复发性流产血栓前状态与肾虚血瘀证相关性及其蛋白组学研究

发布时间：2018-09-18 20:54

【摘要】：复发性流产是育龄女性的多发病及疑难病,病因复杂多样。近年来,因持续高凝状态而导致的血栓形成倾向即血栓前状态(Prethrombotic State, PTS),越来越受到国内外研究者的关注,被认为是导致胎儿死亡及反复流产的重要原因之一。目前,对复发性流产血栓前状态的干预,西医主要以阿司匹林或低分子肝素抗凝治疗为主,但由于存在出血和胃肠道不适等诸多不良反应,使其临床应用受到了较大限制。近年,中医药抗栓效应逐渐得到证实,加之其在治疗复发性流产中的独特优势,应用中医药进行复发性流产血栓前状态的治疗成为了研究的热点之一。我们前期对中医药治疗复发性流产血栓前状态进行的临床观察,结果提示以补肾活血养血中药治疗较为有效,但未进行深入系统的研究。对于复发性流产的治疗,中医学主张“预培其损”,若能明确复发性流产血栓前状态的主要中医证候,对这类人群进行对证治疗,则可以起到“未孕先防”的作用。课题组前期应用补肾活血养血中药治疗复发性流产血栓前状态取得显著临床疗效,且不良反应较小,那么,以药测证,肾虚血瘀证是否为复发性流产血栓前状态的主要证型?如果是,其与非肾虚血瘀证在子宫内膜病理组织学上是否具有差异?在蛋白质组学上又有何不同?为此,我们开展了本项研究,从证候分析,到组织病理学的研究,再到微观蛋白质组的探索,希望能更深入的研究复发性流产血栓前状态这一疾病,对临床防治该病提供一定的临床及实验依据。目的1通过对复发性流产血栓前状态的中医证候研究,探讨其主要证型。2通过研究不同证型复发性流产血栓前状态在子宫内膜容受性及组织病理学上的差异,进一步分析肾虚血瘀证与复发性流产血栓前状态的相关性。3借助蛋白芯片技术研究复发性流产血栓前状态肾虚血瘀证与非肾虚血瘀证蛋白质组学差异,探索该病不同证候间的不同蛋白质表达。方法1参考《中医妇科常见病诊疗指南》、《中医妇科学》及相关文献中制定调查表,建立电子数据库,在严格的质量控制下,对205例复发性流产血栓前状态患者进行电子数据库录入,运用SAS统计软件对数据进行频数统计、聚类分析、多分类logistic回归,探索复发性流产血栓前状态中医证候特征及分布规律,分析其主要证型。2依据第一部分聚类后的中医证候研究,分析在子宫内膜组织上肾虚血瘀证与复发性流产血栓前状态的相关性。分别入组肾虚血瘀证、脾肾两虚证、气血虚弱证及肾精亏虚证患者各15例,共60例患者,黄体期采用一次性宫腔吸管采集内膜组织HE染色观察子宫内膜腺体发育情况、透射电镜下观察胞饮突分布情况；排卵日B超检测子宫内膜血流灌注情况,包括子宫内膜厚度、内膜形态、内膜血流分型、内膜血流搏动指数(PI)、内膜血流阻力指数(RI)；黄体期ELLSA法检测与血栓前状态相关血小板衍生生长因子家族包括血小板生长因子(PDGF)和血管内皮细胞因子(VEGF)及纤溶系统(t-PA, PAI-1),从组织病理学、B超学、分子生物学角度对60例复发性流产血栓前状态患者胞饮突、内膜组织HE染色后结果、内膜厚度、内膜状态、内膜血流、PI、RI、PAI-1、 t-PA、VEGF、PDGF-AA这些指标进行相关性分析,并探讨这些变量指标在不同证型的差异。3在第一部分及第二部分的研究基础上,我们采用血清标本,利用蛋白芯片进行蛋白表达谱分析,探讨血栓前状态引起的复发性流产患者中肾虚血瘀证与非肾虚血瘀证的蛋白表达差异。通过分析确定差异蛋白并定制蛋白芯片,再扩大样本量进行验证。①提取有过正常孕产史妇女的血清样本6例(正常组),血栓前状态引起的复发性流产患者肾虚血瘀证血清样本12例(肾虚血瘀组),血栓前状态引起的复发性流产患者非肾虚血瘀证血清样本11例(非肾虚血瘀组)。②利用raybiotech蛋白芯片(货号：AAH-BLG-1000指标数量：1000)提取正常组、肾虚血瘀组、非肾虚血瘀组的蛋白表达图谱,分析正常组-疾病组、正常组-肾虚血瘀组、肾虚血瘀组-非肾虚血瘀组差异蛋白,以主成分分析图表示每组样本的整体蛋白表达差异。③以聚类图及维恩图(交集图)选出血栓组中特异表达的蛋白,将差异蛋白进行显着性功能分析及KEGG pathway/pathway-net分析,得到与复发性流产血栓前状态相关蛋白。④根据筛选的关键蛋白定制芯片(定制指标数目根据筛选的结果确定),扩大样本量选取肾虚血瘀证组30(12+18)例、非肾虚血瘀证组30(11+19)例样本进行验证。结果1对205例复发性流产血栓前状态患者的症状分布进行频率及聚类分析,其中有87例具备肾虚血瘀型多数证候表现,其次为脾肾两虚证、气血虚弱证、肾精亏虚证；经过频数统计和聚类分析后,得出肾虚血瘀证为复发性流产血栓前状态主证,所占比例为47.28%,出现频率较高的症状为：月经色暗红,腰膝酸软,面色晦暗,胸胁刺痛,耳鸣腰痛,舌紫暗,有瘀斑,脉沉涩。2①不同证候复发性流产血栓前状态患者在子宫内膜容受性形态学上表现：常规HE染色,60例样本中,45例子宫内膜为佳型内膜,其中肾虚血瘀组8例,占17.78%,少于其它三组证型；另有15例子宫内膜为差型内膜,其中肾虚血瘀组7例,占46.67%,多于其它三组证型。采用卡方检验四组组间比较差异不显著(P=0.110.05)。透射电镜下观察,大部分子宫内膜组织可以检测到胞饮突,多数处于发育完全成熟阶段,少数处于发育中阶段或退缩阶段。其中7例无胞饮突发育,肾虚血瘀组3例；32例胞饮突表达少量,肾虚血瘀组9例；15例胞饮突表达中等,肾虚血瘀组2例；6例胞饮突表达丰富,肾虚血瘀组1例；用CMH卡方检验四组组间比较差异不显著(P=0.150.05)。②不同证候复发性流产血栓前状态患者在子宫内膜容受性B超上表现：排卵日测子宫内膜动脉血流,60例患者中,肾虚血瘀组15例,四组组间比较有显著性差异,肾虚血瘀组搏动指数和阻力指数均高于其它三组(P0.01)。在子宫内膜厚度、内膜形态、内膜血流分型统计中,四组组间无显著性差异(P0.05)。③不同证候复发性流产血栓前状态患者在子宫内膜容受性分子生物学上相关性：PAI-1与t-PA呈正相关(r=0.415,P0.01),PAI-1与PDGF-AA呈正相关(r=0.390,P0.01),PAI-1与RI呈正相关(r=0.296,P0.05),PAI-1与内膜血流分型(按照由Ⅰ-Ⅲ顺序编码)呈负相关(r=-0.267, P0.05), t-PA与VEGF呈负相关(r=-0.653, P0.01),t-PA与PDGF-AA呈正相关(r=0.501, P0.01),t-PA与RI呈正相关(r=0.399, P0.01), PDGF-AA与RI呈正相关(r=0.767,P0.01), PDGF-AA与内膜血流分型(按照由Ⅰ-Ⅲ顺序编码)呈负相关(r=-0.570, P0.01), PDGF-AA与胞饮突(按照由丰富-阴性顺序编码)呈正相关(r=0.369, P0.01), PI与RI呈正相关(r=0.508, P0.01), PI与内膜血流分型(按照由Ⅰ-Ⅲ顺序编码)呈负相关(r=-0.308, P0.05), PI与HE染色结果(按照由佳型-差型顺序编码)呈正相关(r=0.440,P0.01),PI与胞饮突(按照由丰富-阴性顺序编码)呈正相关(r=0.360, P0.01), RI与内膜血流分型(按照由Ⅰ-Ⅲ顺序编码)呈负相关(r=-0.762, P0.01), RI与HE染色结果(按照由佳型-差型顺序编码)呈正相关(r=0.354, P0.01), RI与胞饮突(按照由丰富-阴性顺序编码)呈正相关(r=0.519,P0.01),子宫内膜厚度与HE染色结果(按照由佳型-差型顺序编码)呈负相关(r=0.