本文選題：深靜脈血栓　切入點(diǎn)：組織性纖溶酶原激活物　出處：《昆明醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的] DVT危害重,目前藥物治療作用的分子較多,針對(duì)性不強(qiáng)。血栓消退機(jī)制在血栓的溶解中起重要作用,但目前研究較少、需深入探索。本研究在兔DVT模型中,獲取血栓形成前、形成初、完全形成、消退初、消退高峰期、完全消退狀態(tài)下的靜脈血液；在臨床研究中,采集DVT患者血栓消退及不消退狀態(tài)下的靜脈血液。均分離血漿,定量檢測(cè)Pla、Plau的表達(dá)變化的意義。采用相關(guān)生物信息學(xué)分析技術(shù),探討Plat、Plau表達(dá)變化在DVT消退中的具體意義,針對(duì)血栓消退中的內(nèi)皮細(xì)胞、纖維蛋白、血小板的分子層面的聯(lián)系,探討Pla、Plau作為纖溶指標(biāo),指導(dǎo)治療及尋找分子靶向治療新藥的可能性。 [材料和方法] 第一部分：在兔DVT模型血漿中檢測(cè)Plat、Plau的表達(dá) 1.造模及分組：51只日本大耳白兔,雌雄不限、年齡不限,適應(yīng)性喂養(yǎng)3-5天。正常組(8只)：正常飼養(yǎng),抽血取材,將未取材存活的兔隨機(jī)分配另外兩組。造模組(24只)：耳緣靜脈麻醉,開腹,分離暴露下腔靜脈,4-0絲線繞過下腔靜脈,同時(shí)在靜脈表面放置一條4-0絲線。結(jié)扎繞過下腔靜脈的絲線,并將表面的絲線打于結(jié)內(nèi),收緊后抽出表面的絲線可見仍有血流通過結(jié)扎處,關(guān)腹,正常飼養(yǎng)。假手術(shù)組(24只)：隨機(jī)進(jìn)行開腹,不分離暴露血管,關(guān)腹,正常飼養(yǎng)。 2.靜脈血液采集及病理切片：根據(jù)預(yù)實(shí)驗(yàn)所定的特定時(shí)間點(diǎn),分別于造模后2h、6h、24h、7d、14d、21d,隨機(jī)抽取假手術(shù)組、造模組各8只兔,麻醉開腹經(jīng)下腔靜脈(結(jié)扎點(diǎn)上方0.5cm)采血5m1,置于醫(yī)用真空抗凝管,3000rpm離心15min,分離血漿,-80℃冰凍保存；并在每個(gè)時(shí)間點(diǎn)位處死3只兔,取結(jié)扎線下的下腔靜脈及血栓栓體,以4%的多聚甲醛固定后送石蠟切片觀察血栓形成狀態(tài)。 3.ELISA檢測(cè)Plat、Plau的表達(dá):,用ELISA技術(shù)分別檢測(cè)血漿中Plat、Plau蛋白表達(dá)的變化。 4.統(tǒng)計(jì)學(xué)分析：采用SPSS17.0對(duì)數(shù)據(jù)分析,計(jì)量資料采用用均數(shù)±標(biāo)準(zhǔn)差表示,組間比較采用單因素方差分析,P0.05有統(tǒng)計(jì)學(xué)意義。 第二部分：檢測(cè)DVT患者血漿中Plat、Plau的表達(dá) 1.血栓診斷及分組：依照診斷標(biāo)準(zhǔn)(詳見附表2)選取昆明醫(yī)科大學(xué)第一附屬醫(yī)院2013年8月至2014年1月選取血栓患者30例(均未使用纖溶性藥物),每日B超觀察患者血栓變化情況,出院后每?jī)芍蹷超復(fù)查,根據(jù)血栓是否出現(xiàn)消退,實(shí)驗(yàn)分組：正常組(10例)：10例隨機(jī)從20位健康志愿者選��；血栓消退組(10例)：19例患者血栓出現(xiàn)溶解,隨機(jī)選取10例；血栓未消退組(9例)：9例患者觀察12周后血栓未見明顯變化,為血栓未消退組。 2.血液樣本采集：采集各組實(shí)驗(yàn)對(duì)象上肢外周靜脈血,置于醫(yī)用真空抗凝管,3000rpm離心15min,分離血漿,-80℃冰凍保存。 3. ELISA檢測(cè)Plat、Plau的表達(dá)：用ELISA技術(shù)分別檢測(cè)血漿中Plat、Plau蛋白表達(dá)的變化。進(jìn)行血常規(guī)、凝血指標(biāo)等血液學(xué)檢查。 4.統(tǒng)計(jì)學(xué)分析：采用SPSS17.0對(duì)數(shù)據(jù)分析,計(jì)量資料采用用均數(shù)±標(biāo)準(zhǔn)差表示,組間比較采用單因素方差分析,P0.05有統(tǒng)計(jì)學(xué)意義。 第三部分：運(yùn)用基因信息學(xué)探索Pla、Plau在血栓消退中關(guān)鍵的作用 1.應(yīng)用2014Pubmed NCBI數(shù)據(jù)庫(kù),輸入"Plat and DVT ","Plat and arterial thrombosis","Plat and pulmanory embolis","Plau and DVT","Plau and arterial thrombosis ","Plau and pulmanory embolis ",尋找Plat、Plau在動(dòng)靜脈血栓及肺栓塞中作用的相關(guān)信息； 2.在上述前提下,再次檢索" Plat and endothelial cell"," Plau and endothelialcell”,尋找Plat、Plau與內(nèi)皮細(xì)胞在血栓消退中的關(guān)聯(lián)； 3.