全氟化碳对缺血再灌注诱导的肺泡上皮细胞凋亡损伤的保护作用和机制研究

发布时间：2018-06-15 07:56

本文选题：肺移植 + 肺缺血再灌注损伤　；参考：《泸州医学院》2011年硕士论文

【摘要】：目的探索凋亡在肺缺血再灌注损伤中的作用机制,对PFC的生物学保护效应及可能的机制有更深入的理解和阐释,为临床应用PFC治疗LIRI提供较为客观和更充分的理论依据,以期有效改善供体肺保护,研究改良新的供体肺保存液,为临床肺移植手术和体外循环手术地应用提供更大范围的保障。方法 1.建立肺缺血再灌注的细胞模型。A549细胞在不同的时间(6h、12h)被保存于100%氧气及4℃的环境中,并加入低钾右旋糖酐培养以模拟缺血的过程。继而转入放有10%胎牛血清的DMEM/F-12培养基的温暖环境(37℃)孵育2h以模拟再灌注情况。 2.PFC对IRI诱导的A549凋亡损伤的保护作用。应用流式细胞仪(FCM)检测细胞凋亡率化及western blotting法检测caspase-3蛋白的表达。将培养传代的第三、四代A549细胞分为四组:①对照组(control group)组:不作任何干预;②IRI组:仅缺血再灌注处理;③PFC(全氟辛烷)组:按30%体积比(PFC:培养液)在细胞培养中加入全氟辛烷;④PFC+IRI组:按30%体积比(PFC:培养液)在缺血再灌注阶段加入全氟辛烷。FCM缺血阶段时间取6h和12h两个时间点的样本。结果 1.细胞凋亡的FCM结果:肺缺血再灌注条件作用于A549细胞6h和12h后的早期细胞凋亡率分别为10.01%和14.21%;晚期凋亡和坏死率分别为4.93%及5.49%;细胞存活率分别为84.61%与79.59%。即随着时间的推移,细胞凋亡坏死率逐渐上升,而其细胞存活率逐渐下降。与对照组、PFC组和共培养组比较,IRI作用于A549细胞后的6,12h,其早期细胞凋亡率、晚期细胞凋亡和坏死率显著升高,存活率显著下降,且12小时明显重于6小时;与对照组比较,PFC两个时相组的细胞凋亡率和存活率的差异无统计学意义,且两组之间差异也无统计学意义;与对照组和全氟化碳组比较,PFC+IRI组A549细胞的12h早期凋亡率升高,存活率下降,即PFC能明显拮抗缺血再灌注诱导的凋亡作用,但不能完全阻断缺血再灌注对细胞的促凋亡效应。 2. western blotting结果:IRI作用于A549细胞0.5,2,6,12h见caspase-3前体降解的17 kDa和11 kDa两个活性片段;PFC单独作用于A549细胞,该蛋白的活性和水平无明显影响;于PFC+IRI组, caspase-3蛋白的活性显著下降,提示PFC能够显著抑制IRI诱导的此蛋白的激活进而阻断IRI的诱导细胞凋亡的效应。结论 1.缺血再灌注作用于A549细胞可诱导其发生凋亡的损伤改变,这种效应呈时间依赖。 2.PFC单独作用于A549细胞,不诱导其凋亡损伤的发生,当PFC加入缺血再灌注条件下孵育A549细胞时,则可以显著减轻缺血再灌注诱导的A549细胞凋亡的损伤,增加细胞的存活率。
[Abstract]:objective
The mechanism of apoptosis in the lung ischemia reperfusion injury is explored, and the biological protective effect and possible mechanism of PFC are further understood and explained. It provides a more objective and sufficient theoretical basis for the clinical application of PFC to treat LIRI, in order to improve the donor lung protection effectively and to improve the new donor lung preservation solution for clinical pulmonary shift. The application of surgery and cardiopulmonary bypass provides a wider range of protection.
Method
1. the cell model of the lung ischemia reperfusion (6h, 12h) cell was stored at 100% oxygen and 4 degrees in the environment, and the hypokalemic dextran was added to simulate the process of ischemia. Then, the DMEM/F-12 medium with 10% fetal bovine serum was transferred to the warm environment (37 degrees C) to incubate 2H to simulate reperfusion.
The protective effect of 2.PFC on IRI induced A549 apoptosis. The flow cytometry (FCM) was used to detect the apoptosis rate and the expression of caspase-3 protein by Western blotting. The third, fourth generation A549 cells were divided into four groups: (1) the control group (control group) group: no intervention; (2) IRI group: only ischemia reperfusion treatment; (3) PF C (perfluorooctane) group: perfluorooctane was added to cell culture by 30% volume ratio (PFC: Culture); (4) group PFC+IRI: samples of two time points of 6h and 12h were taken at the ischemia reperfusion stage by adding perfluorooctane.FCM ischemia stage at the 30% volume ratio (PFC: medium).
Result
The FCM results of 1. cell apoptosis: the early apoptosis rates of A549 cells 6h and 12h were 10.01% and 14.21%, respectively, and the late apoptosis and necrosis rate were 4.93% and 5.49%, respectively, and the cell survival rate was 84.61% and 79.59%., respectively. Compared with the control group, the PFC group and the co culture group, IRI acted on the 6,12h after A549 cells. The early cell apoptosis rate, the late cell apoptosis and necrosis rate significantly increased, the survival rate decreased significantly, and the 12 hours was significantly higher than 6 hours. Compared with the control group, there was no statistical difference between the apoptosis rate and the survival rate of the PFC two phase group. The difference between the two groups was not statistically significant. Compared with the control group and the perfluorocarbon group, the early apoptosis rate of A549 cells in group PFC+IRI increased and the survival rate decreased. That is, PFC could obviously antagonize the apoptosis induced by ischemia-reperfusion, but it can not completely block the effect of ischemia reperfusion on cell apoptosis.
2. Western blotting results: IRI acts on A549 cell 0.5,2,6,12h to see caspase-3 precursors degraded by 17 kDa and 11 kDa two active fragments; PFC alone acts on A549 cells, and the activity and level of the protein are not significantly affected; the activity of the caspase-3 protein in PFC+IRI group decreases significantly. It also blocked the effect of IRI induced apoptosis.
conclusion
1. the effect of ischemia-reperfusion on A549 cells can induce apoptosis, which is time-dependent.
2.PFC alone acts on A549 cells and does not induce apoptosis. When PFC is incubated with A549 cells under ischemia-reperfusion, the apoptosis of A549 cells induced by ischemia-reperfusion can be significantly reduced and the survival rate of cells is increased.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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