-358,P0.01),子宫内膜血流分型(按照由Ⅰ-Ⅲ顺序编码)与HE染色结果(按照由佳型-差型顺序编码)呈负相关(r=-0.353,P0.01),子宫内膜血流分型(按照由Ⅰ-Ⅲl顺序编码)与胞饮突(按照由丰富-阴性顺序编码)呈负相关(r=-0.699, P0.01), HE染色结果(按照由佳型-差型顺序编码)与胞饮突(按照由丰富-阴性顺序编码)呈正相关(r=0.332,P0.05)。肾虚血瘀证患者VEGF结果低于其它三组,PDGF-AA、t-PA、PAI-1高于其它三组。其中PDGF-AA及t-PA有显著差异(P0.05)。3①复发性流产血栓前状态组与正常组相比,151种细胞因子具有明显差异表达。与正常组相比较,在疾病组血清中8种细胞因子表达显著上调,143种细胞因子表达显著下调,通过对151种细胞因子丰度聚类,复发性流产组的16个细胞因子表达模式相似,且区别于正常组。②肾虚血瘀组与正常组之间,118种细胞因子具有明显差异表达。与正常组相比,在肾虚血瘀组血清中1种细胞因子表达显著上调,117种细胞因子表达显著下调,通过对以上118种细胞因子进行丰度聚类分析,结果显示,肾虚血瘀组的76个细胞因子表达模式相似,且区别于正常组。③肾虚血瘀组与非肾虚血瘀组之间,33种细胞因子具有明显差异表达。与非肾虚血瘀组相比,在肾虚血瘀组血清中7种细胞因子表达显著上调,26种细胞因子表达显著下调,通过对以上33种细胞因子进行丰度聚类分析,结果显示,肾虚血瘀组的20个细胞因子表达模式相似,且区别于非肾虚血瘀组。④另外,复发性流产血栓前状态肾虚血瘀组与非肾虚血瘀组相比较,在信号通路方面细胞因子涉及CXC subfamily、CC subfamily、Hematopoietins、PDGF family、TNFfamily、 TGF-(3family。结论1复发性流产血栓前状态以肾虚血瘀证为主要中医证候。2与其他证型相比,肾虚血瘀证子宫内膜病理组织形态学改变更显著,可能通过影响血管生成系统或纤溶系统造成血管的紧张收缩,形成高凝状态,影响内膜的血液循环及血流灌注,在生理结构上腺体发育不良,胞饮突表达不丰富,最终造成子宫内膜容受性下降,甚至反复流产。3肾虚血瘀证与非肾虚血瘀证在蛋白组学方面有明显差异,33种细胞因子具有明显差异表达,涉及CXC亚族、CC亚族、Hematopoietins、PDGF、TNF、TGF-β家族等7个信号通路,涉及凝血、血小板聚集、炎症、血管形成等,这可能是复发性流产血栓前状态肾虚血瘀证的证候实质。
[Abstract]:Recurrent abortion is a common and difficult disease in women of childbearing age. The etiology of recurrent abortion is complex and varied. In recent years, the tendency of thrombosis caused by persistent hypercoagulable state (PTS) has attracted more and more attention from researchers at home and abroad. It is considered as one of the important causes of fetal death and recurrent abortion. In recent years, the antithrombotic effect of traditional Chinese medicine has been gradually confirmed, and it is unique in the treatment of recurrent abortion. Our previous clinical observation on the prethrombotic state of recurrent abortion with traditional Chinese medicine showed that the treatment with traditional Chinese medicine for invigorating the kidney and activating blood circulation and nourishing blood was more effective, but there was no thorough and systematic study on the treatment of recurrent abortion. Treatment, Chinese medicine advocates "pre-culture its damage", if the main TCM symptoms of recurrent abortion before thrombosis can be clearly identified, the treatment of this group of people can play a "pregnant first prevention" role. If it is different from non-kidney deficiency and blood stasis syndrome in endometrial pathology and histopathology, what is the difference in proteomics? Therefore, we carried out this study, from syndrome analysis to histopathology. Objective 1 To explore the main syndrome types of recurrent abortion by studying the TCM syndromes of the pre-thrombotic state of recurrent abortion. The difference of endometrial receptivity and histopathology of prethrombotic state was analyzed, and the correlation between kidney deficiency and blood stasis syndrome and prethrombotic state of