自噬对氧化低密度脂蛋白诱导滋养细胞炎性反应的保护作用及其与子痫前期发病的关系

发布时间：2018-06-03 03:41

本文选题：子痫前期 + 自噬　；参考：《青岛大学》2017年博士论文

【摘要】：子痫前期是妊娠期妇女特有的常见病症,是妊娠期高血压疾病最严重的阶段,是妊娠期高血压疾病所致母儿死亡的最主要原因。发展中国家,因子痫前期而死亡的孕妇占20%~80%,发达国家,子痫前期的孕妇围生期死亡率是未患该病的5倍。为了探讨子痫前期的病因和发病机制,广大学者从流行病学角度和免疫学等方面都进行了广泛的研究,目前认为该疾病是母体、胎盘、胎儿等众多因素共同作用的结果,主要病因包括子宫螺旋小动脉重铸不足,炎症免疫过度激活,血管内皮细胞受损,遗传因素,营养缺乏和胰岛素抵抗。但疾病的具体发病机制至今仍未完全阐明,目前普遍认为的该疾病病理生理学发生发展的两个主要阶段,即胎盘异常阶段及母体异常阶段,胎盘异常阶段的核心问题是滋养细胞缺血、缺氧造成的氧化应激反应,而母体异常阶段主要是由胎盘局部炎性反应诱发的母体全身炎性反应。尽管子痫前期是全球多发疾病,其临床表现具有多样化,但到目前仍没有有效的预测或预防性治疗措施。临床通用的监测手段仅为孕期新发而孕后自愈性的高血压及尿蛋白的出现,但始终缺乏明确诊断的特异性指标。目前期待治疗措施如降压、扩容、皮质激素的使用等处理疗效甚微,且理论依据不足,明确有效的治疗方法是终止妊娠,从而带来早产等一系列不良后果;与此同时,目前关于子痫前期的治疗中还未曾有关于抗炎治疗的报道,因此我们认为大量研究工作应从氧化应激反应、炎性反应等各方面同时入手来探讨子痫前期的发病机制并为子痫前期的临床治疗提供新的科研思路。自噬是细胞在自噬相关基因(Atg)的调控下由溶酶体参与的一种分解代谢机制,以消化自身受损或功能异常的细胞成分,在细胞增殖、分化过程中维持细胞结构、代谢和功能的平衡,在饥饿或低代谢、缺氧、药物影响的情况下自噬是保证细胞的存活的重要机制。自噬作为抵御病理状态的代偿机制,其调控机制的失调与感染、肿瘤、神经退行性变、心脏疾病等多种疾病的发生、发展有关,并逐渐成为多种疾病领域的研究热点。自噬的发生依赖Atg的启动以及一系列自噬相关蛋白的共同参与,自噬基因的研究最初是在酵母菌中进行的,其中,Beclin 1是哺乳动物体内酵母Atg6的基因同源物,通过II型磷脂酰肌醇-3激酶(PI3K)/Beclin 1途径可激活自噬,Beclin 1通过介导其与自噬相关蛋白的交互作用来完成自噬体的双层膜结构,对于自噬的发生发挥关键性的作用。另外,Beclin 1可与凋亡调节基因Bcl-2产物的蛋白家族相结合,在细胞自噬与凋亡的过程中起到桥梁作用。LC3是哺乳动物体内酵母Atg8的基因同源物,LC3在细胞中以LC3 I与LC3 II两种形式存在,LC3的C末端被Atg4基因剪切后形成胞质型LC3(LC3 I),LC3 I经泛素样加工修饰与磷脂酰乙醇胺结合形成LC3 II并表达在自噬体膜上,LC3 II特异性的结合在自噬体膜上,LC3 II最终被降解后可重新回到细胞质中。因此,LC3 I向LC3 II的转变是自噬发生的重要标志,自噬相关蛋白Beclin 1及LC3的表达是自噬研究的常用检测指标。子痫前期所具有的缺氧、氧化应激及炎症反应等诸多因素都与自噬有着密切关系,据此推测子痫前期的疾病状态可能存在自噬水平的异常。目前关于自噬与子痫前期发病的关系研究较少且没有达成统一的共识。氧化低密度脂蛋白(ox LDL)的生成系低密度脂蛋白(LDL)中大量不饱和脂肪酸在各种氧化条件下生成一种脂类自由基,从而介导产生更多的过氧化脂质引起链式反应,形成多种活性醛,与低密度脂蛋白中的载脂蛋白B结合,产生新的抗原决定簇,形成氧化低密度脂蛋白。氧化低密度脂蛋白及血凝集素样氧化低密度脂蛋白受体1即LOX-1共同作用参与了子痫前期的病理生理变化。较早的研究表明子痫前期患者血浆中氧化低密度脂蛋白水平升高,可能通过引起内皮损伤参与子痫前期的发病。我们之前的研究也表明子痫前期患者血浆中氧化低密度脂蛋白水平升高,且重度子痫前期患者高于轻度子痫前期患者,并通过上调胎盘组织中的LOX-1表达水平参与子痫前期的发病及疾病的进展。通过对滋养细胞的研究发现,LOX-1可能通过影响滋养细胞凋亡而参与子痫前期的发病。我们的研究则表明LOX-1可能通过其介导的氧化应激反应影响滋养细胞血管因子的表达而参与子痫前期的病理生理变化。近期的研究认为氧化低密度脂蛋白与多种细胞自噬有密切关系,其在不同细胞类型中发挥的作用有所不同,可呈浓度依赖的上调脐静脉内皮细胞的自噬水平;而在巨噬细胞及平滑肌细胞中却能抑制自噬的发生,但是关于氧化低密度脂蛋白对于滋养细胞自噬影响的研究却不多见。研究目的:本研究旨在探讨子痫前期患者氧化低密度脂蛋白及胎盘滋养细胞自噬水平的变化以及氧化低密度脂蛋白对滋养细胞自噬及炎症反应的影响,进一步说明自噬对滋养细胞炎性反应的保护作用,为子痫前期的病理生理学研究及疾病的治疗提供新的理论基础。研究方法:1、选择2014年6月至2015年1月在青岛大学附属医院产科住院且经剖宫产分娩的子痫前期孕妇26例为子痫前期组(PE组),选取同期正常晚期妊娠妇女20例为正常孕妇组(NP组)。两组孕妇均于入院后空腹抽取肘静脉血5ml,分离血清,内置于-70℃冰箱保存。两组孕妇均经剖宫产终止妊娠,在娩出胎盘后的30分钟内在无菌条件下获取胎盘标本,避开胎盘母体面的坏死及钙化区,采集两块大小约1 cm×1 cm×1 cm的胎盘组织,经灭菌生理盐水反复漂净残余的血液后,将其中的一块放于1.5 ml的无菌离心管中,15分钟内置于-70℃冰箱保存,以待RNA和蛋白提取,另一块甲醛固定备用。2、采用ELISA法检测两组孕妇血清中ox LDL的水平,以及炎性因子TNF-α和IL-6水平的变化。采用免疫组化法对胎盘组织中TNF-α和IL-6的表达进行定位测定;采用Western Blot法检测各组胎盘组织中TNF-α和IL-6蛋白的表达。3、采用免疫组化法对胎盘组织中自噬相关蛋白(Beclin 1和LC3)进行定位测定;采用real-time PCR法对胎盘组织中Beclin 1和LC3的m RNA进行测定;采用Western Blot法检测各组胎盘组织中Beclin 1和LC3蛋白的表达。4、购置JEG-3细胞系并进行传代培养,经不同浓度ox LDL(25,50,100,150mg/l)以及不同作用时间(6,12,24,48小时)处理后收集细胞,通过免疫荧光法检测不同浓度及不同作用时间下ox LDL对滋养细胞自噬相关蛋白LC3的影响。5、采用JEG-3细胞系,利用不同浓度ox LDL(25,50,100,150mg/l)作用6小时进行研究,通过real-time PCR法检测经ox LDL处理前后滋养细胞中炎性因子(TNF-α和IL-6)m RNA表达的变化。6、采用JEG-3细胞系,将其分成两组,一组利用浓度为100mg/l的ox LDL作用6小时后进行实验,另一组经雷帕霉素100n M预处理预处理1小时后,再利用浓度为100mg/l的ox LDL作用6小时为研究条件,通过real-time PCR法对滋养细胞中炎性因子(TNF-α和IL-6)的m RNA进行测定;采用Western Blot法检测滋养细胞中自噬相关蛋白Beclin 1及LC3的蛋白表达水平的变化。