胎盘血管内皮细胞自噬在妊娠期肝内胆汁淤积症的作用及机制
发布时间：2019-03-03 19:54
【摘要】：背景和目的： 妊娠期肝内胆汁淤积症(intrahepatic cholestasis of pregnancy, ICP)易导致胎儿急性宫内窘迫甚至突然死亡,其病理生理机制不明,常规的产前监测无法预测。故进一步研究ICP病理生理机制,认识ICP导致胎儿死亡的原因,是预防ICP不良妊娠结局的关键。 我们的前期研究发现ICP死胎的胎盘小血管中存在血栓及再通形成的现象,故提出假说：ICP孕母血液中升高的胆酸引起了胎盘血管内皮细胞功能障碍,促使血栓形成,微循环障碍,严重影响母胎氧气和营养物质交换,引起胎儿急性宫内窘迫甚至发生胎儿死亡。即ICP死胎的发生可能与胆酸引起的胎盘血管内皮细胞功能障碍有关。 为验证上述假说,我们进行以下实验：①观察ICP胎盘组织学及超微结构的变化；②免疫组织化学法检测ICP与正常胎盘自噬相关蛋白LC3A/B与血栓形成相关蛋白vWF的表达；③利用体外细胞模型观察胆酸对人血管内皮细胞的影响。 材料与方法： 1.体内试验： ①ICP胎盘组织学观察：收集1999年1月-2012年12月浙江大学医学院附属妇产科医院病理科收藏的有完整临床资料的ICP死胎的胎盘的档案蜡块13例,设为ICP死胎组。选择浙江大学医学院附属妇产科医院活胎分娩的孕妇共40例,其中ICP胎儿活产孕妇20例(ICP活胎组),健康孕妇正常分娩20例为正常妊娠组。光学显微镜下观察各组胎盘组织石蜡切片的HE染色玻片,统计各组胎盘梗死、合体结节细胞增多、绒毛干血管血栓形成及再通发生的比例。 ②ICP胎盘超微结构观察：应用透射电镜扫描观察ICP活胎组与正常妊娠组人胎盘组织中血管内皮细胞的超微结构改变。 ③免疫组织化学法研究：根据前期结果,通过免疫组化法检测ICP与正常胎盘自噬相关蛋白LC3A/B与血栓形成相关蛋白vWF的表达。 2.细胞模型研究 ①用不同浓度梯度(0～100uM)的甘胆酸处理人血管内皮细胞株(HUVEC)24小时。 ②透射电镜扫描观察甘胆酸处理的HUVEC超微结构的变化。 ③通过蛋白免疫印迹方法检测HUVEC自噬相关蛋白LC3Ⅱ与beclin-1的表达量。 结果： 1.ICP胎盘组织学变化： ①正常妊娠组2例有局灶性合体细胞结节增多,未见到梗死、底蜕膜血肿及血栓形成。 ②ICP活胎组20例胎盘病理检查,其中10例有局灶性合体细胞结节增多,7例有梗死,4例见到底蜕膜血肿,1例见到绒毛膜下血栓。 ③ICP死胎组13例胎盘病理检查均有局灶性合体细胞结节增多,其中5例可见梗死伴有小区钙化、绒毛退行性变,5例远端绒毛干血管中可见血栓形成及再通。 2.ICP胎盘组织血管内皮细胞超微结构变化： 正常组胎盘组织中血管内皮细胞内可见W-P小体与大量弹力纤维；ICP活胎组胎盘组织中血管内皮细胞内亦可见到W-P小体与大量弹力纤维,同时可见到自噬小体明显增多,并有向血管腔内分泌的趋势。 3.ICP胎盘自噬相关蛋白LC3A/B与血栓形成相关蛋白vWF的变化 (1)LC3蛋白主要表达于胎盘血管内皮细胞、血管壁平滑肌细胞及胎盘滋养细胞的胞浆。正常妊娠组胎盘血管内皮细胞表达较弱或者不表达。与正常妊娠组比较,ICP活胎组、ICP死胎组胎盘组织的血管内皮细胞中,LC3蛋白表达递次增加,三组间的比较差异有统计学意义(P0.001)。 (2)vWF因子主要表达于胎盘血管内皮细胞的胞浆,正常妊娠组胎盘血管内皮细胞表达较弱。与正常妊娠组比较,ICP活胎组、ICP死胎组胎盘组织中vWF因子表达明显增加,三组间的比较差异有统计学意义(P0.001)。ICP死胎组可见绒毛干的血管中,VWF因子在血栓形成并再通的血管内皮细胞上强表达。 (3)胎盘血管内皮细胞LC3A/B与VWF的表达呈正相关(r=0.65,P0.001)。 4.甘胆酸对HUVEC超微结构的影响： 用100uM甘胆酸与HUVEC细胞共培养24小时,透射电镜扫描观察到HUEVC细胞内自噬小体较对照组明显增多。 5.甘胆酸对HUVEC细胞LC3B-Ⅱ、Beclin-1蛋白表达的影响。 不同浓度(0～100uM)的甘胆酸作用24小时后,HUVEC的LC3B-Ⅱ、Beclin-1表达量随甘胆酸浓度的增加而逐渐增加。其中100uM组LC3B-Ⅱ的表达量显著高于对照组(P0.01)。 结论： 1.ICP死胎组胎盘血管存在血栓及再通现象,提示血栓形成可能是ICP胎儿窘迫甚至死胎发生时胎盘重要的病理变化之一。 2.ICP胎盘血管内皮细胞自噬增加,体外细胞模型研究证实甘胆酸可诱导血管内皮细胞自噬增加,提示自噬增加是ICP重要的病理生理机制之一,可能与胎盘血栓形成、胎儿窘迫、死胎发生有关。
[Abstract]:Background and purpose: Intrahepatic cholestasis of pregnancy (ICP) is easy to cause acute fetal distress and even sudden death of the fetus. To further study the pathophysiological mechanism of ICP, and to know the cause of the death of the fetus by ICP, which is the effect of the prevention of the adverse pregnancy outcome in the ICP. The results suggested that the elevated cholic acid in the maternal blood of ICP induced the dysfunction of the vascular endothelial cells in the placenta, which led to the formation of the thrombus and the micro-circulation. Ring disorder, which seriously affects the exchange of oxygen and nutrients in the mother's fetus, resulting in an acute fetal distress or even a miscarriage of the fetus. The death of a child. That is, the occurrence of an ICP dead fetus may be related to the dysfunction of the placental vascular endothelial cell caused by the cholic acid. In order to verify the above hypothesis, we carried out the following experiments: to observe the changes of the histological and ultrastructure of the ICP placenta; and to detect the relevant protein LC3A/ B and the thrombosis-related protein v of the normal placenta autophagy-related protein LC3A/ B by immunohistochemistry. Expression of WF; in vitro cell model to observe the effect of cholic acid on human vascular endothelial cells The effect of the cells. Materials and methods 1. In-vivo test: the histological observation of the placenta of ICP-ICP: the collection of the records of the placenta of the ICP dead fetus with complete clinical data collected in the Department of Pathology of the Department of Obstetrics and Gynecology of Zhejiang University from January 1999 to December,2012 A total of 40 pregnant women with live birth in the Affiliated Hospital of Obstetrics and Gynecology and Obstetrics and Gynecology of Zhejiang University were selected, of which 20 (ICP) and healthy pregnant women were normal. In 20 cases of normal pregnancy, the HE stained slides of the paraffin sections of each group were observed under the optical microscope, and the placental infarction, the increase of