recurrent abortion was further analyzed. Methods 1 Referring to the Guidelines for Diagnosis and Treatment of Common Gynecologic Diseases in Traditional Chinese Medicine and Gynecology in Traditional Chinese Medicine and related literatures, a questionnaire was made and an electronic database was established. Under strict quality control, 205 patients with recurrent spontaneous abortion with prethrombotic state were entered into the electronic database, and the data were statistically analyzed by SAS statistical software. Analysis, multi-classification logistic regression, explore the characteristics and distribution of TCM syndrome before thrombosis in recurrent abortion, and analyze the main syndrome types. 2 According to the first part of the study of TCM syndrome after clustering, analyze the correlation between the kidney deficiency and blood stasis syndrome in endometrium and the state before thrombosis in recurrent abortion. There were 15 cases of deficiency of both qi and blood, and 60 cases of deficiency of kidney essence. In luteal phase, the endometrial glands were observed by HE staining and the distribution of endocrine drinks was observed by transmission electron microscope. Morphology, Endometrial Blood Flow Classification, Endometrial Blood Flow Pulse Index (PI), Endometrial Blood Flow Resistance Index (RI); Prethrombotic State-related Platelet Derived Growth Factor Family (PDGF), Vascular Endothelial Cell Factor (VEGF) and Fibrinolytic System (t-PA, PAI-1), Histopathology, B Ultrasonography, Molecular Biogenesis Correlation analysis was made on 60 cases of recurrent spontaneous abortion patients with pre-thrombotic state, such as pinocyte process, endometrial tissue HE staining results, intimal thickness, intimal state, intimal blood flow, PI, RI, PAI-1, t-PA, VEGF, PDGF-AA, and the differences of these variables in different syndromes were discussed. We used protein chip to analyze the protein expression profiles of serum samples and explore the protein expression differences between kidney deficiency and blood stasis syndrome and non-kidney deficiency and blood stasis syndrome in patients with recurrent abortion caused by pre-thrombotic state. The serum samples of 6 women (normal group), 12 patients (kidney deficiency and blood stasis group) with recurrent spontaneous abortion caused by prethrombotic state, 11 patients (non-kidney deficiency and blood stasis group) with recurrent spontaneous abortion caused by prethrombotic state were collected. Quantity: 1000) The protein expression profiles of normal group, kidney deficiency and blood stasis group, non-kidney deficiency and blood stasis group were extracted, and the differential proteins of normal group-disease group, normal group-kidney deficiency and blood stasis group, kidney deficiency and blood stasis group-non-kidney deficiency and blood stasis group were analyzed. The principal component analysis (PCA) was used to show the overall protein expression differences of each group. Specifically expressed proteins in the embolic group were analyzed by KEGG pathway/pathway-net and significant functional analysis. Proteins related to the pre-thrombotic state of recurrent abortion were obtained. Results 1 Frequency and cluster analysis were performed on the symptoms of 205 cases of recurrent abortion with prethrombotic state. 