7、统计学方法应用SPSS 17.0进行数据处理,所有测定结果以_x±s表示,两组间比较采用t检验,P㩳0.05为差别有统计学意义。结果:1、通过ELISA法检测结果提示子痫前期组患者血清中ox LDL、TNF-α及IL-6水平较正常对照组明显升高,差异有统计学意义(p0.05)。免疫组化结果提示TNF-α及IL-6在子痫前期组及正常对照组胎盘滋养细胞细胞中均有表达,呈棕色阳性染色,同时,子痫前期组胎盘中染色较深且阳性细胞数目较正常对照组增多。Western Blot法检测TNF-α及IL-6蛋白表达结果提示子痫前期组胎盘组织中炎性因子的表达较正常对照组升高,差异有统计学意义(p0.05)。2、免疫组化结果显示Beclin 1在滋养细胞中呈高于背景的淡棕色阳性染色,主要表达于细胞滋养细胞;LC3高于背景的淡棕色染色主要表达于细胞滋养细胞,在合体滋养细胞中也有表达。在正常对照组(NP组)中Beclin 1及LC3蛋白的阳性细胞数量明显增多且染色增强。real-time PCR法检测结果显示正常对照组胎盘组织中Beclin 1及LC3 m RNA的表达水平明显高于子痫前期组,差异有统计学意义(p0.05)。Western Blot法检测Beclin 1及LC3蛋白表达结果提示正常对照组胎盘组织中自噬相关蛋白的表达较子痫前期组升高,差异有统计学意义(p0.05)。3、在不同浓度ox LDL(25,50,100,150mg/l)以及不同作用时间(6,12,24,48小时)下培养滋养细胞细胞,利用免疫荧光法均不能在细胞内观察到胞浆内红染的LC3点状聚集。当细胞由雷帕霉素100n M预处理1h后,再经ox LDL(100mg/l)刺激的细胞在不同作用时间下均可见到胞浆内红染的LC3点状聚集。经雷帕霉素预处理后再加入ox LDL培养细胞后,经观察得ox LDL作用6h及12h后细胞形态仍是完整的,但作用24小时及48小时即可出现细胞核的肿胀、变形;因此,作用6小时为后续其他实验条件。4、通过real-time PCR法检测TNF-α和IL-6 m RNA的表达发现经50mg/l及100mg/l ox LDL处理后滋养细胞中炎性因子的表达均高于空白对照组,差异有统计学意义(p0.05)。同时,经100mg/l ox LDL培养后滋养细胞中m TOR的表达较空白对照组无明显变化,因此选定100mg/l ox LDL为后续实验条件。5、细胞经雷帕霉素100n M预处理1小时后,再经100mg/l ox LDL培养6小时,Western Blot法检测得Beclin 1及LC3蛋白表达明显高于未经雷帕霉素处理组,差异有统计意义(p0.05);同时real-time PCR法检测TNF-α和IL-6m RNA结果提示炎性因子的表达较未经雷帕霉素处理组明显降低,差异有统计意义(p0.05)。结论及意义:1、子痫前期患者氧化低密度脂蛋白及炎性因子(TNF-α、IL-6)表达水平的升高参与了子痫前期疾病的病理生理过程。2、氧化低密度脂蛋白可引起滋养细胞炎性反应,释放炎性因子;但ox LDL自身不能引起滋养细胞自噬。3、在自噬诱导剂的作用下,增强滋养细胞自噬后可减弱氧化低密度脂蛋白对滋养细胞引起的炎性反应,降低其炎性因子的表达。氧化低密度脂蛋白及炎性因子在子痫前期患者血清中显著升高,细胞实验证实,氧化低密度脂蛋白可诱导滋养细胞炎性因子的表达,相同实验条件下不能诱导滋养细胞自噬的发生,也就是说当氧化低密度脂蛋白通过炎症因素参与子痫前期发病的同时抑制了滋养细胞自噬的能力,因此认为氧化低密度脂蛋白在子痫前期疾病发生的氧化应激反应及炎性反应中起到桥梁作用。同时,自噬对氧化低密度脂蛋白诱导滋养细胞炎性反应具有保护作用,子痫前期胎盘滋养细胞自噬能力的减低不足以保护由氧化低密度脂蛋白引起的炎性反应;因此本研究在氧化低密度脂蛋白及自噬参与子痫前期发病的机制探讨中均具有重要意义;并为子痫前期疾病的治疗提供了新的理论基础。
[Abstract]:Preeclampsia is a common common disease of women in pregnancy. It is the most serious stage of hypertensive disorder in pregnancy. It is the most important cause of maternal mortality in pregnancy induced hypertension. In developing countries, pregnant women who died during preeclampsia and preeclampsia are 20%~80%, and the perinatal mortality of pregnant women in the pre eclampsia period is 5 times as high as that of the disease. In order to explore the etiology and pathogenesis of preeclampsia, many scholars have conducted extensive research on epidemiology and immunology. It is believed that the disease is the result of many factors such as mother, placenta, fetus and so on. The main causes include the insufficiency of the uterine spiral arterioles, the excessive activation of inflammatory immunity, and the intravascular injection. The damage of skin cells, genetic factors, nutritional deficiency and insulin resistance. But the specific pathogenesis of the disease has still not been fully elucidated. At present, the two main stages of pathophysiology of the disease are the placental abnormal stage and the maternal abnormal stage. The core of the placental abnormal stage is the ischemia of trophoblast and hypoxia. The oxidative stress reaction is caused by the maternal systemic inflammatory response, which is mainly caused by the local inflammatory response of the placenta. Although preeclampsia