Rab11蛋白在合体滋养细胞胞外体过度分泌中作用的研究

发布时间：2018-05-04 13:34

本文选题：低氧 + 合体滋养细胞胞外体　；参考：《第三军医大学》2016年硕士论文

【摘要】：背景和目的:子痫前期(preeclampsia-PE)是指妊娠20周后出现的高血压和蛋白尿。若无蛋白尿症状时,PE诊断为高血压合并血小板减少、肝酶升高、肾功能不全、肺水肿、视觉障碍以及头痛神经系统障碍等全身各个系统的疾病。PE影响着3%-10%的妊娠妇女[1],是母体和胎儿发病率和死亡率的首要原因。PE发展为子痫,引起抽搐,在产妇中大约有2.7-8.2/10000会发展为子痫[2]。PE可合并脑出血、肝脏破裂、肺水肿、急慢性肾衰、早产、胎儿宫内生长受限、死胎、死产以及孕产妇的死亡等[2]。终止妊娠是PE唯一有效的治疗方法,产后胎盘娩出后患者症状明显好转,说明胎盘是PE发生、发展的主要因素。胚胎植入后,胎盘形成的过程与子宫内膜微环境相关。早期妊娠时胎盘是在一个相对低氧的环境下形成的,胎盘形成过程,也是氧浓度变化的一个过程,在胚胎着床时,宫腔内的氧含量为3%,而蜕膜和子宫肌层内的氧含量为8%-12%[3]。这个氧浓度梯度促进绒毛外滋养层入侵子宫肌层、蜕膜以及螺旋动脉的重塑。滋养层锚定母体组织,并入侵螺旋动脉内,与血管内皮细胞和平滑肌细胞相互作用,改变螺旋动脉的结构。早孕末期,螺旋动脉管腔内阻塞的滋养层细胞减少直至消失,低阻力、高流量的螺旋动脉形成,母体血液灌注至胎盘绒毛间隙,母胎界面形成。螺旋动脉重塑后胎盘内的氧浓度升高至正常范围。有缺陷的滋养层的入侵和子宫螺旋动脉的重塑障碍引起胎盘的血管收缩和反应性,进而引起缺氧和活性氧的产生,激活低氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α),导致氧化应激。当胎盘缺血、缺氧[4]时,子宫胎盘血供减少,灌注不足,胎盘绒毛状结构被破坏,合成和释放大量血管活性因素,以及促炎性细胞因子、内皮素、合体滋养细胞微泡等胎盘源性不良因子,导致内皮细胞功能障碍,系统性炎症反应,最终引起妊娠20周后高血压、蛋白尿等PE临床症状的发生。目前研究广泛认为胎盘源性不良因子的释放是PE发生、发展的中心通路[5]。合体滋养细胞胞外体(syncytiotrophoblast exosome,STBEX)[6]是合体滋养细胞产生的胎盘源性不良因子的一种,属于细胞间连接物质[7],是一种纳米级囊泡。在正常妊娠时,STBEX作为细胞间连接分子,在绒毛外滋养层和血管平滑肌细胞、内皮细胞间起着信号转导作用,在螺旋动脉的重塑过程中发挥着重要作用。同时,STBEX在胎盘和外周血免疫细胞间进行信号传导,促进母体对胎儿的免疫耐受,在母胎界面血管的形成和成功妊娠中发挥着重要的作用[8]。STBEX最早在妊娠第六周时出现于循环血液中,且随着孕周的增加,STBEX的水平明显升高。但在PE等病理妊娠中,STBEX的作用尚未完全阐明。与同孕周的正常妊娠患者相比,PE患者STBEX的水平明显升高[3]。此外研究发现,STBEX可以激活单核细胞[9],使其分泌IL-1β、IL-6、TNF-α等促炎性细胞因子和趋化因子,造成类似PE严重的系统性炎症反应。STBEX还可以诱导T细胞的增殖而促进免疫反应。STBEX表面的HLA-G5、B7-H1和B7-H3等免疫分子可以激活抗原提呈细胞,调节Th1/Th2免疫平衡[10]。STBEX可通过多种途径作用于母体,既可以引起导致局部的炎性反应又可以调节全身的系统性免疫反应,被认为是母体炎症因子和免疫平衡两方面的重要调控因素,在PE的发病过程中发挥着重要的作用。Rab蛋白是GTP酶Ras超家族中最大的分支[11],在包括真核细胞在内的多种细胞的囊泡转运和信号转导过程中发挥着十分重要的作用。Rab11[12]表达在胎盘组织中,但是其在胎盘中的作用尚未见明确报道。本文旨在探讨PE中,缺氧时Rab11对STBEX过度分泌机制作用研究,以及Rab11蛋白在正常妊娠和PE患者胎盘组织中表达的差异。方法:第一部分:1.用福司可林(Forskolin)融合Bewo细胞,模拟胎盘合体滋养细胞。2.分别在常氧和缺氧条件下培养融合Bewo细胞和对照组细胞,根据是否融合与培养条件的不同,将细胞分为四组。(1)Be Wo常氧组:Be Wo细胞在普通培养基、常氧条件下培养;(2)Be Wo低氧组:Bewo细胞在普通培养基、低氧条件下培养(92%N2、5%CO_2,3%O_2);(3)融合Be Wo常氧组:Be Wo细胞以Forskolin诱导48h后,常氧条件下培养;(4)融合Be Wo低氧组:Be Wo细胞以Forskolin诱导48h后,低氧条件下培养(92%N2、5%CO_2,3%O_2)。3.BCA法检测各组上清液中STBEX蛋白浓度。4.Western-blot检测各组细胞中HIF-1α、Rab11蛋白的表达量。5.用RNA干扰技术沉默HIF-1α后,检测各组细胞Rab11蛋白的表达变化。第二部分:1.搜集重庆市第三军医大学大坪医院产科2014年10月-2015年10月住院产妇,选取子痫前期(PE)患者12例作为实验组,正常晚孕产妇12例作为对照组,收集剖宫产后的胎盘组织。2.分别在常氧条件下和缺氧条件下培养PE胎盘绒毛组织和正常妊娠患者胎盘绒毛组织。分为PE常氧组、PE缺氧组、正常(N)常氧组、正常(N)缺氧组四组。提取各组培养上清液中的STBEX及其蛋白组织,BCA法检测STBEX蛋白浓度。3.酶联免疫吸附试验(ELISA)法测各组上清液中HIF-1α浓度。4.Western-blot检测正常妊娠和PE患者胎盘组织中HIF-1α、Rab11蛋白的表达。结果:1.与Bewo常氧组比较,Bewo低氧组上清液中STBEX蛋白浓度升高,差异显著(p0.05);与融合Bewo常氧组比较,融合Bewo低氧组上清液中STBEX的蛋白浓度升高,差异显著(p0.05);2.融合Bewo低氧组中HIF-1α、Rab11蛋白表达水平显著高于融合Bewo常氧组,差异具有统计学意义,p0.05;Bewo低氧组中HIF-1α、Rab11蛋白表达水平显著高于Bewo常氧组,差异具有统计学意义,p0.05。3.si RNA干扰,沉默融合细胞HIF-1α后,细胞内的Rab11蛋白表达降低。4.分别将PE、正常妊娠胎盘绒毛组织在常氧和缺氧条件下培养后,检测上清液中STBEX蛋白浓度和HIF-1α的水平。与PE常氧组相比,PE缺氧组STBEX蛋白浓度与HIF-1α水平升高,差异显著,p0.05;与正常缺氧组相比,PE缺氧组中STBEX蛋白浓度与HIF-1α水平升高,差异显著,p0.05;与正常常氧组相比,PE常氧组中STBEX蛋白浓度与HIF-1α水平升高,差异显著,p0.05。5.HIF-1α与Rab11蛋白表达在正常妊娠胎盘组织与PE胎盘组织中,与正常胎盘组织相比,PE胎盘组织中HIF-1α、Rab11蛋白的表达量升高,差异具有统计学意义,p0.05。结论:1.低氧条件下,PE胎盘组织中Rab11蛋白表达量明显升高,使STBEX及胎盘囊泡释放增加,可能与PE患者胎盘浅着床有关。2.低氧环境下,融合Bewo细胞中HIF-1α、Rab11蛋白的表达量升高,用RNA干扰技术沉默融合细胞内HIF-1α后,细胞内的Rab11蛋白表达降低。说明HIF-1α可能通过调控Rab11蛋白来调节STBEX的过度分泌,使内皮细胞功能障碍,促发系统性炎症反应,可能在PE的发生中起一定的作用。
