【摘要】：復發(fā)性流產(chǎn)是育齡女性的多發(fā)病及疑難病,病因復雜多樣。近年來,因持續(xù)高凝狀態(tài)而導致的血栓形成傾向即血栓前狀態(tài)(Prethrombotic State, PTS),越來越受到國內(nèi)外研究者的關注,被認為是導致胎兒死亡及反復流產(chǎn)的重要原因之一。目前,對復發(fā)性流產(chǎn)血栓前狀態(tài)的干預,西醫(yī)主要以阿司匹林或低分子肝素抗凝治療為主,但由于存在出血和胃腸道不適等諸多不良反應,使其臨床應用受到了較大限制。近年,中醫(yī)藥抗栓效應逐漸得到證實,加之其在治療復發(fā)性流產(chǎn)中的獨特優(yōu)勢,應用中醫(yī)藥進行復發(fā)性流產(chǎn)血栓前狀態(tài)的治療成為了研究的熱點之一。我們前期對中醫(yī)藥治療復發(fā)性流產(chǎn)血栓前狀態(tài)進行的臨床觀察,結(jié)果提示以補腎活血養(yǎng)血中藥治療較為有效,但未進行深入系統(tǒng)的研究。對于復發(fā)性流產(chǎn)的治療,中醫(yī)學主張“預培其損”,若能明確復發(fā)性流產(chǎn)血栓前狀態(tài)的主要中醫(yī)證候,對這類人群進行對證治療,則可以起到“未孕先防”的作用。課題組前期應用補腎活血養(yǎng)血中藥治療復發(fā)性流產(chǎn)血栓前狀態(tài)取得顯著臨床療效,且不良反應較小,那么,以藥測證,腎虛血瘀證是否為復發(fā)性流產(chǎn)血栓前狀態(tài)的主要證型?如果是,其與非腎虛血瘀證在子宮內(nèi)膜病理組織學上是否具有差異?在蛋白質(zhì)組學上又有何不同?為此,我們開展了本項研究,從證候分析,到組織病理學的研究,再到微觀蛋白質(zhì)組的探索,希望能更深入的研究復發(fā)性流產(chǎn)血栓前狀態(tài)這一疾病,對臨床防治該病提供一定的臨床及實驗依據(jù)。目的1通過對復發(fā)性流產(chǎn)血栓前狀態(tài)的中醫(yī)證候研究,探討其主要證型。2通過研究不同證型復發(fā)性流產(chǎn)血栓前狀態(tài)在子宮內(nèi)膜容受性及組織病理學上的差異,進一步分析腎虛血瘀證與復發(fā)性流產(chǎn)血栓前狀態(tài)的相關性。3借助蛋白芯片技術研究復發(fā)性流產(chǎn)血栓前狀態(tài)腎虛血瘀證與非腎虛血瘀證蛋白質(zhì)組學差異,探索該病不同證候間的不同蛋白質(zhì)表達。方法1參考《中醫(yī)婦科常見病診療指南》、《中醫(yī)婦科學》及相關文獻中制定調(diào)查表,建立電子數(shù)據(jù)庫,在嚴格的質(zhì)量控制下,對205例復發(fā)性流產(chǎn)血栓前狀態(tài)患者進行電子數(shù)據(jù)庫錄入,運用SAS統(tǒng)計軟件對數(shù)據(jù)進行頻數(shù)統(tǒng)計、聚類分析、多分類logistic回歸,探索復發(fā)性流產(chǎn)血栓前狀態(tài)中醫(yī)證候特征及分布規(guī)律,分析其主要證型。2依據(jù)第一部分聚類后的中醫(yī)證候研究,分析在子宮內(nèi)膜組織上腎虛血瘀證與復發(fā)性流產(chǎn)血栓前狀態(tài)的相關性。分別入組腎虛血瘀證、脾腎兩虛證、氣血虛弱證及腎精虧虛證患者各15例,共60例患者,黃體期采用一次性宮腔吸管采集內(nèi)膜組織HE染色觀察子宮內(nèi)膜腺體發(fā)育情況、透射電鏡下觀察胞飲突分布情況；排卵日B超檢測子宮內(nèi)膜血流灌注情況,包括子宮內(nèi)膜厚度、內(nèi)膜形態(tài)、內(nèi)膜血流分型、內(nèi)膜血流搏動指數(shù)(PI)、內(nèi)膜血流阻力指數(shù)(RI)； 黃體期ELLSA法檢測與血栓前狀態(tài)相關血小板衍生生長因子家族包括血小板生長因子(PDGF)和血管內(nèi)皮細胞因子(VEGF)及纖溶系統(tǒng)(t-PA, PAI-1),從組織病理學、B超學、分子生物學角度對60例復發(fā)性流產(chǎn)血栓前狀態(tài)患者胞飲突、內(nèi)膜組織HE染色后結(jié)果、內(nèi)膜厚度、內(nèi)膜狀態(tài)、內(nèi)膜血流、PI、RI、PAI-1、 t-PA、VEGF、PDGF-AA這些指標進行相關性分析,并探討這些變量指標在不同證型的差異。