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

【摘要】：[目的] DVT危害重,目前藥物治療作用的分子較多,針對性不強。血栓消退機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Pla、Plau的表達變化的意義。采用相關生物信息學分析技術,探討Plat、Plau表達變化在DVT消退中的具體意義,針對血栓消退中的內皮細胞、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,指導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Plat、Plau的表達 1.造模及分組：51只日本大耳白兔,雌雄不限、年齡不限,適應性喂養(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,同時在靜脈表面放置一條4-0絲線。結扎繞過下腔靜脈的絲線,并將表面的絲線打于結內,收緊后抽出表面的絲線可見仍有血流通過結扎處,關腹,正常飼養(yǎng)。假手術組(24只)：隨機進行開腹,不分離暴露血管,關腹,正常飼養(yǎng)。 2.靜脈血液采集及病理切片：根據(jù)預實驗所定的特定時間點,分別于造模后2h、6h、24h、7d、14d、21d,隨機抽取假手術組、造模組各8只兔,麻醉開腹經下腔靜脈(結扎點上方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；并在每個時間點位處死3只兔,取結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Plat、Plau的表達:,用ELISA技術分別檢測血漿中Plat、Plau蛋白表達的變化。 4.統(tǒng)計學分析：采用SPSS17.0對數(shù)據(jù)分析,計量資料采用用均數(shù)±標準差表示,組間比較采用單因素方差分析,P0.05有統(tǒng)計學意義。 第二部分：檢測DVT患者血漿中Plat、Plau的表達 1.血栓診斷及分組：依照診斷標準(詳見附表2)選取昆明醫(yī)科大學第一附屬醫(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,出院后每兩周B超復查,根據(jù)血栓是否出現(xiàn)消退,實驗分組：正常組(10例)：10例隨機從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：采集各組實驗對象上肢外周靜脈血,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Plat、Plau的表達：用ELISA技術分別檢測血漿中Plat、Plau蛋白表達的變化。進行血常規(guī)、凝血指標等血液學檢查。 4.統(tǒng)計學分析：采用SPSS17.0對數(shù)據(jù)分析,計量資料采用用均數(shù)±標準差表示,組間比較采用單因素方差分析,P0.05有統(tǒng)計學意義。 第三部分：運用基因信息學探索Pla、Plau在血栓消退中關鍵的作用 1.應用2014Pubmed NCBI數(shù)據(jù)庫,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在動靜脈血栓及肺栓塞中作用的相關信息； 2.在上述前提下,再次檢索" Plat and endothelial cell"," Plau and endothelialcell”,尋找Plat、Plau與內皮細胞在血栓消退中的關聯(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皮細胞膜的具體作用形式； 4.運用2014Kegg Pathway數(shù)據(jù)庫,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通路。 [結果] 一、在兔DVT模型血漿中檢測Plat、Plau的表達變化 1.動物造模成功,在相應的時間點取血漿檢測,Plat蛋白表達在正常組10850.55±1350.29,假手術組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蛋白表達在正常組0.588±0.173,假手術組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在假手術組各個點位間及與正常對照組相比均無統(tǒng)計學差異(P0.05)與正常組及同時間點位假手術組相比,血栓形成前表差異不大(P0.05),血栓形成初、血栓形成后表達均減少(P0.05),血栓消退初及血栓消退中表達均增高(P0.05),血栓完全消退表達變化回落至對照組水平(P0.05)； Plat在假手術組各個點位間及與正常對照組相比均無統(tǒng)計學差異(P0.05),與正常組及同時間點位假手術組相比,血栓形成前表差異不大(P0.05),血栓形成初、血栓形成后表達均減少(P0.05),血栓消退初及血栓消退中表達均增高(P0.05),血栓完全消退表達變化回落至對照組水平(P0.05)。 二、檢測人血中Plat、Plau的表達變化 30例血栓患者中19患者經B超檢查血栓出現(xiàn)消退,1例10周后出現(xiàn)溶解排出實驗,1例失訪,9例未見溶解,12周后采血。 Plat在人血漿中表達正常組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)計學意義(P0.05),血栓未消退組Plat、Plau表達與正常組無明顯差異(P0.05)。第三部分:基因信息學尋找Plat、Plau與內皮細胞在血栓消退中的聯(lián)系 1.在動、靜脈血栓和肺栓中,Plat、Plau可溶解纖維蛋白,促進血栓溶解,血栓形成后可刺激內皮細胞釋放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,針對蛋白水平檢測較少。 2.血管內皮細胞的刺激釋放Plat、Plau,激活內皮細胞表面的纖溶酶原,產生纖溶酶,纖維蛋白水解的肽鏈和胞外基質的溶解,血管退化。 3. Plat、Plau結合內皮細胞膜上受體,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ERK及p38信號通路(ERK也可激活p38),促使內皮細胞生成(血管內皮細胞生長因子)VEGF,抑制血管平滑肌增生、抑制血小板聚集、促使血管新生,促進血管腔再通、血栓機化；Plau經ERK、p38信號通路表達MIP-1a,刺激單核/巨噬細胞分泌MMP-9、MMP-12、MMP-13降解細胞外基質中Ⅰ、Ⅲ型膠原蛋白,促進血栓溶解。 4.內皮細胞受刺激,可經Complement and cogulation cascades Pathway,即補體與凝血途徑信號通路,并在其中起重要作用,使Plat、Plau表達升高,促進血栓溶解；Plau調節(jié)升高,經Heparan sulfate proteoglycans(HSPGs) Pathway即硫酸類肝素蛋白聚酶信號通路,激活MMP-2、MMP-9分泌增加,參與血管新生和細胞外基質降解促進血栓溶解。 [結論] 1. Plat、Plau在兔、人DVT消退中表達均上調,趨勢基本與血栓生物學過程相符合,可反映機體纖溶的狀態(tài),有效指導治療。 2. Plat、Plau主要通過補體和凝血途徑與內皮細胞膜上受體結合,激活纖溶酶水解纖維蛋白多聚體；Plat經ERK及p38信號通路,促使內皮細胞生成VEGF,抑制血管平滑肌增生、抑制血小板聚集、促使血管新生,促進血管腔再通、血栓機化；Plau通過HSPGs途徑激活MMPs系統(tǒng)降解細胞外基質Ⅰ、Ⅲ型膠原蛋白致血栓消退。為進一步在靜脈內皮細胞中通過基因干擾技術研究DVT消退的分子機制提供理論基礎。
[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.

【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R619

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