繼續(xù)檢索“Plat and endothelial cell","Plau and endothelial cell",查找Plat、 Plau在血栓消退過程中與內(nèi)皮細(xì)胞膜的具體作用形式； 4.運(yùn)用2014Kegg Pathway數(shù)據(jù)庫(kù),輸入"Plat and fibrinolysis"、"Plau and fibrinolysis",分析、歸納出Plat、Plau在血栓消退中作用方式及具體信號(hào)通路。 [結(jié)果] 一、在兔DVT模型血漿中檢測(cè)Plat、Plau的表達(dá)變化 1.動(dòng)物造模成功,在相應(yīng)的時(shí)間點(diǎn)取血漿檢測(cè),Plat蛋白表達(dá)在正常組10850.55±1350.29,假手術(shù)組10703.65±1430.35血栓形成前10534.14±1440.05,血栓形成初8165.86±1050.40,血栓完全形成8489.00±±987.56,血栓消退初14042.17±1275.77,血栓消退中16319.64±1563.14,血栓完全消退10997.25±1750.80；Plau蛋白表達(dá)在正常組0.588±0.173,假手術(shù)組0.589±0.177,血栓形成0.567±0.136,血栓形成初0.347±0.097,血栓完全形成0.367±±0.084,血栓消退初0.816±±0.138,血栓消退中1.181±0.156,血栓完全消退0.589±0.154。 Plat在假手術(shù)組各個(gè)點(diǎn)位間及與正常對(duì)照組相比均無統(tǒng)計(jì)學(xué)差異(P0.05)與正常組及同時(shí)間點(diǎn)位假手術(shù)組相比,血栓形成前表差異不大(P0.05),血栓形成初、血栓形成后表達(dá)均減少(P0.05),血栓消退初及血栓消退中表達(dá)均增高(P0.05),血栓完全消退表達(dá)變化回落至對(duì)照組水平(P0.05)； Plat在假手術(shù)組各個(gè)點(diǎn)位間及與正常對(duì)照組相比均無統(tǒng)計(jì)學(xué)差異(P0.05),與正常組及同時(shí)間點(diǎn)位假手術(shù)組相比,血栓形成前表差異不大(P0.05),血栓形成初、血栓形成后表達(dá)均減少(P0.05),血栓消退初及血栓消退中表達(dá)均增高(P0.05),血栓完全消退表達(dá)變化回落至對(duì)照組水平(P0.05)。 二、檢測(cè)人血中Plat、Plau的表達(dá)變化 30例血栓患者中19患者經(jīng)B超檢查血栓出現(xiàn)消退,1例10周后出現(xiàn)溶解排出實(shí)驗(yàn),1例失訪,9例未見溶解,12周后采血。 Plat在人血漿中表達(dá)正常組14.22±2.80,血栓未消退組14.46±3.07,血栓消退組19.95±4.99;Plau在人血中表達(dá)正常組266.79±138.46,血栓未消退組309.95±131.19,血栓消退組511.32±215.65。 血栓消退組與正常組及血栓未消退組相比Plat、Plau升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),血栓未消退組Plat、Plau表達(dá)與正常組無明顯差異(P0.05)。第三部分:基因信息學(xué)尋找Plat、Plau與內(nèi)皮細(xì)胞在血栓消退中的聯(lián)系 1.在動(dòng)、靜脈血栓和肺栓中,Plat、Plau可溶解纖維蛋白,促進(jìn)血栓溶解,血栓形成后可刺激內(nèi)皮細(xì)胞釋放Plat、Plau,發(fā)揮溶解血栓的作用,上述研究多探討臨床藥物療效,針對(duì)蛋白水平檢測(cè)較少。 2.血管內(nèi)皮細(xì)胞的刺激釋放Plat、Plau,激活內(nèi)皮細(xì)胞表面的纖溶酶原,產(chǎn)生纖溶酶,纖維蛋白水解的肽鏈和胞外基質(zhì)的溶解,血管退化。 3. Plat、Plau結(jié)合內(nèi)皮細(xì)胞膜上受體,可激活纖溶酶原,纖溶酶生成增加,使纖溶蛋白多肽鏈水解,Plat經(jīng)ERK及p38信號(hào)通路(ERK也可激活p38),促使內(nèi)皮細(xì)胞生成(血管內(nèi)皮細(xì)胞生長(zhǎng)因子)VEGF,抑制血管平滑肌增生、抑制血小板聚集、促使血管新生,促進(jìn)血管腔再通、血栓機(jī)化；Plau經(jīng)ERK、p38信號(hào)通路表達(dá)MIP-1a,刺激單核/巨噬細(xì)胞分泌MMP-9、MMP-12、MMP-13降解細(xì)胞外基質(zhì)中Ⅰ、Ⅲ型膠原蛋白,促進(jìn)血栓溶解。 4.內(nèi)皮細(xì)胞受刺激,可經(jīng)Complement and cogulation cascades Pathway,即補(bǔ)體與凝血途徑信號(hào)通路,并在其中起重要作用,使Plat、Plau表達(dá)升高,促進(jìn)血栓溶解；Plau調(diào)節(jié)升高,經(jīng)Heparan sulfate proteoglycans(HSPGs) Pathway即硫酸類肝素蛋白聚酶信號(hào)通路,激活MMP-2、MMP-9分泌增加,參與血管新生和細(xì)胞外基質(zhì)降解促進(jìn)血栓溶解。 [結(jié)論] 1. Plat、Plau在兔、人DVT消退中表達(dá)均上調(diào),趨勢(shì)基本與血栓生物學(xué)過程相符合,可反映機(jī)體纖溶的狀態(tài),有效指導(dǎo)治療。 2. Plat、Plau主要通過補(bǔ)體和凝血途徑與內(nèi)皮細(xì)胞膜上受體結(jié)合,激活纖溶酶水解纖維蛋白多聚體；Plat經(jīng)ERK及p38信號(hào)通路,促使內(nèi)皮細(xì)胞生成VEGF,抑制血管平滑肌增生、抑制血小板聚集、促使血管新生,促進(jìn)血管腔再通、血栓機(jī)化；Plau通過HSPGs途徑激活MMPs系統(tǒng)降解細(xì)胞外基質(zhì)Ⅰ、Ⅲ型膠原蛋白致血栓消退。為進(jìn)一步在靜脈內(nèi)皮細(xì)胞中通過基因干擾技術(shù)研究DVT消退的分子機(jī)制提供理論基礎(chǔ)。
[Abstract]:[Objective]
DVT heavy damage, most of the present molecular medical therapy, targeted not strong. The thrombi resolution mechanism play an important role in the dissolution of thrombi, but there has been little research, needs further exploration. This study in a rabbit model of DVT, acquired thrombosis before formation of early, fully formed, fading, fading peak. Complete regression of venous blood condition; in clinical studies, DVT regression and acquisition of thrombosis in patients with venous blood does not subside condition. Both plasma separation, quantitative detection of Pla, the expression of Plau. The significance of using bioinformatics analysis technology of Plat, the specific significance of changes in DVT regression of the expression of Plau according to the regression of thrombosis endothelial cells, fibrin, platelet, the molecular level of Pla, Plau as fibrinolysis, guiding treatment and find the molecular target to the possibility of drugs for the treatment.
[materials and methods]
Part 1: to detect the expression of Plat and Plau in the plasma of rabbit DVT model
1. modeling and grouping: 51 Japanese white rabbits, male or female, irrespective of age, adaptive feeding for 3-5 days. The normal group (8 rats): normal diet, blood samples were not randomly assigned to the other two rabbits survival group. Model group (24 rats): ear vein anesthesia, laparotomy from exposure, inferior vena cava, 4-0 thread around the inferior vena cava, and placed a 4-0 thread in the vein ligation of the inferior vena cava. The surface around the surface of the silk thread, and play on the node, the surface of the thread tightening out there are still some blood flow through the ligation, abdomen, normal feeding. The sham operation group (24): a randomized open, not exposed blood vessels, abdomen, normal feeding.