[Abstract]:Background and purpose: preeclampsia (preeclampsia-PE) refers to hypertension and proteinuria after 20 weeks of pregnancy. If there is no proteinuria, PE is diagnosed as hypertension combined with thrombocytopenia, liver enzyme elevation, renal insufficiency, pulmonary edema, visual impairment, and headache neurologic disorder, and.PE affects the pregnancy of 3%-10%. [1], the primary cause of maternal and fetal morbidity and mortality,.PE develops into eclampsia and causes convulsions. In parturients, about 2.7-8.2/10000 will develop into eclampsia [2].PE with cerebral hemorrhage, liver rupture, pulmonary edema, acute and chronic renal failure, premature birth, fetal intrauterine restriction, stillbirth, stillbirth, and maternal death. Pregnancy is the only effective treatment for PE. After delivery of postpartum placenta, the symptoms of the patient obviously improve, indicating that the placenta is a major factor in the development of PE. After implantation, the process of placenta formation is related to the microenvironment of the endometrium. In early pregnancy, the placenta was formed in a relatively low oxygen environment, the process of placenta formation, and oxygen concentration. A process of degree change, in the embryo implantation, the oxygen content in the uterine cavity is 3%, and the oxygen content in the decidua and the uterine myometrium is 8%-12%[3].. The oxygen concentration gradient promotes the intravascular invasion of the uterine layer, the decidua and the remodeling of the spiral artery. The trophoblast anchoring matrix is woven and intrude into the spiral artery, with the vascular endothelial cells and the vascular endothelial cells. The smooth muscle cells interact and change the structure of the spiral arteries. At the end of the early pregnancy, the trophoblast cells of the intraductal obstruction of the spiral artery are reduced until disappearance, the low resistance, the high flow of spiral arteries, the maternal blood perfusion to the placental villi space, the formation of the maternal fetal interface, and the oxygen concentration in the placenta after the remolding of the spiral vein to the normal range. The intrauterine invasion of the trophoblast and the obstruction of the uterine spiral artery cause the vasoconstriction and reactivity of the placenta, thereby causing hypoxia and reactive oxygen species, and activating the hypoxia inducible factor -1 alpha (Hypoxia-inducible factor-1 alpha, HIF-1 a), causing oxidative stress. When placental ischemia and hypoxia [4], the blood supply of uterus and placenta is reduced and perfusion is insufficient. The villous structure of the placenta is destroyed, the synthesis and release of a large number of vasoactive factors, as well as proinflammatory cytokines, endothelin, and syncytoma microbubbles, such as placental derived adverse factors, lead to endothelial cell dysfunction, systemic inflammatory response, and eventually cause PE clinical symptoms such as hypertension, proteinuria after 20 weeks of pregnancy. It is widely believed that the release of placental dysplasia is the occurrence of PE. The central pathway of development, [5]., syncytiotrophoblast exosome (STBEX) [6], is one of the placental adverse factors produced by syncytial cells, which belongs to the intercellular connective substance [7] and is a nanoscale vesicle. In normal pregnancy, STBEX acts as a kind of vesicle. Intercellular junction molecules, which play an important role in the remodeling process of the spiral arteries, play