is a global multiple disease, its clinical manifestations are diverse, but there is still no effective prediction or preventive treatment at present. The self healing of hypertension and the appearance of urine protein after pregnancy, but there is always a lack of specific diagnostic criteria. At present, the treatment measures, such as decompression, dilatation, and the use of corticosteroids, are very small, and the theoretical basis is insufficient. The clear and effective treatment is to terminate pregnancy and bring a series of adverse consequences, such as premature delivery. At present, there is no report about the treatment of anti - inflammation in preeclampsia, so we think a lot of research should start with the oxidative stress reaction, inflammatory response and other aspects to explore the pathogenesis of preeclampsia and provide new scientific research ideas for the clinical treatment of preeclampsia. Autophagy is autophagy related to autophagy. A mechanism of catabolism involved in the lysosome under the regulation of the gene (Atg) to digest the cellular components of their own damage or function and maintain the balance of cell structure, metabolism and function during cell proliferation and differentiation. Autophagy is an important mechanism for the survival of the cells under the condition of starvation or low metabolism, anoxia, and drug effects. As a compensatory mechanism for resisting pathological state, the disorder of its regulatory mechanism is related to the development of a variety of diseases such as infection, tumor, neurodegenerative change, heart disease, development, and gradually become a hot spot in many fields of disease. The occurrence of autophagy depends on the initiation of Atg and the involvement of a series of autophagy related proteins. The study was originally carried out in yeast, in which Beclin 1 is a gene homologue of yeast Atg6 in mammals, which activates autophagy through the II type phosphatidylinositol -3 kinase (PI3K) /Beclin 1 pathway. Beclin 1 mediates its interaction with autophagy related proteins to complete the bilayer membrane structure of autophagosol, and plays a role in autophagy. In addition, Beclin 1 can be combined with the protein family of the Bcl-2 product of apoptosis regulating gene, which plays a bridge role in the process of autophagy and apoptosis,.LC3 is the gene homologue of yeast Atg8 in mammalian body, LC3 is stored in the two forms of LC3 I and LC3 II, and the C end of LC3 is formed after the Atg4 gene is cut. LC3 (LC3 I), LC3 I is modified with phosphatidyl ethanolamine to form LC3 II and is expressed on the autophagosome membrane, and LC3 II specificity combines on the autophagic membrane. LC3 II is finally degraded to the cytoplasm. Therefore, the transformation of LC3 II is an important sign of autophagy, autophagy associated protein 1 The expression of C3 is a common detection index for autophagy. Many factors such as hypoxia, oxidative stress and inflammatory response in preeclampsia are closely related to autophagy. According to this, it is