3在第一部分及第二部分的研究基礎上,我們采用血清標本,利用蛋白芯片進行蛋白表達譜分析,探討血栓前狀態(tài)引起的復發(fā)性流產(chǎn)患者中腎虛血瘀證與非腎虛血瘀證的蛋白表達差異。通過分析確定差異蛋白并定制蛋白芯片,再擴大樣本量進行驗證。①提取有過正常孕產(chǎn)史婦女的血清樣本6例(正常組),血栓前狀態(tài)引起的復發(fā)性流產(chǎn)患者腎虛血瘀證血清樣本12例(腎虛血瘀組),血栓前狀態(tài)引起的復發(fā)性流產(chǎn)患者非腎虛血瘀證血清樣本11例(非腎虛血瘀組)。②利用raybiotech蛋白芯片(貨號：AAH-BLG-1000指標數(shù)量：1000)提取正常組、腎虛血瘀組、非腎虛血瘀組的蛋白表達圖譜,分析正常組-疾病組、 正常組-腎虛血瘀組、腎虛血瘀組-非腎虛血瘀組差異蛋白,以主成分分析圖表示每組樣本的整體蛋白表達差異。③以聚類圖及維恩圖(交集圖)選出血栓組中特異表達的蛋白,將差異蛋白進行顯著性功能分析及KEGG pathway/pathway-net分析,得到與復發(fā)性流產(chǎn)血栓前狀態(tài)相關蛋白。④根據(jù)篩選的關鍵蛋白定制芯片(定制指標數(shù)目根據(jù)篩選的結(jié)果確定),擴大樣本量選取腎虛血瘀證組30(12+18)例、非腎虛血瘀證組30(11+19)例樣本進行驗證。結(jié)果1對205例復發(fā)性流產(chǎn)血栓前狀態(tài)患者的癥狀分布進行頻率及聚類分析,其中有87例具備腎虛血瘀型多數(shù)證候表現(xiàn),其次為脾腎兩虛證、氣血虛弱證、腎精虧虛證；經(jīng)過頻數(shù)統(tǒng)計和聚類分析后,得出腎虛血瘀證為復發(fā)性流產(chǎn)血栓前狀態(tài)主證,所占比例為47.28%,出現(xiàn)頻率較高的癥狀為：月經(jīng)色暗紅,腰膝酸軟,面色晦暗,胸脅刺痛,耳鳴腰痛,舌紫暗,有瘀斑,脈沉澀。2①不同證候復發(fā)性流產(chǎn)血栓前狀態(tài)患者在子宮內(nèi)膜容受性形態(tài)學上表現(xiàn)：常規(guī)HE染色,60例樣本中,45例子宮內(nèi)膜為佳型內(nèi)膜,其中腎虛血瘀組8例,占17.78%,少于其它三組證型；另有15例子宮內(nèi)膜為差型內(nèi)膜,其中腎虛血瘀組7例,占46.67%,多于其它三組證型。采用卡方檢驗四組組間比較差異不顯著(P=0.110.05)。透射電鏡下觀察,大部分子宮內(nèi)膜組織可以檢測到胞飲突,多數(shù)處于發(fā)育完全成熟階段,少數(shù)處于發(fā)育中階段或退縮階段。其中7例無胞飲突發(fā)育,腎虛血瘀組3例；32例胞飲突表達少量,腎虛血瘀組9例；15例胞飲突表達中等,腎虛血瘀組2例；6例胞飲突表達豐富,腎虛血瘀組1例；用CMH卡方檢驗四組組間比較差異不顯著(P=0.150.05)。②不同證候復發(fā)性流產(chǎn)血栓前狀態(tài)患者在子宮內(nèi)膜容受性B超上表現(xiàn)：排卵日測子宮內(nèi)膜動脈血流,60例患者中,腎虛血瘀組15例,四組組間比較有顯著性差異,腎虛血瘀組搏動指數(shù)和阻力指數(shù)均高于其它三組(P0.01)。在子宮內(nèi)膜厚度、內(nèi)膜形態(tài)、內(nèi)膜血流分型統(tǒng)計中,四組組間無顯著性差異(P0.05)。③不同證候復發(fā)性流產(chǎn)血栓前狀態(tài)患者在子宮內(nèi)膜容受性分子生物學上相關性：PAI-1與t-PA呈正相關(r=0.415,P0.01),PAI-1與PDGF-AA呈正相關(r=0.390,P0.01),PAI-1與RI呈正相關(r=0.296,P0.05),PAI-1與內(nèi)膜血流分型(按照由Ⅰ-Ⅲ順序編碼)呈負相關(r=-0.267, P0.05), t-PA與VEGF呈負相關(r=-0.653, P0.01),t-PA與PDGF-AA呈正相關(r=0.501, P0.01),t-PA與RI呈正相關(r=0.399, P0.01), PDGF-AA與RI呈正相關(r=0.767,P0.01), PDGF-AA與內(nèi)膜血流分型(按照由Ⅰ-Ⅲ順序編碼)呈負相關(r=-0.570, P0.01), PDGF-AA與