线粒体钠钙交换蛋白NCLX对血管内皮细胞的保护作用及其机制探讨
发布时间：2018-08-05 19:41
【摘要】：研究背景:血管内皮功能障碍是糖尿病大血管病变的基础,高血糖导致的线粒体功能障碍是血管内皮损伤的主要机制之一。线粒体钠钙交换蛋白NCLX(mitochondrial Na+/Ca2+exchanger,NCLX)是最新发现的钠钙交换蛋白。2010年NCLX蛋白正式命名,随后发现在心肌、胰腺、神经元等组织都有表达并发挥很重要的作用。NCLX定位在线粒体嵴上,其功能是调控线粒体钙外排。内皮细胞中NCLX的表达,定位,以及功能暂无相关研究。方法:PECAM-1(CD31)标记大鼠主动脉内皮,MitoTracker?标记原代大鼠主动脉内皮细胞(RAECs)线粒体,皮尔逊共存系数(Pearson,s co-localization coefficient)计算MitoTracker?和NCLX蛋白荧光定位的重合率。注射STZ诱导糖尿病大鼠模型。体外培养原代大鼠主动脉内皮细胞,siRNA抑制NCLX表达。在大鼠主动脉内皮细胞株(RAECs line)进行慢病毒感染过表达NCLX。给予高糖或过氧化氢刺激。共聚焦显微镜观察高糖诱导细胞线粒体钙动态变化。共聚焦显微镜、Luminometer化学发光仪、Western Blot和ELISA技术观察细胞ROS、ATP、8-OHdG、eNOS和NLRP3炎症小体及其相关因子改变。结果:1)PECAM-1(CD31)标记大鼠主动脉内皮层免疫荧光显示,大鼠主动脉内皮层表达NCLX蛋白。皮尔逊共存系数显示NCLX定位于原代大鼠主动脉内皮细胞线粒体上。2)在STZ诱导糖尿病大鼠模型中,大鼠内皮层NCLX蛋白表达较正常对照组升高。体外高糖培养原代大鼠主动脉内皮细胞48h,同样发现高糖组NCLX蛋白表达升高(P0.05)。3)共聚焦显微镜显示,在高糖(35 mmol/L)短时(800s)刺激下,抑制NCLX表达的siNCLX组较对照组(siControl组)线粒体钙离子升高幅度增加;线粒体钙外排速率下降(P0.05);siNCLX组产生的瞬时ΔROS较对照组明显增多(P0.05)。4)高糖培养24小时,siNCLX组ROS生成、NLRP3表达、Caspase-1表达与IL-1β分泌均较siControl组显著增高(P0.05)。5)慢病毒感染建立过表达NCLX蛋白大鼠主动脉内皮细胞LV-NCLX组和空载体对照LV-vector组。过氧化氢(100umol/L)处理24小时,LV-NCLX组ATP浓度较LV-vector组高(P0.05)。LV-NCLX组较对照LV-vector组8-OHdG表达显著减少,eNOS表达显著升高(P0.05)。结论:大鼠主动脉内皮层表达线粒体钠钙交换蛋白NCLX,并且定位于内皮细胞线粒体上。NCLX对高糖刺激下的血管内皮线粒体有调节作用:NCLX对线粒体钙外排的调节是其作用机制之一;ROS的产生、NLRP3炎症小体的激活是其重要调节通路;过表达NCLX对血管内皮具有保护作用。推测糖尿病大鼠早期血管内皮细胞NCLX表达升高,有助于维持线粒体的功能和稳定性。
[Abstract]:Background: vascular endothelial dysfunction is the basis of diabetic macroangiopathy. Mitochondrial dysfunction induced by hyperglycemia is one of the main mechanisms of vascular endothelial injury. Mitochondrial natriuretic protein NCLX (mitochondrial Na / Ca 2 exchangers NCLX is the newly discovered natriuretic calcium exchanger protein. NCLX protein was officially named in 2010, and then it was found that it was expressed in myocardium, pancreas, neurons and other tissues and played an important role in the mitochondrial crest. Its function is to regulate mitochondrial calcium efflux. The expression, localization and function of NCLX in endothelial cells have not been studied yet. Methods the rat aorta endothelium was labeled with 10% PECAM-1 (CD31). (RAECs) mitochondria of primary rat aortic endothelial cells were labeled, and Pearson's co-localization coefficient was used to calculate Mito Tracker? Coincidence rate of fluorescence localization with NCLX protein. Diabetic rats were induced by STZ injection. Primary cultured rat aortic endothelial cells inhibited NCLX expression in vitro. NCLX was overexpressed by lentivirus infection in rat aortic endothelial cell line (RAECs line). Give high sugar or hydrogen peroxide stimulation. The dynamic changes of calcium in mitochondria induced by high glucose were observed by confocal microscope. Blot and ELISA techniques were used to observe the changes of the inflammatory corpuscles and their related factors in RossATP ~ (8-OHdGN) Enos and NLRP3. Results PECAM-1 (CD31) labeled rat aortic endodermis showed that NCLX protein was expressed in rat aortic endodermis. Pearson coexistence coefficient showed that NCLX was located on mitochondria of primary rat aortic endothelial cells. In diabetic rats induced by STZ, the expression of NCLX protein in rat endodermis was higher than that in normal control group. After cultured with high glucose for 48h, it was also found that the expression of NCLX protein increased (P0.05) in high glucose group (P0.05). The confocal microscope showed that high glucose (35 mmol/L) was stimulated for a short time (800s). The increase of Ca ~ (2 +) in mitochondria in siNCLX group was higher than that in control group (siControl group). Mitochondrial calcium efflux rate decreased (P0.05) transient 螖 ROS production in siNCLX group was significantly higher than that in control group (P0.05). In 24 hours of high glucose culture, the expression of NLRP3 and the expression of Caspase-1 and IL-1 尾 in siNCLX group were significantly higher than those in siControl group (P0.05). The aortic endothelial cells of white rats were treated with LV-NCLX and empty vector control (LV-vector). The concentration of ATP in LV-NCLX group was significantly higher than that in LV-vector group (P0.05). 8-OHdG expression in LV-NCLX group was significantly lower than that in control LV-vector group (P0.05). Conclusion: the rat aorta endocortex expresses mitochondrial natriuretic calcium exchange protein NCLX, and is located on the mitochondria of endothelial cells. NCLX can regulate the mitochondrial calcium efflux of vascular endothelial cells stimulated by high glucose. The activation of NLRP3 inflammatory corpuscles is an important regulatory pathway. Overexpression of NCLX has protective effect on vascular endothelium. It is speculated that the increase of NCLX expression in vascular endothelial cells in early diabetic rats is helpful to maintain the function and stability of mitochondria.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2
