本文選題：人臍靜脈內(nèi)皮細胞 + 腎素(前體)受體�。� 參考：《大連醫(yī)科大學》2010年碩士論文

【摘要】： 近年來,人們發(fā)現(xiàn)了腎素-血管緊張素系統(tǒng)(renin angitensin system,RAS)中另一活性物質(zhì)：腎素(前體)受體。目前人們克隆出兩種腎素(前體)受體,一種是6-磷酸甘露糖/Ⅱ型胰島素樣生長因子受體(mainnose-6-phosphate/insulin-like growth factor II receptor,M6P/IGF2R),被人們認為在腎素(前體)的清除機制中發(fā)揮重要作用。另一種是功能型受體：腎素(前體)受體[(pro)renin receptor, (P)RR]。(P)RR在心腎疾病中的作用越來越受到人們的關注,現(xiàn)已證實其存在于腎系膜細胞、血管平滑肌細胞、腎臟、心臟及大腦等細胞組織中。該受體與腎素(前體)結合后,可以通過不依賴于傳統(tǒng)的血管緊張素Ⅱ(Angiotensin-Ⅱ,Ang-Ⅱ)途徑,激活細胞內(nèi)信號轉(zhuǎn)導通路,上調(diào)某些基因的表達。絲裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)是細胞內(nèi)的一類絲氨酸/蘇氨酸蛋白激酶,其存在于大多數(shù)細胞內(nèi),可將細胞外刺激信號轉(zhuǎn)導至細胞及其核內(nèi),并引起細胞生物學反應(如細胞增殖、分化、轉(zhuǎn)化及凋亡等),哺乳動物細胞中已鑒定的3個主要的MAPK亞族包括：細胞外信號調(diào)節(jié)蛋白激酶(extracellular signal-regulated kinase 1 and 2,ERK1/2)亞族,p38絲裂原活化蛋白激酶(p38 MAPKs)亞族和c-Jun氨基末端激酶(JNK)亞族,其中細胞外信號調(diào)節(jié)激酶ERK1/2及p38 MAPKs信號轉(zhuǎn)導通路在炎癥和氧化應激中起重要作用。本課題組前期工作已證實(P)RR存在于人臍靜脈內(nèi)皮細胞中(human umbilical vein endothelian cells, HUVECs),而(P)RR在HUVECs中對炎癥因子VCAM-1和氧化應激指標SOD的影響及干擾(P)RR表達是否可抑制(P)RR對VCAM-1及SOD的影響尚無報道,(P)RR致炎癥反應及氧化應激作用是否與信號通路ERK1/2及P38 MAPKs有聯(lián)系還不清楚。擾(P)RR后對ERK1/2及p38 MAPKs信號通路蛋白表達、血管細胞間粘附分子(vascular cell adhesion molecule-1,VCAM-1)蛋白表達及超氧化物歧化酶(superoxide dismutase, SOD)濃度的影響,從而探討其致HUVECs損傷的機制,闡述其與動脈粥樣硬化(atherosclerosis, AS)等伴有血管內(nèi)皮損傷性心血管疾病的關系。 方法： 1.體外培養(yǎng)原代HUVECs,CD34流式細胞鑒定。 2.在HUVECs中以siRNA干擾(P)RR,用RT-PCR方法檢測干擾24,48及72小時后(P)RR的mRNA表達,確定干擾效率最佳時間點。 3.以奧美沙坦和PD123319阻斷Ang-Ⅱ受體AT1、AT2。分別以人重組腎素(renin)及腎素前體(prorenin)處理HUVECs,于處理后5,10,20,35,60,90分鐘收取蛋白,用western blot法檢測ERK1/2及p38信號通路蛋白磷酸化程度,確定腎素及其前體刺激致兩信號通路蛋白磷酸化的最強時間點。 4.以奧美沙坦和PD123319處理HUVECs30分鐘后,分別以人重組腎素及腎素前體刺激HUVECs,分別用western blot法,ELISA法和比色法觀察腎素及其前體是否可使信號通路蛋白ERK1/2及p38磷酸化增強、粘附分子VCAM-1表達增高、抗氧化酶SOD含量降低,以及干擾(P)RR表達后是否可阻斷或減弱腎素及其前體的上述作用。 結果： 1.CD34流式細胞鑒定,HUVECs百分率達98.35%。 2.RT-PCR結果證實(P)RR的mRNA在HUVECs中有表達,siRNA干擾(P)RR后72小時,(P)RR mRNA在HUVECs中表達最弱。 3.經(jīng)腎素前體及腎素處理HUVECs后,信號通路蛋白ERK1/2及p38磷酸化水平增強,在腎素作用于細胞后20分鐘,ERK及p38磷酸化程度最強；分別在腎素前體作用于細胞后60分鐘及35分鐘,ERK及p38磷酸化最強。 4.人重組腎素及腎素前體刺激HUVECs,可增高信號通路蛋白ERK1/2及p38磷酸化水平,上調(diào)粘附分子VCAM-1表達,降低抗氧化酶SOD含量。干擾(P)RR后上述作用減弱。 結論： (P)RR存在于HUVECs中。在HUVECs中,腎素及其前體通過(P)RR途徑可減少抗氧化酶SOD含量,同時激活ERK1/2及p38MAPKs信號通路,上調(diào)炎癥因子VCAM-1蛋白表達,從而引起內(nèi)皮細胞損傷,導致AS等相關疾病的發(fā)生。干擾(P)RR表達,可有效抑制以上作用,發(fā)揮內(nèi)皮保護作用,為臨床治療RAS過度激活所致的伴有內(nèi)皮損傷的疾病提供新的理論依據(jù)。
[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.
Method錛，

本文編號：1914019