suggested that the state of autophagy may exist in preeclampsia. The relationship between autophagy and preeclampsia is less and less than that of the preeclampsia. A unified consensus. The generation of oxidized low density lipoprotein (ox LDL) generation of low density lipoprotein (LDL) produces a fat free radical under various oxidation conditions, which mediates more lipid peroxide induced chain reaction, forms a variety of active aldehydes, and combines with apolipoprotein B in low density lipoprotein, and produces a combination of apolipoprotein B. New antigenic determinants are produced to form oxidized low density lipoprotein. Oxidative low density lipoprotein and blood lectin like oxidized low density lipoprotein receptor 1, LOX-1, are involved in the pathophysiological changes in preeclampsia. Skin injury is involved in the onset of preeclampsia. Our previous study also showed that the plasma levels of oxidized low density lipoprotein in preeclampsia patients were elevated, and severe preeclampsia patients were higher than those with mild preeclampsia, and were involved in the pathogenesis of preeclampsia and the progress of the disease by raising the level of LOX-1 in placenta tissue. Cell culture studies have found that LOX-1 may participate in the onset of preeclampsia by affecting the apoptosis of trophoblastic cells. Our study suggests that LOX-1 may participate in the pathophysiological changes in the preeclampsia through its mediated oxidative stress response and its involvement in the expression of trophoblast vascular factors. Many cells are closely related to autophagy, and they play a different role in different cell types. They can increase the autophagy level of umbilical vein endothelial cells in a concentration dependent manner, but inhibit the occurrence of autophagy in macrophages and smooth muscle cells, but studies on the effect of oxidized low density lipoprotein on the autophagy of trophoblastic cells The purpose of this study is to investigate the changes in the level of autophagy of oxidized low density lipoprotein and placental trophoblast in preeclampsia and the effect of oxidized low density lipoprotein on the autophagy and inflammation of trophoblastic cells, and further explain the protective effect of autophagy on the inflammatory response to trophoblast and the pathophysiology of preeclampsia. The study and the treatment of disease provide a new theoretical basis. 1, 26 cases of preeclampsia (group PE) were selected from June 2014 to January 2015 in the obstetrics department of the Affiliated Hospital of Qiingdao University and cesarean section, and 20 cases of normal pregnant women were selected as normal pregnant women group (group NP). All the two groups of pregnant women were enrolled. The blood 5ml of the elbow vein was extracted on the empty stomach after the hospital, and the serum was separated and stored in the refrigerator of -70 C. The two groups of pregnant women were treated with cesarean section to terminate the pregnancy. The placenta samples were obtained under sterile conditions for 30 