本文選題：胰島細(xì)胞　切入點(diǎn)：缺氧　出處：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的觀察不同時(shí)間持續(xù)缺氧孵育83213胰島β細(xì)胞后,自噬相關(guān)蛋白Beclin1、LC3BII、P62的表達(dá)情況,從而判斷自噬的活性變化。方法1.將符合實(shí)驗(yàn)條件的83213胰島細(xì)胞作為研究對象,隨機(jī)分為常氧組(對照組)、缺氧組(處理組),對照組用常氧(21%O_2)培養(yǎng),缺氧組為持續(xù)缺氧(1%O_2,5%CO_2)2h、3h、6h、12h、24h、48h胰島細(xì)胞,提取各組胰島細(xì)胞的總蛋白并檢測其濃度。2.采用Western Blot蛋白印記方法測定對照組和缺氧組缺氧誘導(dǎo)因子HIF-1a蛋白的表達(dá),以反映常氧組及缺氧組各時(shí)相點(diǎn)HIF-1α的動(dòng)態(tài)變化。3.采用Western Blot蛋白印記方法,檢測常氧組、缺氧組自噬相關(guān)蛋白Beclin1、LC3BⅡ、P62表達(dá)情況,以反映對照組及缺氧處理后各時(shí)相點(diǎn)自噬水平的動(dòng)態(tài)變化。結(jié)果1.與常氧組比較,缺氧2h、3h、6h、12h、24h、48h組HIF-1α蛋白表達(dá)均升高,在24h達(dá)高峰,且差異均有統(tǒng)計(jì)學(xué)意義(P0.05)2.與常氧組比較,缺氧2h胰島細(xì)胞的Beclin1水平升高,于缺氧3h達(dá)高峰,缺氧6h時(shí)有下降趨勢,但仍高于常氧組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);缺氧12h、24h、48h組Beclin1水平均低于常氧組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。與常氧組比較,自噬相關(guān)蛋白LC3BⅡ在缺氧2h、3h、6h升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05),并在3h達(dá)峰,自缺氧12h后LC3BⅡ逐漸下降,且低于常氧組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。與常氧組比較,缺氧2h、3h組P62蛋白降低,缺氧3h與之相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),于缺氧6h逐漸升高,缺氧12h、24h、48h組差異有統(tǒng)計(jì)學(xué)意義(P0.05),并在缺氧24h達(dá)高峰。結(jié)論1.在持續(xù)缺氧情況下,胰島β細(xì)胞的HIF-1α表達(dá)水平增加。2.在一定時(shí)間缺氧情況下,自噬相關(guān)蛋白Beclin1和LC3BⅡ表達(dá)水平增加,P62表達(dá)水平下降,反映胰島β細(xì)胞自噬增強(qiáng);但超過某個(gè)時(shí)間點(diǎn),Beclin1和LC3BⅡ表達(dá)水平下降,P62表達(dá)水平上升,反映隨著缺氧時(shí)間進(jìn)一步延長,自噬不再增強(qiáng),反而減弱。細(xì)胞的生存狀態(tài)與缺氧的時(shí)間和強(qiáng)度有關(guān)。在短時(shí)間缺氧條件下,自噬可能參與對胰島β細(xì)胞的保護(hù)。
[Abstract]:Objective to observe the expression of autophagy associated protein Beclin1, LC3BIIP62, and determine the change of autophagy activity after incubating 83213 islet 尾 cells with hypoxia for different periods. Methods 1. 83213 islet cells, which meet the experimental conditions, were selected as the research objects. They were randomly divided into normoxic group (control group) and hypoxic group (treatment group, control group) cultured with normoxic oxygen 21 and O _ 2. Hypoxia group was treated with continuous hypoxia of 1O _ 2 and 5 cells. The total protein of islet cells in each group was extracted and its concentration was determined. The expression of hypoxia inducible factor (HIF-1a) protein in control group and hypoxia group was determined by Western Blot protein imprinting method. To reflect the dynamic changes of HIF-1 偽 in normoxic group and hypoxia group, the expression of HIF-1 偽 was detected by Western Blot protein imprinting method in normal oxygen group and hypoxia group. Results 1.Compared with the normoxic group, the expression of HIF-1 偽 protein in the hypoxia group (2 h ~ 3 h ~ 6 h ~ 6 h ~ 12 h ~ 24 h ~ 48 h) increased and reached its peak at 24 h, and the difference was statistically significant compared with the normoxic group. 2. The level of Beclin1 in islet cells increased at 2h after hypoxia, peaked at 3h, decreased at 6h, but still higher than that in normoxic group (P 0.05), and the Beclin1 level was lower in 12h ~ 24h ~ 48h hypoxia group than in normoxic group (P < 0.05). Compared with the normoxic group, the level of autophagy associated protein LC3B 鈪，

本文編號：1700285