87 of them had the most symptoms of kidney deficiency and blood stasis, followed by spleen and kidney deficiency, Qi and blood deficiency, kidney essence deficiency. After that, the kidney deficiency and blood stasis syndrome was the main syndrome of recurrent abortion before thrombosis, accounting for 47.28%. The symptoms with high frequency were: dark red menstruation, sore waist and knee, dark complexion, chest and flank tingling pain, tinnitus and lumbago, purple tongue, ecchymosis, pulse depression. Morphological manifestations: routine HE staining, 45 cases of endometrium in 60 samples, including 8 cases of kidney deficiency and blood stasis group, accounting for 17.78%, less than the other three syndrome types; and 15 cases of endometrium is poor type, of which 7 cases of kidney deficiency and blood stasis group, accounting for 46.67%, more than the other three syndrome types. 110.05). Most of the endometrial tissues can be detected by transmission electron microscopy, most of them are in the stage of full maturity, a few are in the stage of development or retraction. There were 2 cases in blood stasis group, 1 case in kidney deficiency and blood stasis group, and 1 case in kidney deficiency and blood stasis group. There was no significant difference between the four groups (P = 0.150.05) by CMH chi-square test. There was no significant difference between the four groups in the statistics of endometrial thickness, endometrial morphology and endometrial blood flow classification (P PAI-1 was positively correlated with t-PA (r = 0.415, P 0.01), PAI-1 was positively correlated with PDGF-AA (r = 0.390, P 0.01), PAI-1 was positively correlated with RI (r = 0.296, P 0.05), PAI-1 was negatively correlated with intimal blood flow typing (coded by I-III sequence) was negatively correl (r =-0.267, P 0.267, P 0.05), t-PAwas negatively correlwith VEGF (r =-0.653, P 0.653, P 0.01), t-PAwas positipositipositively correlwith PDGF-AA (r = 0.656, P 0.01), t-PAI-PAwas positipositipositively correl(t) PDGF-AA was positively correlated with RI (r = 0.399, P 0.01), PDGF-AA was positively correlated with RI (r = 0.767, P 0.01), PDGF-AA was negatively correlated with endometrial blood flow typing (encoded by I-III sequence) (r =-0.570, P 0.01), PDGF-AA was positively correlated with pinocytes (encoded by rich-negative sequence) and PI was positively correlated with RI (r = 0.369, P 0.01). There was a negative correlation between PI and HE staining (r = 0.440, P 0.01), a positive correlation between PI and pinocytes (r = 0.360, P 0.01) and a negative correlation between RI and endometrial blood flow (P 0.01). Correlation (r = - 0.762, P 0.01), RI was positively