minutes after delivery of the placenta, avoiding the necrosis and calcification of the placental maternal surface, and collecting two pieces of placental tissue about 1 cm * 1 cm x 1 cm, and sterilized physiological salt After rinsing the residual blood repeatedly, one of them was placed in a sterile centrifuge tube of 1.5 ml. 15 minutes were stored in the refrigerator of -70 centigrade for RNA and protein extraction. Another block of formaldehyde was fixed to the.2. The level of ox LDL in the serum of two groups of pregnant women, and the changes of TNF- A and IL-6 levels of inflammatory factors were detected by ELISA. Immunohistochemistry was used. The expression of TNF- alpha and IL-6 in placenta tissue was determined by method. The expression of TNF- alpha and IL-6 protein in the placental tissues of each group was detected by Western Blot method. The localization of autophagy related proteins (Beclin 1 and LC3) in placenta tissue was detected by immunohistochemical method, and real-time PCR method was used to detect the Beclin 1 in placenta tissue. The expression of Beclin 1 and LC3 protein in the placental tissues of each group was detected by Western Blot, and JEG-3 cell lines were purchased and cultured. The cells were collected after different concentrations of ox LDL (25,50100150mg/l) and different action time (6,12,24,48 hours). The cells were detected by immunofluorescence and different concentrations and different action times were detected by immunofluorescence. The effect of ox LDL on the autophagy related protein LC3 of trophoblast.5, using JEG-3 cell line and using ox LDL (25,50100150mg/l) at different concentrations for 6 hours, and using real-time PCR method to detect the changes of inflammatory factors in the trophoblast cells before and after ox LDL, and divide them into two groups. A group of ox LDL with concentration of 100mg/l was used for 6 hours, and another group was pretreated by preconditioning with rapamycin 100N M for 1 hours, and then ox LDL with a concentration of 100mg/l was used for 6 hours as the study condition. The real-time PCR method was used to determine the inflammatory factors (TNF- alpha and IL-6) in trophoblast. The changes in the protein expression level of autophagy related protein Beclin 1 and LC3 in trophoblast were detected.7, the statistical method was used for data processing with SPSS 17, all the results were _x + s, the two groups were compared with t test and P 0.05 was statistically significant. Results: 1, the results of ELISA method were used to prompt the sera of the preeclampsia group. The level of ox LDL, TNF- alpha and IL-6 was significantly higher than that in the normal control group. The difference was statistically significant (P0.05). The immunohistochemical results suggested that TNF- alpha and IL-6 were expressed in the placental trophoblast cells of preeclampsia and normal control groups, and the positive staining of the placenta in the placenta was deeper and the number of positive cells was more positive in the preeclampsia group. The expression of TNF- alpha and