an important role in the remodeling process of the spiral arteries in the extracellular trophoblast and vascular smooth muscle cells. At the same time, STBEX signals the immune cells between the placenta and peripheral blood to promote the maternal immune tolerance to the fetus and the formation of the maternal fetal interface vessels. [8].STBEX, which plays an important role in successful pregnancy, appears in circulating blood at the earliest sixth weeks of pregnancy, and the level of STBEX increases with the increase of gestational age. But in PE and other pathological pregnancies, the role of STBEX has not been fully elucidated. The level of STBEX in patients with PE is significantly higher than that of [3]. in the same pregnancy weeks. It has been found that STBEX can activate monocyte [9] to secrete IL-1 beta, IL-6, TNF- alpha and other inflammatory cytokines and chemokines, resulting in a systemic inflammatory response similar to PE,.STBEX can also induce the proliferation of T cells and promote HLA-G5 on the.STBEX surface of the immune response, and B7-H1 and B7-H3 and other immune molecules can activate antigen presenting cells. The regulation of Th1/Th2 immune balance [10].STBEX can act on the mother body in a variety of ways, which can cause both local inflammatory response and systemic immune response. It is considered to be an important regulatory factor in the two aspects of maternal inflammatory factors and immune balance. It plays an important role in the pathogenesis of PE and.Rab protein is GTP The largest branch of the enzyme Ras superfamily, [11], plays a very important role in the process of vesicle transport and signal transduction, including eukaryotic cells, and plays a very important role in the expression of.Rab11[12] in the placenta, but its role in the placenta has not yet been clearly reported. This paper aims to explore the Rab11 over secretion of STBEX in PE. The differences in the expression of Rab11 protein in normal pregnancy and PE placenta tissues were studied. Methods: Part 1: 1. fusion of Bewo cells with foxcolin (Forskolin) was used to simulate the fusion of.2. cells and the control group of Bewo cells and control groups under the condition of normal oxygen and hypoxia, according to the fusion and culture conditions. The cells were divided into four groups. (1) Be Wo hyperoxia group: Be Wo cells were cultured under normal medium and normal oxygen; (2) Be Wo hypoxia group: Bewo cells in ordinary medium and hypoxia condition (92%N2,5%CO_2,3%O_2); (3) fusion Be Wo oxygen group: Be Wo cells were induced under normal oxygen condition and cultured in oxygen condition; (4) convergence 4 After 48h was induced by Forskolin, the concentration of STBEX protein in the supernatant of each group was detected by.3.BCA method under hypoxic condition (92%N2,5%CO_2,3%O_2) and.4.Western-blot was detected in each group of HIF-1 a. The expression of Rab11 protein was detected by RNA interference technique and HIF-1 alpha was used to detect the expression of Rab11 protein in each group. The second part: 1. to collect Chongqing The obstetrics and Gynecology of Daping Hospital, Third Military Medical University, October 2014 -2015 October -2015 years, selected 12 cases of preeclampsia (PE) as experimental group and 12 cases of normal late pregnant and parturient women as control