本文選題：子癇前期 + 腎足細胞�。� 參考：《復旦大學》2014年碩士論文

【摘要】：第一部分Ang-(1-7)對子癇前期足細胞損傷的保護作用目的：近年來腎素-血管緊張素系統(tǒng)(RAS)在子癇前期腎臟損傷中的作用被逐漸認識。Ang-(1-7)是RAS系統(tǒng)一個重要的活性七肽,主要作用于G-蛋白偶聯(lián)的Mas受體發(fā)揮作用,在體內(nèi)主要發(fā)揮拮抗AngⅡ的作用。許多研究顯示足細胞的直接損傷參與了子癇前期蛋白尿的產(chǎn)生,提示子癇前期腎臟損傷是足細胞病。我們前期研究顯示與正常妊娠孕婦相比,子癇前期患者血清及尿液中Ang-(1-7)水平降低,且其水平與足細胞損傷及蛋白尿的發(fā)生密切相關,表明循環(huán)及腎臟局部降低的Ang-(1-7)是子癇前期腎臟損傷的原因之一。在本研究中我們借助體外足細胞培養(yǎng),誘導子癇前期體外足細胞損傷模型,進一步探討Ang-(1-7)在子癇前期腎小球足細胞損傷中的作用。方法：應用體外細胞培養(yǎng)技術,培養(yǎng)條件性永生的人腎臟足細胞,收集子癇前期患者血清及正常妊娠孕婦血清刺激足細胞,并于子癇前期患者血清中加入不同濃度Ang-(1-7)與足細胞共孵育,應用CCK8試劑盒檢測足細胞活性,Western blot檢測足細胞Mas受體的表達,以及足細胞標志蛋白(nephrin、podocin、WT-1)的表達,免疫熒光觀察足細胞骨架蛋白F-actin的變化,流式細胞儀檢測足細胞的凋亡。加入Mas受體拮抗劑A779后,觀察上述變化。結果：1.足細胞活性檢測：與FBS孵育足細胞相比,正常妊娠孕婦及子癇前期患者血清干預足細胞均可使其活性降低,但都高于90%；在FBS中加入不同濃度Ang(1.7)(10-5,10-6,10-8M)及A779(10-5,10-6,10-7M),足細胞活性沒有明顯變化。2.Mas受體的表達：免疫印跡結果顯示人足細胞株表達Mas受體。3.足細胞標志蛋白表達的變化：與正常妊娠組比較,子癇前期組足細胞nephrin,WT-1蛋白表達降低(nephrin:0.42±0.04-fold VS 0.64±0.07-fold of GAPDH,尸 0.005;WT-1:0.28±0.05-fold VS 0.46±0.09-fold of GAPDH,P0.05), podocin蛋白表達未有變化。4.不同濃度Ang-(1-7)對子癇前期足細胞標志蛋白表達的影響：在子癇前期患者血清中加入不同濃度的Ang-(1-7),Ang-(1-7)在10-6M濃度對足細胞有最大保護作用,nephrin.WT-1表達增加(nephrin:0.57±0.07-fold VS 0.42±0.04-fold of GAPDH,P0.05；WT-1:0.41±0.04-fold VS 0.28±0.05=fold of GAPDH,P0.05)。Mas受體拮抗劑A779可拮抗Ang-(1-7)對足細胞的保護作用,與PE+Ang-(1-7)相比,加入A779后,nephrin、WT-1表達減少(nephrin:1.55± 0.20-fol VS 2.1±0.19-fold of GAPDH,P0.01；WT.1:0.60±0.08-fold VS 0.84 ±0.13-fold of GAPDH,P0.01)。5.足細胞標志蛋白在基因水平的表達變化：與正常妊娠組相比,子癇前期組足細胞rephrin、WT=1在mRNA水平表達降低(nephrin:23.28±3.94 VS 7.16± 0.65,P0.005；WT-1:2.01±0.0.42 VS 1.03±2.23,P0.05).加入Ang-(1-7)后,nephrin與WT-1 mRNA表達增加(nephrin:12.78±3.89,P0.005;WT-1:2.23±0.29,P0.05),A779可拮抗上述作用。6.Ang-(1-7)對足細胞骨架蛋白F-actin的影響：與正常妊娠組比較,子癇前期組足細胞骨架蛋白F-actin表達減少,排列紊亂,子癇前期患者血清中加入Ang-(1-7)可緩解子癇前期患者血清引起的足細胞骨架重排。A779可拮抗Ang-(1-7)的作用。7.Ang-(1-7)對足細胞凋亡的影響：與正常妊娠組(2.36+1.050%o)比較,子癇前期組足細胞凋亡增加(8.55±0.68%,P0.001)。子癇前期患者血清中加入Ang-(1-7)后,足細胞凋亡明顯減少(4.47+0.730%)。A779可拮抗Ang-(1-7)的作用。結論：Ang-(1-7)可緩解子癇前期患者血清引起的足細胞損傷。第二部分MAPK通路在Ang-(1-7)對子癇前期足細胞影響中的機制作用目的：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,存在于大多數(shù)細胞內(nèi),能夠將細胞外刺激信號轉導至細胞及其核內(nèi),并引起細胞生物學反應(如細胞增殖、分化、轉化及凋亡等)。研究證實,在大鼠腎小管近端細胞中,Ang-(1-7)可抑制AnⅡ誘導的ERK1/2, p38 MAPK與JNK的磷酸化。在腎臟內(nèi)皮細胞中,Ang-(1-7)可抑制高糖誘導的蛋白合成及p38 MAPK的磷酸化。盡管已有研究證實足細胞可表達ACE2并且在體外培養(yǎng)中可以合成Ang-(1-7),但Ang-(1-7)對足細胞信號通路的作用還未明確。因此本研究目的是探討MAPK通路是否參與Ang-(1-7)對子癇前期足細胞的保護作用。方法：應用體外細胞培養(yǎng)技術,收集正常妊娠孕婦及子癇前期患者血清干預人足細胞,設立正常妊娠組,子癇前期組,子癇前期+Ang-(1-7)、子癇前期+Ang-(1-7)+A779,正常妊娠組+A779進行實驗,應用Western blot檢測MAPK組分ERK1/2、p38、JNK磷酸化的變化。結果：1.MAPK通路變化：與正常妊娠組相比,子癇前期組p38、ERK1/2、JNK磷酸化增力(p-p38:1.69±0.20-fold VS 1.08±0.16-fold of GAPDH, P0.005; pERK:1.92±0.35-fold VS 1.04±0.26-fold of GAPDH, P0.005; pJNK2.35±0.35-fold VS 1.79±0.30-fold of GAPDH, P0.05)。子癇前期患者血清中加入Ang-(1-7)后,可以降低子癇前期患者血清誘導的p38、ERK1/2、JNK磷酸化增加(p-p38:1.23±0.18-fold VS 1.69±0.20-fold of GAPDH,P 0.05; pERK:1.14±0.25-fold VS 0.87±0.18-fold of GAPDH, P0.01; pJNK: 1.90±0.10-fold VS 2.35±0.35-fold of GAPDH,P0.05), A779可以拮抗這一作用。2.各組細胞上清中AngⅡ濃度變化：與正常妊娠組相比,子癇前期組細胞上清中AngⅡ濃度降低(NP:97.10±6.18 VS PE:73.80±0.25, P0.05),PE組加入Ang-(1-7)后,可使AngⅡ濃度進一步降低(PE+Ang-(1-7):63.90±67.54,P0.05),并且A779可拮抗上述作用。結論：Ang-(1-7)可通過降低MAPK通路的磷酸化發(fā)揮對子癇前期足細胞損傷的保護作用。
