离体细胞气泡损伤模型的建立与评估

Establishment and evaluation of a cell bubble contact injury model

目的建立离体细胞气泡损伤模型,用于潜水减压病等气泡损伤相关疾病的发病机制研究。方法研制离体细胞气泡损伤装置(包括气泡生成注射器、细胞培养皿和打孔装置),通过改变气泡生成注射器末端直径大小获得不同直径大小的气泡。以原代培养大鼠肺微血管内皮细胞(PMVEC)为研究对象,通过PI染色和CCK-8细胞活力检测观察细胞损伤与气泡直径和气泡触碰时间之间的量效关系,最终建立稳定的离体细胞气泡损伤模型。结果PI染色结果显示,0.5mm直径气泡触碰1、2、3、4h均未对PMVEC产生明显损伤,1.0、1.5和2.0mm直径气泡触碰1、2、3、4 h均能导致死亡的PMVEC增加,且气泡直径越大、触碰时间越长死亡的PMVEC越多。CCK-8细胞活力检测结果显示,1.0、1.5和2.0 mm直径气泡触碰1、2、3、4 h后PMVEC的活力下降明显,与对照组比较差异有统计学意义(P均<0.01);2.0 mm直径气泡触碰时间>3 h时对PMVEC损伤才较大,触碰3 h时的细胞存活率为(84.27±1.35)%。结论本研究建立了稳定的细胞气泡触碰损伤模型,可用于减压病等气泡损伤相关疾病的研究。就PMVEC而言,直径为2mm的气泡触碰损伤3h可作为最合适的气泡损伤条件。

Objective To establish a cell bubble contact injury model for studying the pathogenesis of bubble-related diseases such as decompression sickness.Methods A cell bubble contact device was designed,including a bubble generating syringe,a cell culture dish,and a punching device.Bubbles with different diameters could be obtained by changing the diameter of the bubble generating syringe end,and the dose-effect relationship between cell damage,bubble diameter and contact time was observed by propidium iodide(PI)staining and cell counting kit 8(CCK-8)cell viability test in primary cultured rat pulmonary microvascular endothelial cells(PMVECs),so as to establish a stable cell bubble contact injury model.Results The results of PI staining showed that there was no obvious damage to PMVECs after 1,2,3,or 4 h of 0.5 mm bubble contacting,while bubbles with diameters of 1.0,1.5,and 2.0 mm increased the death of PMVECs after contacting 1,2,3,and 4 h;and the larger the bubble diameter and the longer the contact time,the more death of PMVECs were.Compared with the control group,CCK-8 cell viability test showed that bubbles with diameters of 1.0,1.5,and 2.0 mm significantly decreased the viability of PMVECs after contacting 1,2,3,and 4 h(all P<0.01).However,the cell damage of PMVECs was obvious only when the 2.0 mm diameter bubble was touched for more than 3 h.The cell viability was(84.27±1.35)%when the bubble with diameter 2.0 mm contacted the PMVECs for 3 h.Conclusion This study establishs a stable cell bubble contact injury model,which can be used to study bubble-related diseases such as decompression sickness.In addition,for PMVECs,3 h of bubble contact with a diameter of 2.0 mm is the optimal condition for bubble-induced injuries.