0.01），示表达明显增加； APC组、DMSO+APC组分别为Control组的5.41±0.69倍和5.56±0.72倍（均p<0.01），并显著高于A/R组（均p<0.01），而APC组、DMSO+APC组之间无显著性差异（p>0.05）；PD98059+APC组为Control组的4.06±0.62倍,但显著低于APC组（p<0.01），与A/R组比较无显著差异（p>0.05）。表明PD98059可取消APC后心肌细胞14-3-3蛋白表达上调。⑤.透射电镜观察：Control组心肌细胞结构保存完好，线粒体嵴排列清晰，核染色质均匀分布，细胞膜结构完整。A/R组心肌细胞线粒体明显肿胀，个别线粒体呈空泡状，嵴消失或变形，肌丝消失，细胞膜结构破坏，细胞核内染色质不均匀，有核边聚现象。APC组心肌细胞超微结构改变轻微，线粒体排列规则、大小均匀，嵴及内外膜清晰完整。核膜完整，较A/R组损伤明显减轻。PD98059+APC组心肌细胞超微结构改变与A/R组相近。结论：1、从细胞水平证实了缺氧预处理后24小时心肌细胞对再次缺氧/复氧损伤具有保护作用； 2、14-3-3蛋白在正常心肌细胞内有基础表达，缺氧/复氧损伤后表达上调；3、缺氧预处理可以通过MAPK信号通路诱导14-3-3蛋白表达；4、14-3-3蛋白参与了心肌细胞缺氧预处理后的延迟保护作用。更多还原

【Abstract】 Objective;To stady whether 14-3-3 protein involves in the delayed protection 24 hr after anoxic preconditioning. Methods; Cultured myocardial cells of neonatal SD rats have been randomly divided into six groups: Control group; PD98059+Control group; anoxia reoxygenation (A/R)group;anoxia preconditioning group(APC); DMSO+APC group; PD98059 +APC group. In each group, the cardiomyocytes were subjected to anoxia (3 hr)followed by reoxygenation (1 hr) 24 hr after anoxic preconditioning on the model of the cultured cardiomyocytes. At the end of experiment, myocardial cells beating rate, cell viability and the activity of LDH in culture were measured,furthermore, The morphology or ultrastructure assessment of ventricular myocytes was observed by inverted microscope or transmission electron microscope, the expression of 14-3-3 protein were measured by western blotting techniques, respectively. Results: ①Beating rate of ventricular myocytes: There was no significance in PD98059+Control group (95±13beat/min)（p>0.05）,compared with that of control group(98±16 beat/min);The beating rate was significantly slow in A/R group(13±3 beat/min) compared with that of control group（p<0.01）; Between APC group(92±10 beat/min) and DMSO+APC group (90±8 beat/min), the beating rate was similar to each other（p>0.05）,and Compared with A/R , The beating rate was significantly increased（p<0.01）; The beating <WP=8>rate of PD98059+APC group (18±6 beat/min) was significantly lower than APC groups.②the cell viability: There was no significance in PD98059+Control group(91.2±12.5%) compared with that of control group(89.6±10.8%)（p>0.05）；The cell viability of A/R(38.9±7.2%) was significantly decreased when compared with that of control group（p<0.01）;The viability of APC (78.2±10.6%) , DMSO+APC (80.6±12.4%) was significantly increased when compared with A/R respectively，（p<0.01）,between APC and DMSO+APC, The cell viability was similar to each other（p>0.05）;The viability of PD98059+APC (57.9±6.8%) was significantly decreased when compared with APC group.③LDH activity:LDH activity of PD98059+Control(3.45±0.42 IU/L) was similar to that of Control group(3.06±0.51 IU/L)（p>0.05）；LDH activity of A/R (30.26±4.88 IU/L) was significantly increased when compared with Control group（p<0.01）； LDH activity of APC（14.12±2.06 IU/L）or DMSO+APC (12.65±2.81 IU/L )was significantly decreased when compared with A/R respectively （p<0.01）,but LDH activity of DMSO+APC was similar to that of APC group（p>0.05）；LDH activity of PD98059+APC (28.52±4.62 IU/L) was similar to that of A/R（p>0.05）but was dramatically increased when compared with APC group（p<0.01）；④Expression of 14-3-3 protein; Western blotting revealed 14-3-3 protein showed a basic expression in Contral group.The expression quantity of 14-3-3 protein in PD98059+Control group was similar to that of Control and 0.98±0.12 fold of Control group（p>0.05）.14-3-3 protein expression of A/R was increased when compared with Control group and 3.52±0.46 fold of Control group（p<0.01）.The 14-3-3 protein contents were increased significantly at APC and DMSO+APC,compared with those in Control and A/R group(p<0.01),they were 5.41±0.69, 5.56±0.72 fold of Control group respectively.but 14-3-3 protein expression of DMSO+APC was similar to that of APC group（p>0.05）. 14-3-3 protein in PD98059+APC <WP=9>group was 4.06±0.62 fold of Control group ,and was dramatically decreased when compared with APC group（p<0.01）but was similar to that of A/R（p<0.05）,which demonstrated PD98059 suppressed upregulation of 14-3-3 protein;⑤ Ultrastructural assessment by the transmission electron microscope: For Control group, the cell membrane was intact, mitochondria scattered loosely throughout the cytoplasm, mitochondria membrane was intact and clear, nuclear chromatin material was uniformly dispersed. The myocytes subjected to A/R injury were characterized by calcified and elongated mitochondria,dispersed nuclear chromatin material,ruptured nuclear membrane,disappeared myo-fibers and更多还原