内质网应激在间歇性低氧小鼠血管内皮功能障碍中的作用

发布时间：2018-08-20 17:14

【摘要】：背景阻塞性睡眠呼吸综合征(obstructive sleep apnea syndrome,OSAS)是一种常见的睡眠呼吸障碍性疾病,是在睡眠过程中反复发生的阻塞性低通气或呼吸暂停。间歇性低氧(intermittent hypoxia,IH)被认为是OSAS的主要特征和重要损伤机制,是引发一系列心血管疾病的关键因素。内质网应激在许多病理生理刺激下(如氧化应激、缺血、缺氧)都可以发生,尤其是参与OSAS相关的疾病如动脉粥样硬化、糖尿病、缺血性心肌病等的发生。内皮细胞凋亡与OSAS患者的血管内皮功能障碍密切相关。除了细胞死亡受体和线粒体依赖的细胞凋亡途径,内质网应激被发现是诱导内皮细胞凋亡的另一种途径。因此,我们推测间歇性低氧可以通过内质网应激介导血管内皮细胞损伤引起血管功能紊乱。目的1.建立IH小鼠模型,应用内质网应激抑制剂,观察IH对小鼠血压和血管舒张功能的影响及IH小鼠胸主动脉中内质网应激相关蛋白的相对表达;2.建立血管内皮细胞IH模型,观察不同程度的间歇性低氧对细胞损伤的影响,并且研究内质网应激在不同程度间歇性低氧导致的细胞凋亡中的作用机制,为防治间歇性低氧导致的血管内皮功能障碍提供依据。方法实验将雄性健康昆明小鼠分为正常对照(Control)组、间歇性低氧(IH)组和内质网应激抑制剂4-苯基丁酸(4-PBA)干预(IH+4-PBA)组,采用套尾法无创测量小鼠尾动脉收缩压(SBP),用血管张力测定技术(myography)检测小鼠胸主动脉的功能,采用蛋白免疫印迹实验(Western Bolt)的方法检测胸主动脉中GRP78和CHOP蛋白表达情况。细胞实验将人脐静脉内皮细胞(HUVECs)分为正常培养(IH 0 h)组、IH 6 h组、IH 12 h组和IH 24 h组,并且对每组应用内质网应激抑制剂4-PBA,用Cell Counting Kit-8(CCK-8)检测细胞的活力,用流式细胞仪检测各组细胞凋亡情况,用酶联免疫吸附测定(ELISA)法测定内皮型一氧化氮合酶(e NOS)的含量。1.内质网应激在间歇性低氧诱发小鼠血压升高中的作用将雄性健康昆明小鼠分为正常对照(Control)组、间歇性低氧(IH)组和间歇性低氧4-PBA干预(IH+4-PBA)组,IH组和IH+4-PBA组放入低氧仓进行间歇性低氧处理,每天8 h,连续28 d,间歇性低氧处理最后一周给予小鼠腹腔注射4-PBA(100mg/kg/d),Control组不放入低氧仓,其余条件和处理组相同。28天后采用套尾法无创测量小鼠尾动脉收缩压(SBP)。2.内质网应激在间歇性低氧引起的小鼠血管内皮功能障碍中的作用采用离体血管张力测定的方法检测三组小鼠胸主动脉内皮依赖性舒张和内皮非依赖性舒张变化情况,观察小鼠动脉的舒缩功能变化。3.间歇性低氧对小鼠胸主动脉中内质网应激相关蛋白表达的影响采用Western Bolt法检测三组小鼠胸主动脉内质网应激相关蛋白GRP78和CHOP蛋白的表达水平。4.内质网应激在间歇性低氧对人脐静脉内皮细胞活力影响中的作用将HUVECs正常培养12 h后,换成无血清培养液。设IH 0 h组、IH6 h组、IH12 h组和IH24 h组,并且对每组应用内质网抑制剂4-PBA(2 mmol/L),用Cell Counting Kit-8(CCK-8)检测细胞的活力,观察间歇性低氧及应用内质网应激抑制剂4-PBA对细胞活力的影响。5.内质网应激在间歇性低氧对人脐静脉内皮细胞凋亡影响中的作用将HUVECs分为IH 0 h组、IH6 h组、IH12 h组和IH24 h组,并且对每组应用内质网抑制剂4-PBA,用流式细胞仪检测细胞的凋亡变化情况。6.内质网应激在间歇性低氧对人脐静脉内皮细胞内e NOS含量影响中的作用用ELISA法测量各组细胞中内皮型一氧化氮合酶(e NOS)的含量,观察间歇性低氧和4-PBA对内皮细胞产生的e NOS含量的影响。结果1.间歇性低氧可以导致小鼠血压升高,其作用可被4-PBA抑制。2.间歇性低氧可以导致小鼠胸主动脉内皮依赖性舒张减弱,4-PBA可改善IH小鼠的胸主动脉内皮依赖性舒张功能;内皮非依赖性舒张无差异。3.间歇性低氧导致小鼠胸主动脉GRP78表达下降,4-PBA导致其表达增加,CHOP蛋白表达增加,4-PBA导致其表达下降。4.间歇性低氧可以使HUVECs活力下降,其作用可被4-PBA抑制;5.间歇性低氧6 h细胞凋亡没有发生变化,长时间间歇性低氧(12 h和24 h)使细胞凋亡增加,应用4-PBA可以抑制间歇性低氧导致的细胞凋亡;6.长时间间歇性低氧(12 h和24 h)可以使内皮细胞产生的e NOS含量减少,其作用可被4-PBA抑制。结论间歇性低氧可引起血管内皮细胞内质网应激,引起细胞凋亡、细胞e NOS水平降低和血管舒张功能减退,导致血压升高。
[Abstract]:BACKGROUND Obstructive sleep apnea syndrome (OSAS) is a common sleep-related respiratory disorder, which occurs repeatedly during sleep with obstructive hypopnea or apnea. Intermittent hypoxia (IH) is considered to be the main feature and important injury mechanism of OSAS, and is the primary cause of OSAS. Endoplasmic reticulum stress can occur under many pathophysiological stimuli, such as oxidative stress, ischemia and hypoxia, especially in OSAS-related diseases such as atherosclerosis, diabetes, ischemic cardiomyopathy. Endothelial cell apoptosis is closely related to vascular endothelial dysfunction in OSAS patients. Endoplasmic reticulum stress has been found to be another way of inducing endothelial cell apoptosis. Therefore, we speculate that intermittent hypoxia may induce vascular dysfunction by endothelial cell injury mediated by endoplasmic reticulum stress. Objective 1. To establish an IH mouse model and apply endoplasmic reticulum stress. Inhibitors, to observe the effects of IH on blood pressure and vasodilator function in mice and the relative expression of ER stress-related proteins in thoracic aorta of IH mice; 2. To establish vascular endothelial cell IH model, to observe the effect of intermittent hypoxia on cell injury, and to study the fine endoplasmic reticulum stress induced by intermittent hypoxia in different degrees. Methods The male healthy Kunming mice were divided into normal control group, intermittent hypoxia (IH) group and endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) intervention group. Systolic blood pressure (SBP) was measured by myography. The expression of GRP78 and CHOP in thoracic aorta was detected by Western Bolt. Human umbilical vein endothelial cells (HUVECs) were divided into normal culture (IH 0 h), IH 6 h, IH 12 h and IH 2 groups by cell experiment. Cell Counting Kit-8 (CCK-8) was used to detect cell viability. Cell apoptosis was detected by flow cytometry. The content of endothelial nitric oxide synthase (e NOS) was measured by enzyme-linked immunosorbent assay (ELISA). 1. Blood pressure was induced by endoplasmic reticulum stress in intermittent hypoxia in mice. Male healthy Kunming mice were divided into normal control group, intermittent hypoxia (IH) group and intermittent hypoxia 4-PBA intervention (IH+4-PBA) group. IH group and IH+4-PBA group were put into hypoxic chamber for intermittent hypoxia treatment, 8 hours a day for 28 days, intermittent hypoxia treatment was given intraperitoneal injection of 4-PBA (100mg/kg/d) for the last week. Endoplasmic reticulum stress was used to detect endothelial dysfunction induced by intermittent hypoxia in mice. Endothelial relaxation of thoracic aorta in three groups was measured by in vitro vascular tension measurement. Effects of intermittent hypoxia on the expression of ER stress-related proteins in thoracic aorta of mice were detected by Western Bolt method. The expression levels of ER stress-related proteins GRP78 and CHOP in thoracic aorta of three groups of mice were detected. Effects of intermittent hypoxia on the viability of human umbilical vein endothelial cells (HUVECs) After 12 hours of normal culture, HUVECs were replaced with serum-free medium. IH 0 h group, IH 6 h group, IH 12 h group and IH 24 h group were set up. Endoplasmic reticulum inhibitor 4-PBA (2 mmol/L) and Cell Counting Kit-8 (CCK-8) were used to detect the viability of HUVECs and observe intermittent hypoxia and stress. The effect of ER stress on the apoptosis of human umbilical vein endothelial cells was studied. 5. The effects of ER stress on the apoptosis of human umbilical vein endothelial cells were divided into IH 0 h group, IH 6 h group, IH 12 h group and IH 24 h group. Effect of endoplasmic reticulum stress on the content of E NOS in human umbilical vein endothelial cells under intermittent hypoxia Intermittent hypoxia can reduce the endothelium-dependent relaxation of thoracic aorta in mice. 4-PBA can improve the endothelium-dependent relaxation of thoracic aorta in IH mice. Endothelial-independent relaxation has no difference. 3. Intermittent hypoxia can decrease the expression of GRP78 in thoracic aorta, 4-PBA can increase the expression of GRP78 and CHOP protein. Intermittent hypoxia can inhibit the activity of HUVECs, which can be inhibited by 4-PBA. 5. Intermittent hypoxia can inhibit the apoptosis of HUVECs for 6 h without any change. Long-term intermittent hypoxia (12 h and 24 h) can increase the apoptosis of HUVECs. 4-PBA can inhibit the apoptosis of HUVECs caused by intermittent hypoxia. Hypoxia (12 h and 24 h) can reduce the content of E NOS produced by endothelial cells, and its effect can be inhibited by 4-PBA. Conclusion Intermittent hypoxia can induce endoplasmic reticulum stress in vascular endothelial cells, induce apoptosis, decrease the level of E NOS and decrease the vasodilator function, and lead to elevated blood pressure.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766

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