correlated with HE staining results (r = 0.354, P 0.01), RI was positively correlated with pinocytes (r = 0.519, P 0.01), and endometrial thickness was negatively correlated with HE staining results (r = 0.358, P 0.01). Membrane blood flow typing (coded by I-III sequence) was negatively correlated with HE staining results (coded by Best-Difference sequence) (r =-0.353, P 0.01), endometrial blood flow typing (coded by I-III sequence) was negatively correlated with pinocytes (coded by Rich-Negative sequence) (r =-0.699, P 0.01), and HE staining results (coded by Best-Difference sequence). The results of VEGF in patients with kidney deficiency and blood stasis syndrome were lower than those in the other three groups. PDGF-AA, t-PA, PAI-1 were higher than those in the other three groups. There were significant differences between PDGF-AA and t-PA (P Compared with the normal group, the expression of 8 kinds of cytokines was significantly up-regulated and 143 kinds of cytokines were significantly down-regulated in the serum of the disease group. Compared with the normal group, the expression of one cytokine was significantly up-regulated and 117 cytokines were significantly down-regulated in the serum of the kidney deficiency and blood stasis group. Compared with non-kidney deficiency and blood stasis group, 7 kinds of cytokines were significantly up-regulated and 26 kinds of cytokines were significantly down-regulated in serum of kidney deficiency and blood stasis group. The expression patterns of 20 cytokines were similar and different from those of non-kidney deficiency and blood stasis group. 4. In addition, compared with non-kidney deficiency and blood stasis group, the cytokines involved in CXC subfamily, CC subfamily, hematopoietins, PDGF family, TNF family, TGF - (3 family) in the signal pathway of recurrent abortion before thrombosis. Prethrombotic state of kidney deficiency and blood stasis syndrome is the main TCM syndrome. 2 Compared with other syndrome types, the endometrial pathological and histomorphological changes of kidney deficiency and blood stasis syndrome are more significant, which may cause tension and contraction of blood vessels by affecting angiogenesis system or fibrinolysis system, forming hypercoagulable state, affecting blood circulation and blood perfusion of endometrium, and in physiological structure. There are obvious differences in proteomics between Kidney-Deficiency and blood-stasis syndrome and non-kidney-deficiency and blood-stasis syndrome. 33 kinds of cytokines have obvious differences in expression, involving seven signaling pathways, such as CXC subgroup, CC subgroup, Hematopoietins, PDGF, TNF, TGF-beta family. It involves blood coagulation, platelet aggregation, inflammation, angiogenesis and so on, which may be the syndrome essence of kidney deficiency and blood stasis syndrome before recurrent abortion thrombosis.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R271.9

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