group. The placental tissue.2. was cultured and cultured PE placental villi and normal placental Plush under normal oxygen condition and hypoxia condition. The hair tissue was divided into PE normal oxygen group, PE hypoxia group, normal (N) normal oxygen group and normal (N) hypoxia group four groups. The STBEX and protein tissue in the culture supernatant were extracted. The BCA assay was used to detect the concentration of STBEX protein in.3. enzyme linked immunosorbent assay (ELISA), and the concentration of HIF-1 a in the supernatant of each group was tested for normal pregnancy and the placenta tissue of the PE patients. The expression of HIF-1 alpha and Rab11 protein. Results: 1. compared with Bewo normal oxygen group, the concentration of STBEX protein in the supernatant of Bewo hypoxia group was higher, the difference was significant (P0.05), and the concentration of STBEX in the supernatant of Bewo hypoxia group was higher than that in the fusion Bewo normal oxygen group, and the difference was significant (P0.05), and the expression level of HIF-1 alpha in the 2. fusion Bewo hypoxia group was significant. The difference was statistically significant higher than that of the fusion Bewo normoxic group. The expression level of HIF-1 alpha in Bewo hypoxia group was significantly higher than that of Bewo normal oxygen group. The difference was statistically significant. P0.05.3.si RNA interference, and the expression of Rab11 protein in the cells with p0.05.3.si RNA interference and the decrease of Rab11 protein expression in the cells were PE and normal placental villi in normal pregnancy. The concentration of STBEX protein in the supernatant and the level of HIF-1 alpha in the supernatant were detected under the condition of hypoxia and hypoxia. Compared with the PE normal oxygen group, the concentration of STBEX protein and the level of HIF-1 a were higher, the difference was significant, P0.05. Compared with the normal hypoxia group, the concentration of STBEX protein and HIF-1 a in the hypoxia group were higher, the difference was significant, P0.05, and the normal oxygen group. The concentration of STBEX protein and HIF-1 alpha in the PE hyperoxia group were higher, and the difference was significant. The expression of p0.05.5.HIF-1 A and Rab11 protein in normal placental tissue and PE placenta was higher than that of normal placenta. The expression of HIF-1 A and Rab11 protein in PE placenta increased, and the difference was statistically significant, p0.05. conclusion: 1. hypoxic conditions, PE The expression of Rab11 protein in placental tissue increased significantly, which increased the release of STBEX and placental vesicles. It may be associated with the.2. hypoxia environment of the placental superficial placenta of PE patients, the expression of HIF-1 alpha and Rab11 protein in the fusion Bewo cells increased. The expression of Rab11 protein in the cell was decreased after the RNA interference technique was silent and fused in the cell HIF-1 alpha. It may regulate the oversecretion of STBEX by regulating the Rab11 protein, which may cause dysfunction of endothelial cells and promote systemic inflammatory response, which may play a role in the occurrence of PE.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.244

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