本文编号：2166817
[Abstract]:Background: vascular endothelial dysfunction is the basis of diabetic macroangiopathy. Mitochondrial dysfunction induced by hyperglycemia is one of the main mechanisms of vascular endothelial injury. Mitochondrial natriuretic protein NCLX (mitochondrial Na / Ca 2 exchangers NCLX is the newly discovered natriuretic calcium exchanger protein. NCLX protein was officially named in 2010, and then it was found that it was expressed in myocardium, pancreas, neurons and other tissues and played an important role in the mitochondrial crest. Its function is to regulate mitochondrial calcium efflux. The expression, localization and function of NCLX in endothelial cells have not been studied yet. Methods the rat aorta endothelium was labeled with 10% PECAM-1 (CD31). (RAECs) mitochondria of primary rat aortic endothelial cells were labeled, and Pearson's co-localization coefficient was used to calculate Mito Tracker? Coincidence rate of fluorescence localization with NCLX protein. Diabetic rats were induced by STZ injection. Primary cultured rat aortic endothelial cells inhibited NCLX expression in vitro. NCLX was overexpressed by lentivirus infection in rat aortic endothelial cell line (RAECs line). Give high sugar or hydrogen peroxide stimulation. The dynamic changes of calcium in mitochondria induced by high glucose were observed by confocal microscope. Blot and ELISA techniques were used to observe the changes of the inflammatory corpuscles and their related factors in RossATP ~ (8-OHdGN) Enos and NLRP3. Results PECAM-1 (CD31) labeled rat aortic endodermis showed that NCLX protein was expressed in rat aortic endodermis. Pearson coexistence coefficient showed that NCLX was located on mitochondria of primary rat aortic endothelial cells. In diabetic rats induced by STZ, the expression of NCLX protein in rat endodermis was higher than that in normal control group. After cultured with high glucose for 48h, it was also found that the expression of NCLX protein increased (P0.05) in high glucose group (P0.05). The confocal microscope showed that high glucose (35 mmol/L) was stimulated for a short time (800s). The increase of Ca ~ (2 +) in mitochondria in siNCLX group was higher than that in control group (siControl group). Mitochondrial calcium efflux rate decreased (P0.05) transient 螖 ROS production in siNCLX group was significantly higher than that in control group (P0.05). In 24 hours of high glucose culture, the expression of NLRP3 and the expression of Caspase-1 and IL-1 尾 in siNCLX group were significantly higher than those in siControl group (P0.05). The aortic endothelial cells of white rats were treated with LV-NCLX and empty vector control (LV-vector). The concentration of ATP in LV-NCLX group was significantly higher than that in LV-vector group (P0.05). 8-OHdG expression in LV-NCLX group was significantly lower than that in control LV-vector group (P0.05). Conclusion: the rat aorta endocortex expresses mitochondrial natriuretic calcium exchange protein NCLX, and is located on the mitochondria of endothelial cells. NCLX can regulate the mitochondrial calcium efflux of vascular endothelial cells stimulated by high glucose. The activation of NLRP3 inflammatory corpuscles is an important regulatory pathway. Overexpression of NCLX has protective effect on vascular endothelium. It is speculated that the increase of NCLX expression in vascular endothelial cells in early diabetic rats is helpful to maintain the function and stability of mitochondria.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2
【参考文献】
相关期刊论文 前1条
1 NITA Lulia I.;HERSHFINKEL Michal;SEKLER Israel;;Life after the birth of the mitochondrial Na~+/Ca~(2+) exchanger,NCLX[J];Science China(Life Sciences);2015年01期
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