【摘要】：研究背景:血管內(nèi)皮功能障礙是糖尿病大血管病變的基礎(chǔ),高血糖導(dǎo)致的線粒體功能障礙是血管內(nèi)皮損傷的主要機(jī)制之一。線粒體鈉鈣交換蛋白NCLX(mitochondrial Na+/Ca2+exchanger,NCLX)是最新發(fā)現(xiàn)的鈉鈣交換蛋白。2010年NCLX蛋白正式命名,隨后發(fā)現(xiàn)在心肌、胰腺、神經(jīng)元等組織都有表達(dá)并發(fā)揮很重要的作用。NCLX定位在線粒體嵴上,其功能是調(diào)控線粒體鈣外排。內(nèi)皮細(xì)胞中NCLX的表達(dá),定位,以及功能暫無(wú)相關(guān)研究。方法:PECAM-1(CD31)標(biāo)記大鼠主動(dòng)脈內(nèi)皮,MitoTracker?標(biāo)記原代大鼠主動(dòng)脈內(nèi)皮細(xì)胞(RAECs)線粒體,皮爾遜共存系數(shù)(Pearson,s co-localization coefficient)計(jì)算MitoTracker?和NCLX蛋白熒光定位的重合率。注射STZ誘導(dǎo)糖尿病大鼠模型。體外培養(yǎng)原代大鼠主動(dòng)脈內(nèi)皮細(xì)胞,siRNA抑制NCLX表達(dá)。在大鼠主動(dòng)脈內(nèi)皮細(xì)胞株(RAECs line)進(jìn)行慢病毒感染過(guò)表達(dá)NCLX。給予高糖或過(guò)氧化氫刺激。共聚焦顯微鏡觀察高糖誘導(dǎo)細(xì)胞線粒體鈣動(dòng)態(tài)變化。共聚焦顯微鏡、Luminometer化學(xué)發(fā)光儀、Western Blot和ELISA技術(shù)觀察細(xì)胞ROS、ATP、8-OHdG、eNOS和NLRP3炎癥小體及其相關(guān)因子改變。結(jié)果:1)PECAM-1(CD31)標(biāo)記大鼠主動(dòng)脈內(nèi)皮層免疫熒光顯示,大鼠主動(dòng)脈內(nèi)皮層表達(dá)NCLX蛋白。皮爾遜共存系數(shù)顯示NCLX定位于原代大鼠主動(dòng)脈內(nèi)皮細(xì)胞線粒體上。2)在STZ誘導(dǎo)糖尿病大鼠模型中,大鼠內(nèi)皮層NCLX蛋白表達(dá)較正常對(duì)照組升高。體外高糖培養(yǎng)原代大鼠主動(dòng)脈內(nèi)皮細(xì)胞48h,同樣發(fā)現(xiàn)高糖組NCLX蛋白表達(dá)升高(P0.05)。3)共聚焦顯微鏡顯示,在高糖(35 mmol/L)短時(shí)(800s)刺激下,抑制NCLX表達(dá)的siNCLX組較對(duì)照組(siControl組)線粒體鈣離子升高幅度增加;線粒體鈣外排速率下降(P0.05);siNCLX組產(chǎn)生的瞬時(shí)ΔROS較對(duì)照組明顯增多(P0.05)。4)高糖培養(yǎng)24小時(shí),siNCLX組ROS生成、NLRP3表達(dá)、Caspase-1表達(dá)與IL-1β分泌均較siControl組顯著增高(P0.05)。5)慢病毒感染建立過(guò)表達(dá)NCLX蛋白大鼠主動(dòng)脈內(nèi)皮細(xì)胞LV-NCLX組和空載體對(duì)照LV-vector組。過(guò)氧化氫(100umol/L)處理24小時(shí),LV-NCLX組ATP濃度較LV-vector組高(P0.05)。LV-NCLX組較對(duì)照LV-vector組8-OHdG表達(dá)顯著減少,eNOS表達(dá)顯著升高(P0.05)。結(jié)論:大鼠主動(dòng)脈內(nèi)皮層表達(dá)線粒體鈉鈣交換蛋白NCLX,并且定位于內(nèi)皮細(xì)胞線粒體上。NCLX對(duì)高糖刺激下的血管內(nèi)皮線粒體有調(diào)節(jié)作用:NCLX對(duì)線粒體鈣外排的調(diào)節(jié)是其作用機(jī)制之一;ROS的產(chǎn)生、NLRP3炎癥小體的激活是其重要調(diào)節(jié)通路;過(guò)表達(dá)NCLX對(duì)血管內(nèi)皮具有保護(hù)作用。推測(cè)糖尿病大鼠早期血管內(nèi)皮細(xì)胞NCLX表達(dá)升高,有助于維持線粒體的功能和穩(wěn)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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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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本文編號(hào)：2166817