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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接通透性减低,庚醇、卡维地洛也有不同程度的降低缝隙连接通讯的作用。庚醇、卡维地洛通过改变缝隙连接通讯以及其他方面的作用来减少心肌细胞的死亡和凋亡。
OBJECTIVE: To investigate the changes of gap junctional junctions in cardiomyocytes during different ischemia-reperfusion periods and to investigate the relationship between gap junctional cardiomyocytes and cardiomyocyte necrosis and apoptosis as well as the intervention of carvedilol and heptanol. Methods: The experiment was performed at the Clinical Research Institute of China-Japan Friendship Hospital from October 2004 to March 2005. Cultured 7d cardiomyocytes hypoxia for 30min after switching to normal sugaring DMEM medium for 1, 2, 3, 6h, resulting in reoxygenation injury. Dosing regimen: Divided into three groups; untreated group, heptanol group, carvedilol group. Heptanol with DMEM (containing 5% fetal bovine serum) dubbed 1,2mmol / L concentration, carvedilol dubbed 0.001g / L concentration, respectively, the drug was added to the test cells, cultured cells after 30min to do Related experiments. No medication without medication. Using cell scratch technique, the different groups were observed under an inverted fluorescence microscope, and the circulation of Roche’s yellow through the gap was observed at different times of hypoxia-reperfusion. Flow cytometry was used to observe the apoptosis of cells at different time of hypoxia-reperfusion. Results: ① The gap junctional communication of myocardium decreased obviously at 30 min hypoxia, the fluorescence was only transmitted to the second row of cells near the scratch, and the normal cell fluorescence reached the cells outside the four columns. Re-oxygen 1h recovery to the third column, and later gradually returned to normal. The use of heptanol significantly slows the transmission of fluorescence is only transmitted to the second column of cells, hypoxia and reoxygenation conduction no significant change. The use of carvedilol conduction change is not obvious. ② There was no significant difference in apoptosis index between normal and ischemic groups at 30 min (P> 0.05), and at 1 h after reoxygenation, the apoptotic index was 27.4%, which was significantly different from normal (P <0.01). Necrosis of cardiomyocytes in the 2h after reoxygenation difference was significant (P <0.01). After using carvedilol and heptanol, apoptosis index decreased by 55.11% and 21.53% respectively at 1 hour after reperfusion. (3) The pulsation frequency of cardiomyocytes cultured for 7 days was (120 ± 18) times / min. When the anoxia was 30 minutes, the pulsation was weakened and the frequency was (56 ± 16) times / min. ) Times / min, the difference was significant (P <0.01). After adding 2mmol / L heptanol, the pulsation of cardiomyocytes was obviously weakened, which was about (64 ± 20) times / min, with significant difference (P <0.001). Adding 0.001g / L carvedilol beats also have varying degrees of reduction (P <0.05). During hypoxia-reperfusion, the number of beats in the two intervention groups changed little. CONCLUSION: Myocardial cells have a reduced permeability of gap junctions during hypoxia. Heptyl alcohol and carvedilol also reduce the effects of gap junctional intercellular communication. Heptyl alcohol, carvedilol, reduces cardiomyocyte death and apoptosis by altering gap junctional communication and other effects.