[Abstract]:The protective effect of Ang- (1-7) on preeclamptic foot cell injury in the first part: the role of renin angiotensin system (RAS) in renal injury in preeclampsia has been gradually recognized in recent years..Ang- (1-7) is an important active seven peptide in RAS system, which mainly acts on the Mas receptor of G- egg white couple and plays a major role in the body. The effect of anti Ang II. A number of studies have shown that the direct injury of podocytes participates in the production of preeclampsia proteinuria, suggesting that the renal injury in preeclampsia is podocyte disease. Our previous study showed that compared with normal pregnant women, the serum and urine Ang- (1-7) level of preeclampsia patients decreased, and their levels were associated with podocyte injury and proteinuria. Ang- (1-7) is one of the causes of renal injury in preeclampsia. In this study, we used in vitro podocyte culture to induce an eclamppooppodal injury model and further explore the role of Ang- (1-7) in the preeclampsia glomerulonephritis injury. The cultured human renal podocytes were cultured with the conditioned and immortalized human kidney. The serum of preeclampsia and the serum of normal pregnant women were collected to stimulate the podocyte cells. The sera of the preeclampsia were added to the sera of the preeclampsia patients with Ang- (1-7) and the podocytes were incubated with the CCK8 kit, and the Western blot was used to detect the Mas receptor of the podocyte. Expression, expression of nephrin, podocin, WT-1, change of podocyte skeleton protein F-actin by immunofluorescence, and flow cytometry to detect the apoptosis of foot cells. After adding Mas receptor antagonist A779, the changes were observed. Results: 1. podocytes viability detection: normal pregnancy pregnant women compared with FBS incubated foot cells The intervention of sera in preeclampsia patients could reduce the activity of podocyte, but it was higher than 90%; Ang (10-5,10-6,10-8M) and A779 (10-5,10-6,10-7M) were added to FBS in different concentrations. The activity of podocyte did not significantly change the expression of.2.Mas receptor: the result of immunoblotting showed that the human foot cell line expressed the Mas receptor.3. podocyte protein. Changes in expression: compared with normal pregnancy group, the expression of nephrin, WT-1 protein in preeclampsia group decreased (nephrin:0.42 + 0.04-fold VS 0.64 + 0.07-fold of GAPDH, corpse 0.005, WT-1:0.28 + 0.05-fold VS 0.46 + 0.09-fold), and there was no change in the protein expression (1-7) of preeclampsia foot cell mark The effect of Ang- (1-7) in serum of preeclampsia patients, Ang- (1-7) in 10-6M concentration has the greatest protective effect on foot cells, and the expression of nephrin.WT-1 is increased (nephrin:0.57 + 0.07-fold VS 0.42 + 0.04-fold of GAPDH, P0.05; WT-1:0.41 + 0.28 Antagonist A779 could antagonize the protective effect of Ang- (1-7) on podocytes. Compared with PE+Ang- (1-7), nephrin, WT-1 expression decreased after adding A779 (nephrin:1.55 + 0.20-fol VS 2.1 + 0.19-fold of GAPDH). The expression of rephrin and WT=1 in the preeclampsia group decreased at the level of mRNA (nephrin:23.28 + 3.94 VS 7.16 + 0.65, P0.005; WT-1:2.01 + 0.0.42 VS 1.03 + 2.23, P0.05). The effect of the foot cytoskeleton protein F-actin: compared with the normal pregnancy group, the expression of cytoskeleton protein F-actin in preeclampsia group was reduced and the arrangement of Ang- (1-7) was added to the sera of preeclampsia patients, which could relieve the serum cytoskeleton rearrangement caused by the serum of preeclampsia patients and the effect of Ang- (1-7) on the.7.Ang- (1-7) to the podocyte (1-7). The effect of apoptosis: compared with the normal pregnancy group (2.36+1.050%o), the apoptosis of podocyte in preeclampsia group increased (8.55 + 0.68%, P0.001). After Ang- (1-7) was added to the serum of preeclampsia, the apoptosis of podocytes decreased significantly (4.47+0.730%).A779 can antagonize Ang- (1-7). Conclusion: Ang- (1-7) can relieve the serum level caused by preeclampsia patients. Cellular damage. The second part of MAPK pathway in the effect of Ang- (1-7) on preeclamptic podocytes: mitogen-activated protein kinases (MAPKs) is a kind of serine / threonine protein kinase in cell, which exists in most cells and can transduce extracellular stimulation signal to cell and cell. Ang- (1-7) inhibits the phosphorylation of An II induced ERK1/2, p38 MAPK and JNK in the proximal end cells of rat renal tubules. In renal endothelial cells, Ang- (1-7) inhibits high glucose induced protein synthesis and p38 MAPK phosphorylation. ACE2 and Ang- (1-7) can be synthesized in vitro, but the role of Ang- (1-7) on the signalling pathway is not clear. Therefore, the purpose of this study is to explore whether MAPK pathway is involved in the protection of preeclampsia foot cells by Ang- (1-7). Pregnant women and preeclampsia patients intervened with human podocytes, set up normal pregnancy group, preeclampsia group, preeclampsia +Ang- (1-7), +Ang- (1-7) +A779 in preeclampsia, normal pregnancy group +A779, and Western blot were used to detect ERK1/2, p38, JNK phosphorylation of MAPK components. Results: 1.MAPK pathway changes: with normal pregnancy group P38, ERK1/2, JNK phosphorylation increased (p-p38:1.69 + 0.20-fold VS 1.08 + 0.16-fold of GAPDH, P0.005, pERK:1.92 + 0.35-fold), which could reduce preeclampsia patients with pre eclampsia (1-7). Serum induced p38, ERK1/2, JNK phosphorylation increased (p-p38:1.23 + 0.18-fold VS 1.69 + 0.20-fold of GAPDH, P 0.05, pERK:1.14 + 0.25-fold 0.87). The concentration of Ang II in the cell supernatant of preeclampsia group decreased (NP:97.10 + 6.18 VS PE:73.80 + 0.25, P0.05), and PE group added Ang- (1-7) to reduce the concentration of Ang II (PE+Ang- (1-7): 63.90 + 67.54, P0.05), and A779 could antagonize the above effect. Conclusion: Ang- (1-7) can reduce the phosphorylation of the pathway. Protective effect of poddal injury in preeclampsia.

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.244

【共引文獻】

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