2. venous blood collection and pathological section: according to the specific time point of the pre experiment, respectively after modeling 2h, 6h, 24h, 7d, 14d, 21d, were randomly selected from the sham operation group, model group, 8 rabbits anesthetized open inferior vena cava (0.5cm ligation above) blood 5m1 by vacuum in medicine 3000rpm anticoagulant tube, centrifugal separation of plasma, 15min, -80 and C frozen preservation; 3 rabbits were killed at each time point, and the inferior vena cava thrombus body take ligation line, with 4% paraformaldehyde fixation paraffin sections to observe thrombosis.
3.ELISA was used to detect the expression of Plat and Plau: the changes in the expression of Plat and Plau protein in plasma were detected by ELISA technique.
4. statistical analysis: using SPSS17.0 for data analysis, measurement data is expressed by mean + standard deviation. Comparison between groups is based on one-way ANOVA, P0.05 has statistical significance.
The second part: detection of the expression of Plat and Plau in plasma of DVT patients
1. thrombosis diagnosis and grouping: according to the diagnostic criteria (see Table 2) from the First Affiliated Hospital of Kunming Medical University from August 2013 to January 2014 were 30 cases of patients with thrombosis (no fibrinolytic drugs), daily ultrasound to observe the changes in patients with thrombosis, each of the two Zhou Bchao hospital after the review, according to whether the thrombosis subsided, experimental groups: normal group (10 cases of:10 cases) randomly from 20 healthy volunteers were selected; thrombus extinction group (10 cases):19 patients with thrombosis dissolved, randomly selected 10 cases of thrombosis; no extinction group (9 cases):9 patients were observed after 12 weeks of thrombosis. There were no obvious changes for thrombosis did not subside.
2. collection of blood samples: collecting the peripheral venous blood of the upper limbs of each group, placed in the medical vacuum anticoagulant tube, 3000rpm centrifuged 15min, separated plasma, and cryopreserved at -80 C.
3. ELISA was used to detect the expression of Plat and Plau: the changes in the expression of Plat and Plau in plasma were detected by ELISA technique. Blood routine, coagulation index and other hematological examinations were carried out.
4. statistical analysis: using SPSS17.0 for data analysis, measurement data is expressed by mean + standard deviation. Comparison between groups is based on one-way ANOVA, P0.05 has statistical significance.
The third part: using gene informatics to explore the key role of Pla, Plau in thrombus decline
1. application of 2014Pubmed NCBI database, enter "Plat and DVT", "Plat and," Plat arterial thrombosis "and Pulmanory embolis", "Plau and DVT", "Plau and," Plau arterial thrombosis "and Pulmanory embolis", for Plat, Plau related information on the role of venous thrombosis and pulmonary embolism;
2., under the above premise, we retrieve "Plat and endothelial cell" and "Plau and endothelialcell" again, looking for Plat, Plau and endothelial cells in thrombosis regression.
3., continue to search "Plat and endothelial cell", "Plau and endothelial cell" to find Plat and Plau in the process of thrombus regression and the specific form of interaction with endothelial cell membrane.
4., we used 2014Kegg Pathway database to input "Plat and fibrinolysis" and "Plau and fibrinolysis". We concluded the action mode and specific signaling pathway of Plat and Plau in thrombus regression.
[results]
1. Changes in the expression of Plat and Plau in the plasma of rabbit DVT model
1. animal models have been made from the detection of plasma at the corresponding time point, the expression of Plat protein in normal group was 10850.55 + 1350.29, 10703.65 + sham operation group 1430.35 thrombosis 10534.14 + 1440.05, 8165.86 + 1050.40 early thrombosis, complete thrombosis of 8489 + + 987.56, 14042.17 + 1275.77 early thrombosis subsided, thrombosis in extinction 16319.64 + 1563.14, 10997.25 + 1750.80 thrombus disappeared completely; the expression of Plau protein in normal group and sham operation group 0.588 + 0.173, 0.589 + 0.177, 0.567 + 0.136 thrombosis, thrombosis at 0.347 + 0.097, 0.367 + 0.084 + complete thrombosis, thrombosis disappeared at the beginning of 0.816 + 0.138 + 1.181 + 0.156, thrombusi resolution. Thrombus disappeared completely in 0.589 + 0.154.
