缺血后再灌注过程中的凋亡和坏死是导致损伤的细胞水平的关键机制。在缺血阶段和再灌注阶段,细胞内钙离子过载都是在病理状况发展之前发生的。在这方面,旨在减少缺血/再灌注损伤的策略之一与钙通道阻滞剂的使用有关.这项研究的目的是研究肽毒素的作用,钙通道阻断剂ω-六毒素-Hv1a,不同类型的上皮细胞在体外重建缺血/再灌注条件下的死亡特点。
■在这项研究中,我们使用CHO-K1上皮细胞培养。细胞凋亡的变化,坏死,细胞指数,在体外模拟缺血/再灌注过程时,通过添加钙通道阻滞剂毒素来评估钙离子浓度。缺血和再灌注损伤是通过氧气和营养剥夺,然后在完全营养培养基中再灌注来实现的。使用多峰读板器-荧光计进行测量。
■细胞凋亡增加,坏死,并在建立缺血/再灌注模型时记录钙离子浓度。凋亡和坏死水平的降低,以及钙离子浓度达到生理水平或接近生理水平,当在再灌注阶段以50nM的浓度添加毒素时注意到。细胞指数显示在毒素存在下更快的恢复。
■实验数据证实了肽钙通道阻滞剂对缺血后再灌注过程中上皮细胞状态的有益作用的假设,可以考虑作为再灌注前器官适应的策略进行进一步研究。
Apoptosis and necrosis during reperfusion after ischemia are key mechanisms at the cellular level leading to damage. The development of pathological conditions is preceded by intracellular calcium ion overload both at the stage of ischemia and at the stage of reperfusion. In this regard, one of the strategies aimed at reducing damage during ischemia/reperfusion is associated with the use of calcium channel blockers. The aim of the study was to study the effect of a peptide toxin, a calcium channel blocker ω-hexatoxin-Hv1a, on different types of epithelial cell death during in vitro reconstruction of ischemia/reperfusion conditions characteristic of organ transplantation.
In this study, we used CHO-K1 epithelial cell culture. Changes in apoptosis, necrosis, cell index, and calcium ion concentration were assessed when modeling ischemia/reperfusion processes in vitro with the addition of a calcium channel blocker toxin. Ischemic and reperfusion injury was achieved by oxygen and nutrient deprivation followed by reperfusion in a complete nutrient medium. The measurements were performed using a multimodal plate reader-fluorimeter.
An increase in apoptosis, necrosis, and the concentration of calcium ions was recorded when modeling ischemia/reperfusion processes. A decrease in the level of apoptosis and necrosis, as well as the concentration of calcium ions to a physiological level or a level close to physiological, was noted when the toxin was added at a concentration of 50 nM at the reperfusion stage. The cell index showed a faster restoration in the presence of the toxin.
The experimental data confirm the hypothesis of a beneficial effect of peptide calcium channel blockers on the state of epithelial cells during reperfusion after ischemia and can be considered for further study as a strategy for organ adaptation before reperfusion.