APC通过内质网应激抑制内皮细胞凋亡研究

发布时间：2018-05-27 12:17

本文选题：内质网应激 + 活化蛋白C　；参考：《南京大学》2012年博士论文

【摘要】：背景与目的内毒素诱导ALI (endotoxin-induced acute lung injury)是当前呼吸危重病研究热点之一。内毒素可直接和间接导致肺血管内皮细胞的损伤与凋亡。近年来,活化蛋白C因具备抗凝、抗炎、抗凋亡、保护内皮屏障等多种生物学功能而用于严重脓毒血症治疗,并显著改善该类患者预后。研究证实,活化蛋白C抗凋亡作用与其参与线粒体凋亡途径以及死亡受体凋亡途径有关,但是内质网(endoplasmic reticulum, ER)是否也参与活化蛋白C的抗凋亡机制尚未见报道。本研究旨在探讨APC干预内皮细胞凋亡模型后,是否能够诱导ER应激及其与APC抗凋亡作用的关系,以及是否能够通过诱导ER应激而产生GSK-3β介导的抗凋亡效应,从而提出APC抗凋亡效应的新机制,为APC在内毒素诱导ALI干预治疗中的应用提供更多实验室依据。方法1.利用流式细胞仪及Caspase-3活性检测,观察不同剂量与时间的脂多糖(Lipopolysaccharide, LPS)刺激对HUVECs (human umbilical vein endothelial cell, HUVEC)凋亡的影响,构建LPS诱导的HUVECs凋亡模型；2.利用Western blot技术检测150nM APC干预HUVECs后糖调节蛋白78(glucose-regulated protein 78, GRP78)表达情况,利用流式细胞仪凋亡检测技术观察APC对LPS诱导HUVECs凋亡的影响；3.利用流式细胞仪检测技术及Caspase-3活性检测,观察ER钙离子释放阻滞剂TMB-8 (8-[N, N-diethyla-mino]-octyl-3,4,5-trimethoxy-benzoate, TMB-8),对150nM APC抑制脂多糖(Lipopoly saccharide, LPS)诱导HUVECs凋亡的影响；4.利用Western blot检测技术,观察APC干预LPS诱导HUVECs凋亡后GSK-3β与p53蛋白的变化情况；5.利用RT-PCR与Western blot技术,对预先设计合成好的GSK-3p-siRNA进行验证；利用流式细胞仪检测技术观察沉默GSK-3β基因表达对APC抑制LPS诱导HUVECs凋亡作用的影响。结果(1)以0.1、1、10、20μg/ml LPS刺激HUVECs,可见0.1μg/ml LPS镜下观变化不易察觉,1、10、20μg/ml LPS均可导致显著的细胞形态变化,但10、20μg/mlLPS处理]HUVECs24小时后死亡明显增多；(2)采用150nM APC对LPS预处理24小时的HUVECs进行干预,6小时可见GRP78表达明显升高,并持续至24小时,各时间点GRP78表达与对照组比较均有统计学意义(P0.05),与单纯APC刺激HUVECsGRP78表达比较也具有统计学意义(P0.05); (3) LPS预处理HUVECs24小时后,同时给予150nM APC及50umol/L的TMB-8,6、12、24小时流式细胞仪检测结果中凋亡细胞比例以及Caspase-3活性检测与对照组比较均无统计学意义(P0.05)。(4)1μg/ml LPS处理HUVECs24小时后GSK-3β表达略有增加,p53表达显著增加,在给予150nM APC干预后可见GSK-3β表达显著增加(P0.05),p53表达明显下降。(5)在GSK-3p-siRNA转染细胞中,Caspase-3活性及流式细胞仪检测结果均显示与GSK-3p正常表达细胞比较存在统计学意义(P0.05)；而对GSK-3p-siRNA转染细胞给予APC干预后的Caspase-3活性检测结果与无APC干预的对照组比较无统计学意义(P0.05)。结论本研究明确了APC可作为一种内质网应激原：(1)刺激内皮细胞产生的UPR,可能在APC抑制LPS诱导HUVECs凋亡中发挥细胞保护作用；(2)刺激HUVECs后诱导的短暂内质网源性钙离子释放,对LPS诱导IUVECs凋亡并未产生影响;(3)诱导产生内质网应激,并通过GSK-3β介导实现抑制LPS诱导的内皮细胞凋亡效应。
[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.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R563.8

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