缺血再灌注后心肌细胞缝隙连接通讯改变与其凋亡及坏死的关系  

Relationship of the gap junctional intercellular communication of myocardial cells with the apoptosis and necrosis following ischemia/reperfusion

作　　者：曾玉杰[1] 冯义柏[2] 于世龙[2] 柯元南[1] 王勇[1] 朱丹丹[1] 李志远[1] 姜磊[1] 李楠[1] 

机构地区：[1]中日友好医院心内科,北京市100029 [2]华中科技大学同济医学院附属协和医院心内科,湖北省武汉市430022

出　　处：《中国组织工程研究与临床康复》2007年第16期3074-3077,3085,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:了解不同缺血再灌注时间心肌细胞缝隙连接通讯改变情况,分析心肌细胞缝隙连接通讯与心肌细胞坏死、凋亡的关系以及卡维地洛、庚醇的干预作用。方法:实验于2004-10/2005-03在中日友好医院临床研究所完成。取培育7d心肌细胞缺氧30min后换用正常含糖的DMEM培养基培养1,2,3,6h,造成再给氧损伤。给药方案:分为三组;未用药组,庚醇组,卡维地洛组。庚醇用DMEM(含5%的小牛血清)配成1,2mmol/L的浓度,卡维地洛配成0.001g/L的浓度,分别加入药物于受试细胞,培养细胞30min后再做相关实验。未用药组不加药。采用细胞划痕技术,在倒置荧光显微镜下直观地观察不同组别、缺氧再灌注不同时间罗氏黄经过缝隙连接流通情况。利用流式细胞仪观察缺氧再灌注不同时间细胞凋亡情况。结果:①在缺氧30min时心肌缝隙连接通讯明显下降,荧光仅传至划痕附近第二列细胞,正常细胞荧光则传至四列以外的细胞。再供氧1h时恢复到第三列,以后逐步恢复到正常。运用庚醇后传导明显减慢荧光仅传至第二列细胞,缺氧和再供氧时传导也无明显变化。运用卡维地洛后传导改变不明显。②正常和缺血30min时凋亡指数差异无显著性(P>0.05),再供氧1h凋亡指数为27.4%,与正常相比差异显著(P<0.01)。坏死的心肌细胞在再供氧2h后差异有显著性(P<0.01)。运用卡维地洛、庚醇后凋亡指数在再灌注1h时分别较未用药组减少了55.11%,21.53%。③培养7d的心肌细胞搏动频率为(120±18)次/min,缺氧30min时,搏动减弱,次数为(56±16)次/min,再供氧1h时,搏动次数为(118±22)次/min,两者差异显著(P<0.01)。加入浓度为2mmol/L的庚醇后,心肌细胞搏动明显减弱,约为(64±20)次/min,差异显著(P<0.001)。加入0.001g/L卡维地洛搏动次数也有不同程度的减低(P<0.05)。在缺氧再灌注过程中,两个干预组搏动次数变化不大。结论:心肌细胞在缺氧时缝隙连�AIM： To investigate the gap junctional intercellular communication in different ischemia/reperfusion time, analyze the relation of gap junctional communication with myocytes necrosis and apoptosis, and study the interventional effect of carvedilol and heptanol. METHODS： The experiment was performed at Clinic Institute, China Japan Friendship Hospital from October 2004 to March 2005. Cultured 7-day myocardial cells were obtained, and then cultured for 1, 2, 3 and 6 hours with DMEM medium containing normal glucose after hypoxia for 30 minutes to induce re-hypoxia injury. Administration schedule： there were 3 groups, namely non-administration group, heptanol group and carvedilol group. Heptanol mixed with DMEM （5% calf serum） into the concentration of 1, 2 mmol/L, while carvedilol into 0.001 g/L concentration. Cells were cultured for 30 minutes before corresponding trails were conducted. No drug was treated in the non-administration group. Lucifer yellow CH （LY） could pass through gap junction to neighboring cells, which were directly observed under inverted fluorescent microscope from different groups and at different hypoxia/reperfusion by scrape loading and dye transfer （SLDT）. The apoptosis induced by ischemia/reperfusion were observed by flow cytometry. RESULTS： （1）The gap junctional intercellular communication was markedly inhibited in myocyte ischemia for 30 minutes, and the dye was only transferred to second neighboring cells. In normal, the fluorescence could be seen beyond the fourth row cells, and then recovered to the third row at 1-hour reperfusion, and the transmission of dye recovered step by step during reperfusion. In heptanol treated cells, transmission remarkably reduced, and fluorescence only recovered to the second row. There was no significant change at hypoxia and reperfusion, so did it after treating with carvedilol.（2） The indexes of apoptosis had no difference between normal and ischemic cells at minute 30 （P 〉 0.05）. The apoptotic indexes were 27.4% in

关 键 词：心肌细胞凋亡 缝隙连接通讯 划痕标记染料示踪技术 卡维地洛 庚醇 

分 类 号：R542.2[医药卫生—心血管疾病]