Ang-（1-7）对子痫前期足细胞损伤的保护作用及其机制研究

发布时间：2018-05-04 20:57

本文选题：子痫前期 + 肾足细胞　；参考：《复旦大学》2014年硕士论文

【摘要】：第一部分Ang-(1-7)对子痫前期足细胞损伤的保护作用目的：近年来肾素-血管紧张素系统(RAS)在子痫前期肾脏损伤中的作用被逐渐认识。Ang-(1-7)是RAS系统一个重要的活性七肽,主要作用于G-蛋白偶联的Mas受体发挥作用,在体内主要发挥拮抗AngⅡ的作用。许多研究显示足细胞的直接损伤参与了子痫前期蛋白尿的产生,提示子痫前期肾脏损伤是足细胞病。我们前期研究显示与正常妊娠孕妇相比,子痫前期患者血清及尿液中Ang-(1-7)水平降低,且其水平与足细胞损伤及蛋白尿的发生密切相关,表明循环及肾脏局部降低的Ang-(1-7)是子痫前期肾脏损伤的原因之一。在本研究中我们借助体外足细胞培养,诱导子痫前期体外足细胞损伤模型,进一步探讨Ang-(1-7)在子痫前期肾小球足细胞损伤中的作用。方法：应用体外细胞培养技术,培养条件性永生的人肾脏足细胞,收集子痫前期患者血清及正常妊娠孕妇血清刺激足细胞,并于子痫前期患者血清中加入不同浓度Ang-(1-7)与足细胞共孵育,应用CCK8试剂盒检测足细胞活性,Western blot检测足细胞Mas受体的表达,以及足细胞标志蛋白(nephrin、podocin、WT-1)的表达,免疫荧光观察足细胞骨架蛋白F-actin的变化,流式细胞仪检测足细胞的凋亡。加入Mas受体拮抗剂A779后,观察上述变化。结果：1.足细胞活性检测：与FBS孵育足细胞相比,正常妊娠孕妇及子痫前期患者血清干预足细胞均可使其活性降低,但都高于90%；在FBS中加入不同浓度Ang(1.7)(10-5,10-6,10-8M)及A779(10-5,10-6,10-7M),足细胞活性没有明显变化。2.Mas受体的表达：免疫印迹结果显示人足细胞株表达Mas受体。3.足细胞标志蛋白表达的变化：与正常妊娠组比较,子痫前期组足细胞nephrin,WT-1蛋白表达降低(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蛋白表达未有变化。4.不同浓度Ang-(1-7)对子痫前期足细胞标志蛋白表达的影响：在子痫前期患者血清中加入不同浓度的Ang-(1-7),Ang-(1-7)在10-6M浓度对足细胞有最大保护作用,nephrin.WT-1表达增加(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受体拮抗剂A779可拮抗Ang-(1-7)对足细胞的保护作用,与PE+Ang-(1-7)相比,加入A779后,nephrin、WT-1表达减少(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.足细胞标志蛋白在基因水平的表达变化：与正常妊娠组相比,子痫前期组足细胞rephrin、WT=1在mRNA水平表达降低(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与WT-1 mRNA表达增加(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)对足细胞骨架蛋白F-actin的影响：与正常妊娠组比较,子痫前期组足细胞骨架蛋白F-actin表达减少,排列紊乱,子痫前期患者血清中加入Ang-(1-7)可缓解子痫前期患者血清引起的足细胞骨架重排。A779可拮抗Ang-(1-7)的作用。7.Ang-(1-7)对足细胞凋亡的影响：与正常妊娠组(2.36+1.050%o)比较,子痫前期组足细胞凋亡增加(8.55±0.68%,P0.001)。子痫前期患者血清中加入Ang-(1-7)后,足细胞凋亡明显减少(4.47+0.730%)。A779可拮抗Ang-(1-7)的作用。结论：Ang-(1-7)可缓解子痫前期患者血清引起的足细胞损伤。第二部分MAPK通路在Ang-(1-7)对子痫前期足细胞影响中的机制作用目的：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,存在于大多数细胞内,能够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)。研究证实,在大鼠肾小管近端细胞中,Ang-(1-7)可抑制AnⅡ诱导的ERK1/2, p38 MAPK与JNK的磷酸化。在肾脏内皮细胞中,Ang-(1-7)可抑制高糖诱导的蛋白合成及p38 MAPK的磷酸化。尽管已有研究证实足细胞可表达ACE2并且在体外培养中可以合成Ang-(1-7),但Ang-(1-7)对足细胞信号通路的作用还未明确。因此本研究目的是探讨MAPK通路是否参与Ang-(1-7)对子痫前期足细胞的保护作用。方法：应用体外细胞培养技术,收集正常妊娠孕妇及子痫前期患者血清干预人足细胞,设立正常妊娠组,子痫前期组,子痫前期+Ang-(1-7)、子痫前期+Ang-(1-7)+A779,正常妊娠组+A779进行实验,应用Western blot检测MAPK组分ERK1/2、p38、JNK磷酸化的变化。结果：1.MAPK通路变化：与正常妊娠组相比,子痫前期组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)。子痫前期患者血清中加入Ang-(1-7)后,可以降低子痫前期患者血清诱导的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可以拮抗这一作用。2.各组细胞上清中AngⅡ浓度变化：与正常妊娠组相比,子痫前期组细胞上清中AngⅡ浓度降低(NP:97.10±6.18 VS PE:73.80±0.25, P0.05),PE组加入Ang-(1-7)后,可使AngⅡ浓度进一步降低(PE+Ang-(1-7):63.90±67.54,P0.05),并且A779可拮抗上述作用。结论：Ang-(1-7)可通过降低MAPK通路的磷酸化发挥对子痫前期足细胞损伤的保护作用。
[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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.244

【共引文献】