胞飲突(按照由豐富-陰性順序編碼)呈正相關(r=0.369, P0.01), PI與RI呈正相關(r=0.508, P0.01), PI與內(nèi)膜血流分型(按照由Ⅰ-Ⅲ順序編碼)呈負相關(r=-0.308, P0.05), PI與HE染色結(jié)果(按照由佳型-差型順序編碼)呈正相關(r=0.440,P0.01),PI與胞飲突(按照由豐富-陰性順序編碼)呈正相關(r=0.360, P0.01), RI與內(nèi)膜血流分型(按照由Ⅰ-Ⅲ順序編碼)呈負相關(r=-0.762, P0.01), RI與HE染色結(jié)果(按照由佳型-差型順序編碼)呈正相關(r=0.354, P0.01), RI與胞飲突(按照由豐富-陰性順序編碼)呈正相關(r=0.519,P0.01),子宮內(nèi)膜厚度與HE染色結(jié)果(按照由佳型-差型順序編碼)呈負相關(r=0.-358,P0.01),子宮內(nèi)膜血流分型(按照由Ⅰ-Ⅲ順序編碼)與HE染色結(jié)果(按照由佳型-差型順序編碼)呈負相關(r=-0.353,P0.01),子宮內(nèi)膜血流分型(按照由Ⅰ-Ⅲl順序編碼)與胞飲突(按照由豐富-陰性順序編碼)呈負相關(r=-0.699, P0.01), HE染色結(jié)果(按照由佳型-差型順序編碼)與胞飲突(按照由豐富-陰性順序編碼)呈正相關(r=0.332,P0.05)。腎虛血瘀證患者VEGF結(jié)果低于其它三組,PDGF-AA、t-PA、PAI-1高于其它三組。其中PDGF-AA及t-PA有顯著差異(P0.05)。3①復發(fā)性流產(chǎn)血栓前狀態(tài)組與正常組相比,151種細胞因子具有明顯差異表達。與正常組相比較,在疾病組血清中8種細胞因子表達顯著上調(diào),143種細胞因子表達顯著下調(diào),通過對151種細胞因子豐度聚類,復發(fā)性流產(chǎn)組的16個細胞因子表達模式相似,且區(qū)別于正常組。②腎虛血瘀組與正常組之間,118種細胞因子具有明顯差異表達。與正常組相比,在腎虛血瘀組血清中1種細胞因子表達顯著上調(diào),117種細胞因子表達顯著下調(diào),通過對以上118種細胞因子進行豐度聚類分析,結(jié)果顯示,腎虛血瘀組的76個細胞因子表達模式相似,且區(qū)別于正常組。③腎虛血瘀組與非腎虛血瘀組之間,33種細胞因子具有明顯差異表達。與非腎虛血瘀組相比,在腎虛血瘀組血清中7種細胞因子表達顯著上調(diào),26種細胞因子表達顯著下調(diào),通過對以上33種細胞因子進行豐度聚類分析,結(jié)果顯示,腎虛血瘀組的20個細胞因子表達模式相似,且區(qū)別于非腎虛血瘀組。④另外,復發(fā)性流產(chǎn)血栓前狀態(tài)腎虛血瘀組與非腎虛血瘀組相比較,在信號通路方面細胞因子涉及CXC subfamily、CC subfamily、Hematopoietins、PDGF family、TNFfamily、 TGF-(3family。結(jié)論1復發(fā)性流產(chǎn)血栓前狀態(tài)以腎虛血瘀證為主要中醫(yī)證候。2與其他證型相比,腎虛血瘀證子宮內(nèi)膜病理組織形態(tài)學改變更顯著,可能通過影響血管生成系統(tǒng)或纖溶系統(tǒng)造成血管的緊張收縮,形成高凝狀態(tài),影響內(nèi)膜的血液循環(huán)及血流灌注,在生理結(jié)構(gòu)上腺體發(fā)育不良,胞飲突表達不豐富,最終造成子宮內(nèi)膜容受性下降,甚至反復流產(chǎn)。3腎虛血瘀證與非腎虛血瘀證在蛋白組學方面有明顯差異,33種細胞因子具有明顯差異表達,涉及CXC亞族、CC亞族、Hematopoietins、PDGF、TNF、TGF-β家族等7個信號通路,涉及凝血、血小板聚集、炎癥、血管形成等,這可能是復發(fā)性流產(chǎn)血栓前狀態(tài)腎虛血瘀證的證候?qū)嵸|(zhì)。
[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.
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R271.9

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