本文選題：低氧 + 合體滋養(yǎng)細胞胞外體　； 參考：《第三軍醫(yī)大學》2016年碩士論文

【摘要】：背景和目的:子癇前期(preeclampsia-PE)是指妊娠20周后出現(xiàn)的高血壓和蛋白尿。若無蛋白尿癥狀時,PE診斷為高血壓合并血小板減少、肝酶升高、腎功能不全、肺水腫、視覺障礙以及頭痛神經(jīng)系統(tǒng)障礙等全身各個系統(tǒng)的疾病。PE影響著3%-10%的妊娠婦女[1],是母體和胎兒發(fā)病率和死亡率的首要原因。PE發(fā)展為子癇,引起抽搐,在產(chǎn)婦中大約有2.7-8.2/10000會發(fā)展為子癇[2]。PE可合并腦出血、肝臟破裂、肺水腫、急慢性腎衰、早產(chǎn)、胎兒宮內(nèi)生長受限、死胎、死產(chǎn)以及孕產(chǎn)婦的死亡等[2]。終止妊娠是PE唯一有效的治療方法,產(chǎn)后胎盤娩出后患者癥狀明顯好轉(zhuǎn),說明胎盤是PE發(fā)生、發(fā)展的主要因素。胚胎植入后,胎盤形成的過程與子宮內(nèi)膜微環(huán)境相關(guān)。早期妊娠時胎盤是在一個相對低氧的環(huán)境下形成的,胎盤形成過程,也是氧濃度變化的一個過程,在胚胎著床時,宮腔內(nèi)的氧含量為3%,而蛻膜和子宮肌層內(nèi)的氧含量為8%-12%[3]。這個氧濃度梯度促進絨毛外滋養(yǎng)層入侵子宮肌層、蛻膜以及螺旋動脈的重塑。滋養(yǎng)層錨定母體組織,并入侵螺旋動脈內(nèi),與血管內(nèi)皮細胞和平滑肌細胞相互作用,改變螺旋動脈的結(jié)構(gòu)。早孕末期,螺旋動脈管腔內(nèi)阻塞的滋養(yǎng)層細胞減少直至消失,低阻力、高流量的螺旋動脈形成,母體血液灌注至胎盤絨毛間隙,母胎界面形成。螺旋動脈重塑后胎盤內(nèi)的氧濃度升高至正常范圍。有缺陷的滋養(yǎng)層的入侵和子宮螺旋動脈的重塑障礙引起胎盤的血管收縮和反應性,進而引起缺氧和活性氧的產(chǎn)生,激活低氧誘導因子-1α(Hypoxia-inducible factor-1α,HIF-1α),導致氧化應激。當胎盤缺血、缺氧[4]時,子宮胎盤血供減少,灌注不足,胎盤絨毛狀結(jié)構(gòu)被破壞,合成和釋放大量血管活性因素,以及促炎性細胞因子、內(nèi)皮素、合體滋養(yǎng)細胞微泡等胎盤源性不良因子,導致內(nèi)皮細胞功能障礙,系統(tǒng)性炎癥反應,最終引起妊娠20周后高血壓、蛋白尿等PE臨床癥狀的發(fā)生。目前研究廣泛認為胎盤源性不良因子的釋放是PE發(fā)生、發(fā)展的中心通路[5]。合體滋養(yǎng)細胞胞外體(syncytiotrophoblast exosome,STBEX)[6]是合體滋養(yǎng)細胞產(chǎn)生的胎盤源性不良因子的一種,屬于細胞間連接物質(zhì)[7],是一種納米級囊泡。在正常妊娠時,STBEX作為細胞間連接分子,在絨毛外滋養(yǎng)層和血管平滑肌細胞、內(nèi)皮細胞間起著信號轉(zhuǎn)導作用,在螺旋動脈的重塑過程中發(fā)揮著重要作用。同時,STBEX在胎盤和外周血免疫細胞間進行信號傳導,促進母體對胎兒的免疫耐受,在母胎界面血管的形成和成功妊娠中發(fā)揮著重要的作用[8]。STBEX最早在妊娠第六周時出現(xiàn)于循環(huán)血液中,且隨著孕周的增加,STBEX的水平明顯升高。但在PE等病理妊娠中,STBEX的作用尚未完全闡明。與同孕周的正常妊娠患者相比,PE患者STBEX的水平明顯升高[3]。此外研究發(fā)現(xiàn),STBEX可以激活單核細胞[9],使其分泌IL-1β、IL-6、TNF-α等促炎性細胞因子和趨化因子,造成類似PE嚴重的系統(tǒng)性炎癥反應。STBEX還可以誘導T細胞的增殖而促進免疫反應。STBEX表面的HLA-G5、B7-H1和B7-H3等免疫分子可以激活抗原提呈細胞,調(diào)節(jié)Th1/Th2免疫平衡[10]。STBEX可通過多種途徑作用于母體,既可以引起導致局部的炎性反應又可以調(diào)節(jié)全身的系統(tǒng)性免疫反應,被認為是母體炎癥因子和免疫平衡兩方面的重要調(diào)控因素,在PE的發(fā)病過程中發(fā)揮著重要的作用。Rab蛋白是GTP酶Ras超家族中最大的分支[11],在包括真核細胞在內(nèi)的多種細胞的囊泡轉(zhuǎn)運和信號轉(zhuǎn)導過程中發(fā)揮著十分重要的作用。