Plat in the sham operation group of each point and compared with the normal control group had no statistical difference (P0.05) compared with the normal group and sham operation group at the same time point, a little difference table (P0.05), thrombosis early after decreased thrombosis (P0.05), thrombus regression were increased the early expression and thrombus regression (P0.05), thrombus completely subsided expression dropped to the level of control group (P0.05);
Plat in the sham operation group of each point and compared with the normal control group had no statistical difference (P0.05), compared with normal group and sham operation group at the same time point, a little difference table (P0.05), thrombosis early after decreased thrombosis (P0.05), and early thrombosis resolution the regression of thrombus expression was significantly increased (P0.05), thrombus completely subsided expression dropped to the level of control group (P0.05).
Two, the changes in the expression of Plat and Plau in human blood
In 30 patients with thrombus, 19 patients underwent B ultrasound examination of thrombus decline, 1 cases were dissolved and discharged after 10 weeks, 1 cases were lost, 9 cases were not dissolved, and the blood was collected after 12 weeks.
Plat expression in human plasma was 14.22 + 2.80 in normal group, 14.46 + 3.07 in thrombosis group, 19.95 + 4.99 in thrombus regression group, Plau in human blood group, 266.79 + 138.46 in normal group, 309.95 309.95 131.19 in thrombus group, and 511.32 215.65. in thrombus regression group.
The thrombi resolution group and normal group and no thrombus regression group compared to Plat, Plau increased, the difference was statistically significant (P0.05), thrombus did not subside in group Plat, the expression of Plau had no significant difference with normal group (P0.05). The third part: the gene information search for Plat, Plau and endothelial cells in thrombus regression of contact
1., in mobile venous thrombosis and pulmonary embolus, Plat and Plau can dissolve fibrin and promote thrombolysis. After thrombus formation, Plat and Plau can stimulate endothelial cells to play the role of thrombolysis.
2., vascular endothelial cells stimulate the release of Plat and Plau, activate fibrinolytic enzyme on the surface of endothelial cells, produce fibrinolytic enzyme, fibrin hydrolyzed peptide chain and extracellular matrix dissolution, and vascular degeneration.
3. Plat, Plau combined with endothelial cell membrane receptor, activation of plasminogen, plasmin generation increased, the fibrinolytic protein polypeptide chain hydrolysis, Plat by ERK and p38 signaling pathway (ERK can also activate p38), promote endothelial growth (vascular endothelial growth factor) VEGF, inhibiting vascular smooth muscle proliferation, inhibition of platelet aggregation, promote angiogenesis, promote vascular recanalization, thrombus; Plau by ERK, the expression of p38 signaling pathway of MIP-1a stimulated monocytes / macrophages to secrete MMP-9, MMP-12, MMP-13 degradation in the extracellular matrix of type III collagen, accelerate clot lysis.
4. endothelial cells stimulated by Complement and Cogulation, cascades Pathway, is the complement and coagulation pathway signaling pathway, and plays an important role, in which Plat, Plau expression increased, promote thrombolysis; regulation of Plau increased, by Heparan (HSPGs) sulfate proteoglycans Pathway that heparan sulfate protein enzyme pathway, activation of MMP-2 and increase the secretion of MMP-9, and involved in angiogenesis and extracellular matrix degradation and promote thrombolysis.
[Conclusion]
1. Plat, Plau up-regulated in the rabbit and human DVT regression, the trend is basically consistent with the biological process of thrombus, which can reflect the state of fibrinolysis and effectively guide the treatment.
2. Plat, Plau mainly through the combination of the complement and coagulation pathways and endothelial cell membrane receptor, plasminogen activation multimers hydrolyze fibrin; Plat by ERK and p38 signaling pathway, promote endothelial cells generate VEGF, inhibit the proliferation of vascular smooth muscle, inhibit platelet aggregation, promote blood vessel newborn, promote vascular recanalization, thrombus Plau; through the activation of HSPGs MMPs system to degrade the extracellular matrix of type III collagen induced thrombosis disappeared. It provides the theoretical basis for further study the molecular mechanism by RNA interference DVT regression in vein endothelial cells.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R619

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2 易江華,林淦松;髖部手術(shù)前血漿蛋白水平與術(shù)后深靜脈血栓形成的關(guān)系[J];骨與關(guān)節(jié)損傷雜志;2003年10期

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8 呂厚山，徐斌;人工關(guān)節(jié)置換術(shù)后下肢深靜脈血栓形成[J];中華骨科雜志;1999年03期

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10 萬雙林,趙凱,施培華,范順武;骨科住院病人下肢深靜脈血栓形成的診治[J];浙江醫(yī)學(xué);2001年01期

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