【作者】 陈杰；

【导师】 何明；

【作者基本信息】 江西医学院 ， 药理学， 2003， 硕士

【摘要】 目的：研究细胞内外氯离子变化对心肌A/R损伤的影响。方法：采用1—3 d SD乳鼠, 原代心肌细胞培养，培养第4 d进行缺氧/复氧实验。分别给予氯通道抑制剂SITS( 0.1 mmol·L－1)或DIDS(0.1 mmol·L－1)、钙离子阻断剂Ver (10 mmol·L－1)及去除细胞外孵育液中的Cl-或Ca2+（即Cl--free或Ca2+-free）等干预。随机分为14组： Control组、Cl--free Control组、DIDS Control组、SITS Control组、Ca2+-free Control组、A/R组、Cl--free组、DIDS组、SITS组、Ver组、Cl--free+SITS组、Ver+ Cl--free组、Ca2+-free组、Ca2+-free+ Cl--free组。各组进行以下指标观察：①心肌细胞搏动频率；②细胞存活率（MTT法）；③孵育液中LDH活性；④透射电镜观察细胞超微结构。结果：①Control组心肌细胞搏动频率相对恒定(98±16 beat·min-1)，节律规则，细胞存活率高(92±14.2%)，LDH活性低(2.17±1.47 IU·L-1) ，线粒体内外膜清晰完整，嵴排列清晰。②与Control组比较，Cl--free Control组、DIDS Control组、SITS Control组、Ca2+-free Control组对心肌细胞的影响无显著性差异（p>0.05），而A/R组细胞搏动频率(13±3 beat·min-1)显著减慢、节律不齐，部分细胞停搏；细胞存活率（38.3±3.6%）显著下降、LDH活性(107.1±24.8 IU·L-1)显著升高；线粒体明显肿胀，嵴变形或消失，核膜结构破坏。③DIDS组与A/R组比较，细胞搏动频率(21±5 beat·min-1)、细胞存活率 (44.2±3.2%)、LDH活性的改变(88.0±23.3 IU·L-1)无显著性差异（p＞0.05），线粒体明显肿胀，嵴消失或变形，与A/R组损伤相似。④Cl--free(77±10 beat·min-1)、Cl--free+Ver(88±10 beat·min-1)、<WP=5>Ver(74±9 beat·min-1)、SITS(95±10 beat·min-1)、Ca2+-free(94±10 beat·min-1)、Ca2+-free+Cl--free(94±10 beat·min-1)、Cl--free+SITS(95±10 beat·min-1)各组之间对心肌细胞搏动频率无明显影响（p>0.05），与A/R组比较有显著性差异（p<0.01）；Cl--free(60.8±5.2%)、Cl--free + Ver(74.8±6.3%) 、SITS(68.2±6.3%) 、Ver(65.8±5.3%)、Ca2+-free(74±5.3%)、Ca2+-free+Cl--free（85.3±13.5%）、SITS+ Cl--free (76±6.1%)组分别与A/R组比较，细胞存活率有显著升高（p<0.05或p<0.01）；Cl--free(41.8±13.2 IU·L-1) 、 Cl--free+Ver (20.7±6.7 IU·L-1)、SITS(27.2±4.3 IU·L-1) 、Ver(31.0±8.5 IU·L-1) 、Ca2+-free(22.6±5.4 IU·L-1)、Ca2+-free+Cl--free（12.0±5.5 IU·L-1）、SITS+ Cl--free (15.2±2.6 IU·L-1 )组分别与A/R组比较，LDH活性显著降低（p<0.05或p<0.01）; Cl--free 、SITS 、SITS+ Cl--free组与A/R组比较，细胞超微结构损伤较轻，线粒体内外膜及嵴清晰完整；⑤与Cl--free组比较,Cl--free +Ver、Ca2+-free+Cl--free、SITS+ Cl--free组细胞存活率显著升高（p< 0.05），Cl--free+Ver 、Ca2+-free+Cl--free、SITS+ Cl--free组LDH显著降低（p< 0.05）；⑥Cl--free+Ver组与Ver组比较，细胞存活率高，LDH活性低，有显著性差异（P<0.05）。Ca2+-free+ Cl--free组与Ca2+-free组比较，细胞存活率高，LDH活性低，有显著差异（P<0.05）。Cl--free+SITS组与SITS组比较，细胞存活率高，LDH活性低，有显著差异（P<0.05）。结论：1、细胞外Cl-在心肌细胞A/R损伤中起重要作用。2、细胞器内氯储存小体Cl-的释放可能参与心肌细胞A/R损伤。3、SITS对心肌细胞A/R损伤有显著的保护作用而DIDS可加重心肌细胞A/R损伤。4、细胞内Cl-和Ca2+浓度升高可协同造成心肌细胞A/R损伤。更多还原