Rab11[12]表達在胎盤組織中,但是其在胎盤中的作用尚未見明確報道。本文旨在探討PE中,缺氧時Rab11對STBEX過度分泌機制作用研究,以及Rab11蛋白在正常妊娠和PE患者胎盤組織中表達的差異。方法:第一部分:1.用福司可林(Forskolin)融合Bewo細胞,模擬胎盤合體滋養(yǎng)細胞。2.分別在常氧和缺氧條件下培養(yǎng)融合Bewo細胞和對照組細胞,根據(jù)是否融合與培養(yǎng)條件的不同,將細胞分為四組。(1)Be Wo常氧組:Be Wo細胞在普通培養(yǎng)基、常氧條件下培養(yǎng);(2)Be Wo低氧組:Bewo細胞在普通培養(yǎng)基、低氧條件下培養(yǎng)(92%N2、5%CO_2,3%O_2);(3)融合Be Wo常氧組:Be Wo細胞以Forskolin誘導48h后,常氧條件下培養(yǎng);(4)融合Be Wo低氧組:Be Wo細胞以Forskolin誘導48h后,低氧條件下培養(yǎng)(92%N2、5%CO_2,3%O_2)。3.BCA法檢測各組上清液中STBEX蛋白濃度。4.Western-blot檢測各組細胞中HIF-1α、Rab11蛋白的表達量。5.用RNA干擾技術(shù)沉默HIF-1α后,檢測各組細胞Rab11蛋白的表達變化。第二部分:1.搜集重慶市第三軍醫(yī)大學大坪醫(yī)院產(chǎn)科2014年10月-2015年10月住院產(chǎn)婦,選取子癇前期(PE)患者12例作為實驗組,正常晚孕產(chǎn)婦12例作為對照組,收集剖宮產(chǎn)后的胎盤組織。2.分別在常氧條件下和缺氧條件下培養(yǎng)PE胎盤絨毛組織和正常妊娠患者胎盤絨毛組織。分為PE常氧組、PE缺氧組、正常(N)常氧組、正常(N)缺氧組四組。提取各組培養(yǎng)上清液中的STBEX及其蛋白組織,BCA法檢測STBEX蛋白濃度。3.酶聯(lián)免疫吸附試驗(ELISA)法測各組上清液中HIF-1α濃度。4.Western-blot檢測正常妊娠和PE患者胎盤組織中HIF-1α、Rab11蛋白的表達。結(jié)果:1.與Bewo常氧組比較,Bewo低氧組上清液中STBEX蛋白濃度升高,差異顯著(p0.05);與融合Bewo常氧組比較,融合Bewo低氧組上清液中STBEX的蛋白濃度升高,差異顯著(p0.05);2.融合Bewo低氧組中HIF-1α、Rab11蛋白表達水平顯著高于融合Bewo常氧組,差異具有統(tǒng)計學意義,p0.05;Bewo低氧組中HIF-1α、Rab11蛋白表達水平顯著高于Bewo常氧組,差異具有統(tǒng)計學意義,p0.05。3.si RNA干擾,沉默融合細胞HIF-1α后,細胞內(nèi)的Rab11蛋白表達降低。4.分別將PE、正常妊娠胎盤絨毛組織在常氧和缺氧條件下培養(yǎng)后,檢測上清液中STBEX蛋白濃度和HIF-1α的水平。與PE常氧組相比,PE缺氧組STBEX蛋白濃度與HIF-1α水平升高,差異顯著,p0.05;與正常缺氧組相比,PE缺氧組中STBEX蛋白濃度與HIF-1α水平升高,差異顯著,p0.05;與正常常氧組相比,PE常氧組中STBEX蛋白濃度與HIF-1α水平升高,差異顯著,p0.05。5.HIF-1α與Rab11蛋白表達在正常妊娠胎盤組織與PE胎盤組織中,與正常胎盤組織相比,PE胎盤組織中HIF-1α、Rab11蛋白的表達量升高,差異具有統(tǒng)計學意義,p0.05。結(jié)論:1.低氧條件下,PE胎盤組織中Rab11蛋白表達量明顯升高,使STBEX及胎盤囊泡釋放增加,可能與PE患者胎盤淺著床有關(guān)。2.低氧環(huán)境下,融合Bewo細胞中HIF-1α、Rab11蛋白的表達量升高,用RNA干擾技術(shù)沉默融合細胞內(nèi)HIF-1α后,細胞內(nèi)的Rab11蛋白表達降低。說明HIF-1α可能通過調(diào)控Rab11蛋白來調(diào)節(jié)STBEX的過度分泌,使內(nèi)皮細胞功能障礙,促發(fā)系統(tǒng)性炎癥反應,可能在PE的發(fā)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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R714.244

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