IL-6 protein detected by.Western Blot method in normal control group showed that the expression of inflammatory factors in placental tissue of preeclampsia group was higher than that of normal control group, and the difference was statistically significant (P0.05).2. The immunohistochemical results showed that Beclin 1 was a light brown positive staining in the trophoblastic cells which was higher than the background, mainly expressed in the cells. In the normal control group (group NP), the number of Beclin 1 and LC3 protein positive cells increased significantly and the staining enhanced.Real-time PCR assay showed the Beclin 1 and LC3 m RNA in the normal control group of the placental tissue. The expression level of the preeclampsia group was significantly higher than that in the preeclampsia group (P0.05) the expression of Beclin 1 and LC3 protein by.Western Blot method suggested that the expression of autophagy related protein in the normal control group was higher than the preeclampsia group, and the difference was statistically significant (P0.05).3, at different concentrations ox LDL (25,50100150mg/l) and not The cytotrophoblast cells were cultured at the same time of action (6,12,24,48 hours), and the cytoplasmic red dye LC3 spot aggregation could not be observed by immunofluorescence. When the cells were pretreated with rapamycin 100N M, the cells stimulated by ox LDL (100mg/l) could see the LC3 dot aggregation of erythrocyte in the cytoplasm at different time. After pretreated with rapamycin, after adding ox LDL to ox LDL cells, it was observed that the cell morphology was still intact after the action of 6h and 12h, but the cell nucleus could be swelled and deformed for 24 hours and 48 hours. Therefore, the action of 6 hours was the subsequent other experimental conditions.4, and the expression of TNF- A and IL-6 m was detected by real-time PCR method. The expression of inflammatory factors in the trophoblastic cells after 50mg/l and 100mg/l ox LDL was higher than that in the blank control group. The difference was statistically significant (P0.05). At the same time, the expression of M TOR in the trophoblastic cells after 100mg/l ox LDL was not significantly changed. Therefore, 100mg/l ox was selected as a follow-up experimental condition, and the cells were treated by rapamycin. The expression of Beclin 1 and LC3 protein detected by Western Blot method was significantly higher than that without rapamycin treatment group after 1 hours of pretreatment with 100mg/l ox LDL, and the difference was statistically significant (P0.05), while real-time PCR method was statistically significant (P0.05), while real-time PCR assay showed that the expression of the inflammatory factor was significantly lower than that of the non rapamycin treatment group. The difference was statistically significant (P0.05). Conclusion and significance: 1, the expression of oxidized low density lipoprotein and TNF- (IL-6) in preeclampsia patients
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.244

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