缺氧再复氧对心肌细胞缝隙连接通信影响的研究

Study on the Gap Junctional Intercellular Communication in Hypoxia and Reprovided Oxygen Myocardial Cells

目的研究不同缺氧再复氧时间心肌细胞缝隙连接(Gap junction,GJ)通信改变情况,以及庚醇(Heptanol,HT)对GJ通信的影响。方法利用荧光染料Lucifer yellow CH(LY)不能透过细胞膜,但可以经过细胞GJ在细胞间进行传递的特点,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LY经过GJ流通情况,及HT干预后GJ通信改变情况。结果在缺氧30min时心肌GJ通信明显下降,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HT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。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)。当运用HT后心肌细胞搏动明显减弱,约为(64±20)次/min,差异显著(P(0.001)。在缺氧再灌注过程中,HT搏动次数变化不大。结论心肌细胞在缺氧时GJ通透性减低,HT也有不同程度的降低GJ通信的作用。

Objective To investigate the communication of myocardial cell gap junction in hypoxia and reprovided oxygen by scrape-loading and dye transfer（SLDT） and influence on it by HT. Methods Lucifer yellow CH（LY） can pass through GJ to neighboring cells, but not through cell membrane. On the basis of it, we examined the change of the gap junctional intercellular communication during cardiac myocytes hypoxia and reprovided oxygen by SLDT. The effects of HT on the communication were studied. Results Normally, the fluorescence can be seen beyond the fourth row cells. The gap junctional intercellular communication was inhibited in myocyte hypoxia for 30min, the dye was only transferred to second neighboring cells. The transmission of dye recovered step by step during reprovided oxygen. In HT treated cells, there was similar inhibition of communication with hypoxia cells. The neonatal cardiac myocytes exhibited synchronous contraction with a relatively constant rate（ 120 ± 18） beats/min at day 7 of culture. The frequency of beat at 30min of hypoxia was （56 ± 16） beats/min（P 〈 0. 01）. In presence of 2 mmol/L heptanol, the frequency was（64 ± 20） beat/min. The inhibitory effects of heptanol were reversible after washing out of heptanol. Conclusion The gap junctional communication reduces in hypoxia myocytes. HT could change the conductibility of GJ.