the body nodule cells and the blood vessel blood of the villi were counted. The proportion of the formation and recanalization of the plug: the observation of the ultrastructure of the ICP placenta: the observation of the ICP live fetal group and the normal pregnancy group by transmission electron microscopy Ultrastructural changes of vascular endothelial cells. Chemical study of vascular endothelial cells: According to the early results, the plasma-related protein LC3A/ B of ICP and normal placenta was detected by immunohistochemistry. An egg related to the formation of a thrombus The expression of white vWF.2. The cell model was treated with a different concentration gradient (0-100 uM) of glycocholic acid. human vascular endothelial cell strain (HUVEC) for 24 h. transmission electron microscopy Changes of the ultrastructure of HUVEC treated with chenodeoxycholic acid. HUVEC was detected by Western blot. autophagy Off-protein LC3 鈪，
本文编号：2434040
[Abstract]:Background and purpose: Intrahepatic cholestasis of pregnancy (ICP) is easy to cause acute fetal distress and even sudden death of the fetus. To further study the pathophysiological mechanism of ICP, and to know the cause of the death of the fetus by ICP, which is the effect of the prevention of the adverse pregnancy outcome in the ICP. The results suggested that the elevated cholic acid in the maternal blood of ICP induced the dysfunction of the vascular endothelial cells in the placenta, which led to the formation of the thrombus and the micro-circulation. Ring disorder, which seriously affects the exchange of oxygen and nutrients in the mother's fetus, resulting in an acute fetal distress or even a miscarriage of the fetus. The death of a child. That is, the occurrence of an ICP dead fetus may be related to the dysfunction of the placental vascular endothelial cell caused by the cholic acid. In order to verify the above hypothesis, we carried out the following experiments: to observe the changes of the histological and ultrastructure of the ICP placenta; and to detect the relevant protein LC3A/ B and the thrombosis-related protein v of the normal placenta autophagy-related protein LC3A/ B by immunohistochemistry. Expression of WF; in vitro cell model to observe the effect of cholic acid on human vascular endothelial cells The effect of the cells. Materials and methods 1. In-vivo test: the histological observation of the placenta of ICP-ICP: the collection of the records of the placenta of the ICP dead fetus with complete clinical data collected in the Department of Pathology of the Department of Obstetrics and Gynecology of Zhejiang University from January 1999 to December,2012 A total of 40 pregnant women with live birth in the Affiliated Hospital of Obstetrics and Gynecology and Obstetrics and Gynecology of Zhejiang University were selected, of which 20 (ICP) and healthy pregnant women were normal. In 20 cases of normal pregnancy, the HE stained slides of the paraffin sections of each group were observed under the optical microscope, and the placental infarction, the increase of the body nodule cells and the blood vessel blood of the villi were counted. The proportion of the formation and recanalization of the plug: the observation of the ultrastructure of the ICP placenta: the observation of the ICP live fetal group and the normal pregnancy group by transmission electron microscopy Ultrastructural changes of vascular endothelial cells. Chemical study of vascular endothelial cells: According to the early results, the plasma-related protein LC3A/ B of ICP and normal placenta was detected by immunohistochemistry. An egg related to the formation of a thrombus The expression of white vWF.2. The cell model was treated with a different concentration gradient (0-100 uM) of glycocholic acid. human vascular endothelial cell strain (HUVEC) for 24 h. transmission electron microscopy Changes of the ultrastructure of HUVEC treated with chenodeoxycholic acid. HUVEC was detected by Western blot. autophagy Off-protein LC3 鈪，
本文编号：2434040
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