本文選題：肺移植 + 肺缺血再灌注損傷　； 參考：《瀘州醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的 探索凋亡在肺缺血再灌注損傷中的作用機(jī)制,對(duì)PFC的生物學(xué)保護(hù)效應(yīng)及可能的機(jī)制有更深入的理解和闡釋,為臨床應(yīng)用PFC治療LIRI提供較為客觀和更充分的理論依據(jù),以期有效改善供體肺保護(hù),研究改良新的供體肺保存液,為臨床肺移植手術(shù)和體外循環(huán)手術(shù)地應(yīng)用提供更大范圍的保障。 方法 1.建立肺缺血再灌注的細(xì)胞模型。A549細(xì)胞在不同的時(shí)間(6h、12h)被保存于100%氧氣及4℃的環(huán)境中,并加入低鉀右旋糖酐培養(yǎng)以模擬缺血的過(guò)程。繼而轉(zhuǎn)入放有10%胎牛血清的DMEM/F-12培養(yǎng)基的溫暖環(huán)境(37℃)孵育2h以模擬再灌注情況。 2.PFC對(duì)IRI誘導(dǎo)的A549凋亡損傷的保護(hù)作用。應(yīng)用流式細(xì)胞儀(FCM)檢測(cè)細(xì)胞凋亡率化及western blotting法檢測(cè)caspase-3蛋白的表達(dá)。將培養(yǎng)傳代的第三、四代A549細(xì)胞分為四組:①對(duì)照組(control group)組:不作任何干預(yù);②IRI組:僅缺血再灌注處理;③PFC(全氟辛烷)組:按30%體積比(PFC:培養(yǎng)液)在細(xì)胞培養(yǎng)中加入全氟辛烷;④PFC+IRI組:按30%體積比(PFC:培養(yǎng)液)在缺血再灌注階段加入全氟辛烷。FCM缺血階段時(shí)間取6h和12h兩個(gè)時(shí)間點(diǎn)的樣本。 結(jié)果 1.細(xì)胞凋亡的FCM結(jié)果:肺缺血再灌注條件作用于A549細(xì)胞6h和12h后的早期細(xì)胞凋亡率分別為10.01%和14.21%;晚期凋亡和壞死率分別為4.93%及5.49%;細(xì)胞存活率分別為84.61%與79.59%。即隨著時(shí)間的推移,細(xì)胞凋亡壞死率逐漸上升,而其細(xì)胞存活率逐漸下降。與對(duì)照組、PFC組和共培養(yǎng)組比較,IRI作用于A549細(xì)胞后的6,12h,其早期細(xì)胞凋亡率、晚期細(xì)胞凋亡和壞死率顯著升高,存活率顯著下降,且12小時(shí)明顯重于6小時(shí);與對(duì)照組比較,PFC兩個(gè)時(shí)相組的細(xì)胞凋亡率和存活率的差異無(wú)統(tǒng)計(jì)學(xué)意義,且兩組之間差異也無(wú)統(tǒng)計(jì)學(xué)意義;與對(duì)照組和全氟化碳組比較,PFC+IRI組A549細(xì)胞的12h早期凋亡率升高,存活率下降,即PFC能明顯拮抗缺血再灌注誘導(dǎo)的凋亡作用,但不能完全阻斷缺血再灌注對(duì)細(xì)胞的促凋亡效應(yīng)。 2. western blotting結(jié)果:IRI作用于A549細(xì)胞0.5,2,6,12h見caspase-3前體降解的17 kDa和11 kDa兩個(gè)活性片段;PFC單獨(dú)作用于A549細(xì)胞,該蛋白的活性和水平無(wú)明顯影響;于PFC+IRI組, caspase-3蛋白的活性顯著下降,提示PFC能夠顯著抑制IRI誘導(dǎo)的此蛋白的激活進(jìn)而阻斷IRI的誘導(dǎo)細(xì)胞凋亡的效應(yīng)。 結(jié)論 1.缺血再灌注作用于A549細(xì)胞可誘導(dǎo)其發(fā)生凋亡的損傷改變,這種效應(yīng)呈時(shí)間依賴。 2.PFC單獨(dú)作用于A549細(xì)胞,不誘導(dǎo)其凋亡損傷的發(fā)生,當(dāng)PFC加入缺血再灌注條件下孵育A549細(xì)胞時(shí),則可以顯著減輕缺血再灌注誘導(dǎo)的A549細(xì)胞凋亡的損傷,增加細(xì)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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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