【摘要】：背景和目的： 妊娠期肝內(nèi)膽汁淤積癥(intrahepatic cholestasis of pregnancy, ICP)易導(dǎo)致胎兒急性宮內(nèi)窘迫甚至突然死亡,其病理生理機(jī)制不明,常規(guī)的產(chǎn)前監(jiān)測無法預(yù)測。故進(jìn)一步研究ICP病理生理機(jī)制,認(rèn)識ICP導(dǎo)致胎兒死亡的原因,是預(yù)防ICP不良妊娠結(jié)局的關(guān)鍵。 我們的前期研究發(fā)現(xiàn)ICP死胎的胎盤小血管中存在血栓及再通形成的現(xiàn)象,故提出假說：ICP孕母血液中升高的膽酸引起了胎盤血管內(nèi)皮細(xì)胞功能障礙,促使血栓形成,微循環(huán)障礙,嚴(yán)重影響母胎氧氣和營養(yǎng)物質(zhì)交換,引起胎兒急性宮內(nèi)窘迫甚至發(fā)生胎兒死亡。即ICP死胎的發(fā)生可能與膽酸引起的胎盤血管內(nèi)皮細(xì)胞功能障礙有關(guān)。 為驗證上述假說,我們進(jìn)行以下實驗：①觀察ICP胎盤組織學(xué)及超微結(jié)構(gòu)的變化；②免疫組織化學(xué)法檢測ICP與正常胎盤自噬相關(guān)蛋白LC3A/B與血栓形成相關(guān)蛋白vWF的表達(dá)；③利用體外細(xì)胞模型觀察膽酸對人血管內(nèi)皮細(xì)胞的影響。 材料與方法： 1.體內(nèi)試驗： ①ICP胎盤組織學(xué)觀察：收集1999年1月-2012年12月浙江大學(xué)醫(yī)學(xué)院附屬婦產(chǎn)科醫(yī)院病理科收藏的有完整臨床資料的ICP死胎的胎盤的檔案蠟塊13例,設(shè)為ICP死胎組。選擇浙江大學(xué)醫(yī)學(xué)院附屬婦產(chǎn)科醫(yī)院活胎分娩的孕婦共40例,其中ICP胎兒活產(chǎn)孕婦20例(ICP活胎組),健康孕婦正常分娩20例為正常妊娠組。光學(xué)顯微鏡下觀察各組胎盤組織石蠟切片的HE染色玻片,統(tǒng)計各組胎盤梗死、合體結(jié)節(jié)細(xì)胞增多、絨毛干血管血栓形成及再通發(fā)生的比例。 ②ICP胎盤超微結(jié)構(gòu)觀察：應(yīng)用透射電鏡掃描觀察ICP活胎組與正常妊娠組人胎盤組織中血管內(nèi)皮細(xì)胞的超微結(jié)構(gòu)改變。 ③免疫組織化學(xué)法研究：根據(jù)前期結(jié)果,通過免疫組化法檢測ICP與正常胎盤自噬相關(guān)蛋白LC3A/B與血栓形成相關(guān)蛋白vWF的表達(dá)。 2.細(xì)胞模型研究 ①用不同濃度梯度(0～100uM)的甘膽酸處理人血管內(nèi)皮細(xì)胞株(HUVEC)24小時。 ②透射電鏡掃描觀察甘膽酸處理的HUVEC超微結(jié)構(gòu)的變化。 ③通過蛋白免疫印跡方法檢測HUVEC自噬相關(guān)蛋白LC3Ⅱ與beclin-1的表達(dá)量。 結(jié)果： 1.ICP胎盤組織學(xué)變化： ①正常妊娠組2例有局灶性合體細(xì)胞結(jié)節(jié)增多,未見到梗死、底蛻膜血腫及血栓形成。 ②ICP活胎組20例胎盤病理檢查,其中10例有局灶性合體細(xì)胞結(jié)節(jié)增多,7例有梗死,4例見到底蛻膜血腫,1例見到絨毛膜下血栓。 ③ICP死胎組13例胎盤病理檢查均有局灶性合體細(xì)胞結(jié)節(jié)增多,其中5例可見梗死伴有小區(qū)鈣化、絨毛退行性變,5例遠(yuǎn)端絨毛干血管中可見血栓形成及再通。 2.ICP胎盤組織血管內(nèi)皮細(xì)胞超微結(jié)構(gòu)變化： 正常組胎盤組織中血管內(nèi)皮細(xì)胞內(nèi)可見W-P小體與大量彈力纖維；ICP活胎組胎盤組織中血管內(nèi)皮細(xì)胞內(nèi)亦可見到W-P小體與大量彈力纖維,同時可見到自噬小體明顯增多,并有向血管腔內(nèi)分泌的趨勢。 3.ICP胎盤自噬相關(guān)蛋白LC3A/B與血栓形成相關(guān)蛋白vWF的變化 (1)LC3蛋白主要表達(dá)于胎盤血管內(nèi)皮細(xì)胞、血管壁平滑肌細(xì)胞及胎盤滋養(yǎng)細(xì)胞的胞漿。正常妊娠組胎盤血管內(nèi)皮細(xì)胞表達(dá)較弱或者不表達(dá)。與正常妊娠組比較,ICP活胎組、ICP死胎組胎盤組織的血管內(nèi)皮細(xì)胞中,LC3蛋白表達(dá)遞次增加,三組間的比較差異有統(tǒng)計學(xué)意義(P0.001)。 (2)vWF因子主要表達(dá)于胎盤血管內(nèi)皮細(xì)胞的胞漿,正常妊娠組胎盤血管內(nèi)皮細(xì)胞表達(dá)較弱。與正常妊娠組比較,ICP活胎組、ICP死胎組胎盤組織中vWF因子表達(dá)明顯增加,三組間的比較差異有統(tǒng)計學(xué)意義(P0.001)。ICP死胎組可見絨毛干的血管中,VWF因子在血栓形成并再通的血管內(nèi)皮細(xì)胞上強(qiáng)表達(dá)。 (3)胎盤血管內(nèi)皮細(xì)胞LC3A/B與VWF的表達(dá)呈正相關(guān)(r=0.65,P0.001)。 4.甘膽酸對HUVEC超微結(jié)構(gòu)的影響： 用100uM甘膽酸與HUVEC細(xì)胞共培養(yǎng)24小時,透射電鏡掃描觀察到HUEVC細(xì)胞內(nèi)自噬小體較對照組明顯增多。 5.甘膽酸對HUVEC細(xì)胞LC3B-Ⅱ、Beclin-1蛋白表達(dá)的影響。 不同濃度(0～100uM)的甘膽酸作用24小時后,HUVEC的LC3B-Ⅱ、Beclin-1表達(dá)量隨甘膽酸濃度的增加而逐漸增加。其中100uM組LC3B-Ⅱ的表達(dá)量顯著高于對照組(P0.01)。 結(jié)論： 1.ICP死胎組胎盤血管存在血栓及再通現(xiàn)象,提示血栓形成可能是ICP胎兒窘迫甚至死胎發(fā)生時胎盤重要的病理變化之一。 2.ICP胎盤血管內(nèi)皮細(xì)胞自噬增加,體外細(xì)胞模型研究證實甘膽酸可誘導(dǎo)血管內(nèi)皮細(xì)胞自噬增加,提示自噬增加是ICP重要的病理生理機(jī)制之一,可能與胎盤血栓形成、胎兒窘迫、死胎發(fā)生有關(guān)。
[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，

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