本文選題：內(nèi)質(zhì)網(wǎng)應(yīng)激 + 活化蛋白C��； 參考：《南京大學(xué)》2012年博士論文

【摘要】：背景與目的內(nèi)毒素誘導(dǎo)ALI (endotoxin-induced acute lung injury)是當(dāng)前呼吸危重病研究熱點(diǎn)之一。內(nèi)毒素可直接和間接導(dǎo)致肺血管內(nèi)皮細(xì)胞的損傷與凋亡。近年來,活化蛋白C因具備抗凝、抗炎、抗凋亡、保護(hù)內(nèi)皮屏障等多種生物學(xué)功能而用于嚴(yán)重膿毒血癥治療,并顯著改善該類患者預(yù)后。研究證實(shí),活化蛋白C抗凋亡作用與其參與線粒體凋亡途徑以及死亡受體凋亡途徑有關(guān),但是內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum, ER)是否也參與活化蛋白C的抗凋亡機(jī)制尚未見報(bào)道。本研究旨在探討APC干預(yù)內(nèi)皮細(xì)胞凋亡模型后,是否能夠誘導(dǎo)ER應(yīng)激及其與APC抗凋亡作用的關(guān)系,以及是否能夠通過誘導(dǎo)ER應(yīng)激而產(chǎn)生GSK-3β介導(dǎo)的抗凋亡效應(yīng),從而提出APC抗凋亡效應(yīng)的新機(jī)制,為APC在內(nèi)毒素誘導(dǎo)ALI干預(yù)治療中的應(yīng)用提供更多實(shí)驗(yàn)室依據(jù)。方法1.利用流式細(xì)胞儀及Caspase-3活性檢測(cè),觀察不同劑量與時(shí)間的脂多糖(Lipopolysaccharide, LPS)刺激對(duì)HUVECs (human umbilical vein endothelial cell, HUVEC)凋亡的影響,構(gòu)建LPS誘導(dǎo)的HUVECs凋亡模型；2.利用Western blot技術(shù)檢測(cè)150nM APC干預(yù)HUVECs后糖調(diào)節(jié)蛋白78(glucose-regulated protein 78, GRP78)表達(dá)情況,利用流式細(xì)胞儀凋亡檢測(cè)技術(shù)觀察APC對(duì)LPS誘導(dǎo)HUVECs凋亡的影響；3.利用流式細(xì)胞儀檢測(cè)技術(shù)及Caspase-3活性檢測(cè),觀察ER鈣離子釋放阻滯劑TMB-8 (8-[N, N-diethyla-mino]-octyl-3,4,5-trimethoxy-benzoate, TMB-8),對(duì)150nM APC抑制脂多糖(Lipopoly saccharide, LPS)誘導(dǎo)HUVECs凋亡的影響；4.利用Western blot檢測(cè)技術(shù),觀察APC干預(yù)LPS誘導(dǎo)HUVECs凋亡后GSK-3β與p53蛋白的變化情況；5.利用RT-PCR與Western blot技術(shù),對(duì)預(yù)先設(shè)計(jì)合成好的GSK-3p-siRNA進(jìn)行驗(yàn)證；利用流式細(xì)胞儀檢測(cè)技術(shù)觀察沉默GSK-3β基因表達(dá)對(duì)APC抑制LPS誘導(dǎo)HUVECs凋亡作用的影響。結(jié)果(1)以0.1、1、10、20μg/ml LPS刺激HUVECs,可見0.1μg/ml LPS鏡下觀變化不易察覺,1、10、20μg/ml LPS均可導(dǎo)致顯著的細(xì)胞形態(tài)變化,但10、20μg/mlLPS處理]HUVECs24小時(shí)后死亡明顯增多；(2)采用150nM APC對(duì)LPS預(yù)處理24小時(shí)的HUVECs進(jìn)行干預(yù),6小時(shí)可見GRP78表達(dá)明顯升高,并持續(xù)至24小時(shí),各時(shí)間點(diǎn)GRP78表達(dá)與對(duì)照組比較均有統(tǒng)計(jì)學(xué)意義(P0.05),與單純APC刺激HUVECsGRP78表達(dá)比較也具有統(tǒng)計(jì)學(xué)意義(P0.05); (3) LPS預(yù)處理HUVECs24小時(shí)后,同時(shí)給予150nM APC及50umol/L的TMB-8,6、12、24小時(shí)流式細(xì)胞儀檢測(cè)結(jié)果中凋亡細(xì)胞比例以及Caspase-3活性檢測(cè)與對(duì)照組比較均無統(tǒng)計(jì)學(xué)意義(P0.05)。(4)1μg/ml LPS處理HUVECs24小時(shí)后GSK-3β表達(dá)略有增加,p53表達(dá)顯著增加,在給予150nM APC干預(yù)后可見GSK-3β表達(dá)顯著增加(P0.05),p53表達(dá)明顯下降。(5)在GSK-3p-siRNA轉(zhuǎn)染細(xì)胞中,Caspase-3活性及流式細(xì)胞儀檢測(cè)結(jié)果均顯示與GSK-3p正常表達(dá)細(xì)胞比較存在統(tǒng)計(jì)學(xué)意義(P0.05)；而對(duì)GSK-3p-siRNA轉(zhuǎn)染細(xì)胞給予APC干預(yù)后的Caspase-3活性檢測(cè)結(jié)果與無APC干預(yù)的對(duì)照組比較無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論本研究明確了APC可作為一種內(nèi)質(zhì)網(wǎng)應(yīng)激原：(1)刺激內(nèi)皮細(xì)胞產(chǎn)生的UPR,可能在APC抑制LPS誘導(dǎo)HUVECs凋亡中發(fā)揮細(xì)胞保護(hù)作用；(2)刺激HUVECs后誘導(dǎo)的短暫內(nèi)質(zhì)網(wǎng)源性鈣離子釋放,對(duì)LPS誘導(dǎo)IUVECs凋亡并未產(chǎn)生影響;(3)誘導(dǎo)產(chǎn)生內(nèi)質(zhì)網(wǎng)應(yīng)激,并通過GSK-3β介導(dǎo)實(shí)現(xiàn)抑制LPS誘導(dǎo)的內(nèi)皮細(xì)胞凋亡效應(yīng)。
[Abstract]:Background and objective endotoxin induced ALI (endotoxin-induced acute lung injury) is one of the hotspots in the current research of respiratory critical diseases. Endotoxin can directly and indirectly lead to damage and apoptosis of pulmonary vascular endothelial cells. In recent years, activated protein C has been used for many biological functions, such as anticoagulation, anti-inflammatory, anti apoptosis, protection of endothelial barrier and other biological functions. It has been proved that the anti apoptosis effect of activated protein C is related to its involvement in mitochondrial apoptosis pathway and death receptor apoptosis pathway, but whether endoplasmic reticulum (ER) is also involved in the anti apoptosis mechanism of activated protein C has not yet been