【Abstract】 Objective: To investigate the change of extracellular and/or intracellular chloride concentration on primary cultured rat ventricular myocytes subjected to A/R injury. Methods: We performed 180 minutes simulated ischemia in cultured neonatal SD rat ventricular myocytes, followed by 120 minutes simulated reperfusion. Cultured myocardial cells of neonatal SD rats have been randomly divided into fourteen groups: Control group, Cl--free control group, DIDS control group, SITS control group, Ca2+-free control group, A/R group, Cl--free group, DIDS group, SITS group, Ver group, Cl--free+SITS group, Ver+ Cl--free group, Ca2+-free group, Ca2+-free+Cl--free group. Myocardial cells beating rate were counted under inverted microscope, cell viability and the activity of LDH in culture were measured at the end of experiment. The morphology or ultrastructure assessment of ventricular myocytes was observed by inverted microscope or transmission electron microscope, respectively. Results: ①The beating rate (98±16 beat·min-1)of ventricular myocytes in Control group was consistent and regular, cell viability(92.2±5.6%)and LDH activity(2.17±1.47 IU·L-1)was considered normal, control myocytes had well-preserved ultrastructure. Mitochondria were elongated or oval in shape, mitochondria membrane was intact and <WP=7>clear.②There was no significant about cell beating rate, cell viability and LDH activity in Cl--free Control, DIDS Control, SITS Control or Ca2+-free Control group (p>0.05), compared with that of Control group. ③the beating rate(13±3 beat·min-1) became significantly slow, myocytes beating rhythm became obviously inconsistent, few of myocytes no longer beat spontaneously. the cell viability (38±3.6%) decreased markedly and LDH activity (107.0±24.8 IU·L-1) increased sharply in A/R group compared with that of Control group（p<0.01）, A/R myocytes had showed that most of the mitochondria were markedly abnormal in shape and greater in size, with abnormal cristae or areas of loss of matrix. In some mitochondria, the cristae and matrix was cleared out.③The cell beating rate (21±5 beat·min-1), the cell viability (44.2±3.2%)and LDH release (89.0±23.3 IU·L-1) in DIDS group were similar to those of A/R group （p>0.05）.The change of mito- chondria in DIDS group was similar to that of A/R group. ④the beating rates of myocytes in Cl--free (77±10 beat·min-1), Cl--free+Ver (88±10 beat·min-1), Ver (74±9 beat·min-1), SITS (95±10 beat·min-1), Ca2+-free(94±10 beat·min-1),Ca2+-free+Cl--free(94±10 beat·min-1)and Cl--free+SITS(95±10 beat·min-1) was similar to each other（p>0.05）,The beating rate in above seven groups was significantly increased ,Compared with that of A/R（p<0.05 or p<0.01）.The cell viability of Cl--free(60.8±5.2%),Cl--free+Ver(74.8±6.3%),SITS(68.2±6.6%),Ver(65.2±5.3%), Ca2+-free(74.2±5.3%),Ca2+-free+Cl--free（85.3±13.5%）or SITS+ Cl--free <WP=8>(78.3±12.5%)increased significantly ,compared with that of A/R （p<0.05 or p<0.01）,respectively, LDH activity of Cl--free(41.8±13.2 IU·L-1),Cl--free+Ver(20.7±6.7IU·L-1),SITS(14.0±2.6 IU·L-1) ,Ver(31.8±8.5 IU·L-1) ,Ca2+-free(22.6±5.4 IU·L-1),Ca2+-free+Cl--free（12.0±5.5 IU·L-1）or SITS+Cl--free(15.2±2.6 IU·L-1 )significantly decreased, compared with A/R respectively（p<0.05 or p<0.01）,The cellular structures were extremely well preserved in SITS, Cl--free or SITS+Cl--free cells subjected to the A/R. ⑤The cell viability of Cl--free +Ver ,Ca2+-free+ Cl--free or SITS+ Cl--free increased significantly, compared with Cl--free（p<0.05）,LDH activity of Cl--free +Ver,Ca2+-free+ Cl--free or SITS+ Cl--free decreased significantly compared with Cl--free（p<0.05）.⑥the cell viability in Ca2+-free+ Cl--free group increased, compared with that of Ca2+-free（p<0.05）. the cell viability in Cl--free+Ver group increased, compared with that of Ver（p<0.05）.the cell viability in Cl--free+SITS group was increased, compared with that of SITS（p<0.05）.LDH activity in Ca2+-free+ Cl--free group declined， compared with that of Ca2+-free（p<0.05）更多还原