肾素（前体）受体系统致人脐静脉内皮细胞损伤机制的研究

发布时间：2018-05-20 09:10

  本文选题：人脐静脉内皮细胞 + 肾素(前体)受体　； 参考：《大连医科大学》2010年硕士论文

【摘要】： 近年来,人们发现了肾素-血管紧张素系统(renin angitensin system,RAS)中另一活性物质：肾素(前体)受体。目前人们克隆出两种肾素(前体)受体,一种是6-磷酸甘露糖/Ⅱ型胰岛素样生长因子受体(mainnose-6-phosphate/insulin-like growth factor II receptor,M6P/IGF2R),被人们认为在肾素(前体)的清除机制中发挥重要作用。另一种是功能型受体：肾素(前体)受体[(pro)renin receptor, (P)RR]。(P)RR在心肾疾病中的作用越来越受到人们的关注,现已证实其存在于肾系膜细胞、血管平滑肌细胞、肾脏、心脏及大脑等细胞组织中。该受体与肾素(前体)结合后,可以通过不依赖于传统的血管紧张素Ⅱ(Angiotensin-Ⅱ,Ang-Ⅱ)途径,激活细胞内信号转导通路,上调某些基因的表达。丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)是细胞内的一类丝氨酸/苏氨酸蛋白激酶,其存在于大多数细胞内,可将细胞外刺激信号转导至细胞及其核内,并引起细胞生物学反应(如细胞增殖、分化、转化及凋亡等),哺乳动物细胞中已鉴定的3个主要的MAPK亚族包括：细胞外信号调节蛋白激酶(extracellular signal-regulated kinase 1 and 2,ERK1/2)亚族,p38丝裂原活化蛋白激酶(p38 MAPKs)亚族和c-Jun氨基末端激酶(JNK)亚族,其中细胞外信号调节激酶ERK1/2及p38 MAPKs信号转导通路在炎症和氧化应激中起重要作用。本课题组前期工作已证实(P)RR存在于人脐静脉内皮细胞中(human umbilical vein endothelian cells, HUVECs),而(P)RR在HUVECs中对炎症因子VCAM-1和氧化应激指标SOD的影响及干扰(P)RR表达是否可抑制(P)RR对VCAM-1及SOD的影响尚无报道,(P)RR致炎症反应及氧化应激作用是否与信号通路ERK1/2及P38 MAPKs有联系还不清楚。扰(P)RR后对ERK1/2及p38 MAPKs信号通路蛋白表达、血管细胞间粘附分子(vascular cell adhesion molecule-1,VCAM-1)蛋白表达及超氧化物歧化酶(superoxide dismutase, SOD)浓度的影响,从而探讨其致HUVECs损伤的机制,阐述其与动脉粥样硬化(atherosclerosis, AS)等伴有血管内皮损伤性心血管疾病的关系。 方法： 1.体外培养原代HUVECs,CD34流式细胞鉴定。 2.在HUVECs中以siRNA干扰(P)RR,用RT-PCR方法检测干扰24,48及72小时后(P)RR的mRNA表达,确定干扰效率最佳时间点。 3.以奥美沙坦和PD123319阻断Ang-Ⅱ受体AT1、AT2。分别以人重组肾素(renin)及肾素前体(prorenin)处理HUVECs,于处理后5,10,20,35,60,90分钟收取蛋白,用western blot法检测ERK1/2及p38信号通路蛋白磷酸化程度,确定肾素及其前体刺激致两信号通路蛋白磷酸化的最强时间点。 4.以奥美沙坦和PD123319处理HUVECs30分钟后,分别以人重组肾素及肾素前体刺激HUVECs,分别用western blot法,ELISA法和比色法观察肾素及其前体是否可使信号通路蛋白ERK1/2及p38磷酸化增强、粘附分子VCAM-1表达增高、抗氧化酶SOD含量降低,以及干扰(P)RR表达后是否可阻断或减弱肾素及其前体的上述作用。 结果： 1.CD34流式细胞鉴定,HUVECs百分率达98.35%。 2.RT-PCR结果证实(P)RR的mRNA在HUVECs中有表达,siRNA干扰(P)RR后72小时,(P)RR mRNA在HUVECs中表达最弱。 3.经肾素前体及肾素处理HUVECs后,信号通路蛋白ERK1/2及p38磷酸化水平增强,在肾素作用于细胞后20分钟,ERK及p38磷酸化程度最强；分别在肾素前体作用于细胞后60分钟及35分钟,ERK及p38磷酸化最强。 4.人重组肾素及肾素前体刺激HUVECs,可增高信号通路蛋白ERK1/2及p38磷酸化水平,上调粘附分子VCAM-1表达,降低抗氧化酶SOD含量。干扰(P)RR后上述作用减弱。 结论： (P)RR存在于HUVECs中。在HUVECs中,肾素及其前体通过(P)RR途径可减少抗氧化酶SOD含量,同时激活ERK1/2及p38MAPKs信号通路,上调炎症因子VCAM-1蛋白表达,从而引起内皮细胞损伤,导致AS等相关疾病的发生。干扰(P)RR表达,可有效抑制以上作用,发挥内皮保护作用,为临床治疗RAS过度激活所致的伴有内皮损伤的疾病提供新的理论依据。
[Abstract]:In recent years, another active substance in the renin - angiotensin system (renin angitensin system, RAS), the renin (precursor) receptor, has been found. Two kinds of renin (precursor) receptors are cloned, one is 6- phosphoric mannose / type II insulin-like growth factor receptor (mainnose-6-phosphate/insulin-like growth factor II receptor, M6P/IGF2R) is believed to play an important role in the mechanism of renin (precursor) scavenging. The other is the functional receptor: the renin (pro) renin receptor, (P) RR]. (P) RR in the heart and kidney disease is becoming more and more concerned. It has been confirmed in the renal mesangial cells, vascular smooth muscle cells, kidneys, and heart. In combination with the renin (precursor), the receptor can activate the intracellular signal transduction pathway by not relying on the traditional angiotensin II (Angiotensin- II) (Ang- II) pathway. The mitogen activated protein kinase (mitogen-activated protein kinases, MAPKs) is a class of cells. Serine / threonine protein kinase (serine / threonine protein kinase), which exists in most cells, can transduce extracellular stimulation signals into cells and their nuclei, and causes cell biological reactions (such as cell proliferation, differentiation, transformation and apoptosis). The 3 major MAPK subgroups identified in mammalian cells include extracellular signal regulated protein kinase (Extracel) Lular signal-regulated kinase 1 and 2, ERK1/2) subgroup, p38 mitogen activated protein kinase (p38 MAPKs) subgroup and c-Jun amino terminal kinase (JNK) subgroup, in which extracellular signal regulated kinase ERK1/2 and p38 signal transduction pathways play a role in inflammation and oxidative stress. In umbilical vein endothelial cells (human umbilical vein endothelian cells, HUVECs), and (P) RR in HUVECs on the inflammatory factor VCAM-1 and oxidative stress indicator SOD, and whether the interference (P) expression has no effect on the effect of the inflammatory reaction and oxidative stress The relationship between MAPKs and ERK1/2 and p38 MAPKs signaling pathway protein expression, the expression of intercellular adhesion molecules (vascular cell adhesion molecule-1, VCAM-1) and the concentration of superoxide dismutase (superoxide) after disturbing (P) RR are discussed. Atherosclerosis (AS) is associated with vascular endothelial injury and cardiovascular disease.
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