reported. The aim of this study is to explore the APC stem. Whether the preendothelial cell apoptosis model can induce ER stress and its relationship with the anti apoptosis effect of APC, and whether it can induce the anti apoptosis effect mediated by GSK-3 beta by inducing ER stress, and then propose a new mechanism of APC anti apoptosis effect, and provide more laboratory basis for the application of APC in inducing ALI dry pretherapy. Method 1. the effects of Lipopolysaccharide (LPS) on apoptosis of HUVECs (human umbilical vein endothelial cell, HUVEC) were observed by flow cytometry and Caspase-3 activity, and HUVECs apoptosis model was constructed by LPS induced HUVECs. 2. The expression of protein 78 (glucose-regulated protein 78, GRP78) was observed by flow cytometry, and the effect of APC on LPS induced HUVECs apoptosis was observed. 3. the flow cytometry and Caspase-3 activity detection were used to observe the TMB-8 (8- [N, N-diethyla-mino]-octyl-3,4,5-trimethoxy-benzoate, ER) calcium release blocker. B-8), the effect of 150nM APC on the inhibition of lipopolysaccharide (Lipopoly saccharide, LPS) induced HUVECs apoptosis; 4. using Western blot detection technique to observe the change of APC intervention LPS inducible HUVECs apoptosis after HUVECs apoptosis; 5. The effect of silent GSK-3 beta gene expression on the inhibitory effect of APC on the inhibition of HUVECs apoptosis induced by LPS was observed by cytometer. Results (1) HUVECs was stimulated with 0.1,1,10,20 mu g/ml LPS, and the apparent changes under 0.1 micron LPS were not easy to be detected. (2) (2) 150nM APC was used for the intervention of HUVECs for 24 hours pretreated by LPS. The expression of GRP78 was obviously increased in 6 hours and lasted for 24 hours. The expression of GRP78 in each time point was statistically significant compared with the control group (P0.05), and was also statistically significant (P0.05) compared with the pure APC stimulation HUVECsGRP78 table (P0.05); (3) LPS pretreatment HUV. After ECs24 hours, the ratio of apoptotic cells and Caspase-3 activity detected by TMB-8,6,12,24 hourly flow cytometer with 150nM APC and 50umol/L were not statistically significant (P0.05). (4) GSK-3 beta expression was slightly increased after 1 u g/ml LPS processing HUVECs24 hours, and p53 expression increased significantly. The expression of GSK-3 beta was significantly increased (P0.05), and the expression of p53 decreased significantly. (5) in GSK-3p-siRNA transfected cells, Caspase-3 activity and flow cytometry showed that there was a statistical significance (P0.05) compared with GSK-3p normal expression cells (P0.05), while GSK-3p-siRNA transfected cells were given APC dry prognosis of Caspase-3 activity detection results. Compared with the control group without APC intervention, there was no statistical significance (P0.05). Conclusion this study showed that APC could be used as an endoplasmic reticulum stress source: (1) the stimulation of UPR produced by endothelial cells may play a cellular protective role in the inhibition of LPS induced HUVECs apoptosis by APC; (2) the release of transient endoplasmic reticulum induced calcium induced by HUVECs and the lure of LPS induced by HUVECs. The apoptosis of IUVECs was not affected. (3) endoplasmic reticulum stress was induced, and the apoptosis induced by LPS was inhibited by GSK-3 beta.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R563.8

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