Plat、Plau激活内皮细胞膜上纤溶酶促血栓消退的研究

发布时间：2018-03-18 05:12

本文选题：深静脉血栓　切入点：组织性纤溶酶原激活物　出处：《昆明医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：[目的] DVT危害重,目前药物治疗作用的分子较多,针对性不强。血栓消退机制在血栓的溶解中起重要作用,但目前研究较少、需深入探索。本研究在兔DVT模型中,获取血栓形成前、形成初、完全形成、消退初、消退高峰期、完全消退状态下的静脉血液；在临床研究中,采集DVT患者血栓消退及不消退状态下的静脉血液。均分离血浆,定量检测Pla、Plau的表达变化的意义。采用相关生物信息学分析技术,探讨Plat、Plau表达变化在DVT消退中的具体意义,针对血栓消退中的内皮细胞、纤维蛋白、血小板的分子层面的联系,探讨Pla、Plau作为纤溶指标,指导治疗及寻找分子靶向治疗新药的可能性。 [材料和方法] 第一部分：在兔DVT模型血浆中检测Plat、Plau的表达 1.造模及分组：51只日本大耳白兔,雌雄不限、年龄不限,适应性喂养3-5天。正常组(8只)：正常饲养,抽血取材,将未取材存活的兔随机分配另外两组。造模组(24只)：耳缘静脉麻醉,开腹,分离暴露下腔静脉,4-0丝线绕过下腔静脉,同时在静脉表面放置一条4-0丝线。结扎绕过下腔静脉的丝线,并将表面的丝线打于结内,收紧后抽出表面的丝线可见仍有血流通过结扎处,关腹,正常饲养。假手术组(24只)：随机进行开腹,不分离暴露血管,关腹,正常饲养。 2.静脉血液采集及病理切片：根据预实验所定的特定时间点,分别于造模后2h、6h、24h、7d、14d、21d,随机抽取假手术组、造模组各8只兔,麻醉开腹经下腔静脉(结扎点上方0.5cm)采血5m1,置于医用真空抗凝管,3000rpm离心15min,分离血浆,-80℃冰冻保存；并在每个时间点位处死3只兔,取结扎线下的下腔静脉及血栓栓体,以4%的多聚甲醛固定后送石蜡切片观察血栓形成状态。 3.ELISA检测Plat、Plau的表达:,用ELISA技术分别检测血浆中Plat、Plau蛋白表达的变化。 4.统计学分析：采用SPSS17.0对数据分析,计量资料采用用均数±标准差表示,组间比较采用单因素方差分析,P0.05有统计学意义。第二部分：检测DVT患者血浆中Plat、Plau的表达 1.血栓诊断及分组：依照诊断标准(详见附表2)选取昆明医科大学第一附属医院2013年8月至2014年1月选取血栓患者30例(均未使用纤溶性药物),每日B超观察患者血栓变化情况,出院后每两周B超复查,根据血栓是否出现消退,实验分组：正常组(10例)：10例随机从20位健康志愿者选取；血栓消退组(10例)：19例患者血栓出现溶解,随机选取10例；血栓未消退组(9例)：9例患者观察12周后血栓未见明显变化,为血栓未消退组。 2.血液样本采集：采集各组实验对象上肢外周静脉血,置于医用真空抗凝管,3000rpm离心15min,分离血浆,-80℃冰冻保存。 3. ELISA检测Plat、Plau的表达：用ELISA技术分别检测血浆中Plat、Plau蛋白表达的变化。进行血常规、凝血指标等血液学检查。 4.统计学分析：采用SPSS17.0对数据分析,计量资料采用用均数±标准差表示,组间比较采用单因素方差分析,P0.05有统计学意义。第三部分：运用基因信息学探索Pla、Plau在血栓消退中关键的作用 1.应用2014Pubmed NCBI数据库,输入"Plat and DVT ","Plat and arterial thrombosis","Plat and pulmanory embolis","Plau and DVT","Plau and arterial thrombosis ","Plau and pulmanory embolis ",寻找Plat、Plau在动静脉血栓及肺栓塞中作用的相关信息； 2.在上述前提下,再次检索" Plat and endothelial cell"," Plau and endothelialcell”,寻找Plat、Plau与内皮细胞在血栓消退中的关联； 3.继续检索“Plat and endothelial cell","Plau and endothelial cell",查找Plat、 Plau在血栓消退过程中与内皮细胞膜的具体作用形式； 4.运用2014Kegg Pathway数据库,输入"Plat and fibrinolysis"、"Plau and fibrinolysis",分析、归纳出Plat、Plau在血栓消退中作用方式及具体信号通路。 [结果] 一、在兔DVT模型血浆中检测Plat、Plau的表达变化 1.动物造模成功,在相应的时间点取血浆检测,Plat蛋白表达在正常组10850.55±1350.29,假手术组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蛋白表达在正常组0.588±0.173,假手术组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在假手术组各个点位间及与正常对照组相比均无统计学差异(P0.05)与正常组及同时间点位假手术组相比,血栓形成前表差异不大(P0.05),血栓形成初、血栓形成后表达均减少(P0.05),血栓消退初及血栓消退中表达均增高(P0.05),血栓完全消退表达变化回落至对照组水平(P0.05)； Plat在假手术组各个点位间及与正常对照组相比均无统计学差异(P0.05),与正常组及同时间点位假手术组相比,血栓形成前表差异不大(P0.05),血栓形成初、血栓形成后表达均减少(P0.05),血栓消退初及血栓消退中表达均增高(P0.05),血栓完全消退表达变化回落至对照组水平(P0.05)。二、检测人血中Plat、Plau的表达变化 30例血栓患者中19患者经B超检查血栓出现消退,1例10周后出现溶解排出实验,1例失访,9例未见溶解,12周后采血。 Plat在人血浆中表达正常组14.22±2.80,血栓未消退组14.46±3.07,血栓消退组19.95±4.99;Plau在人血中表达正常组266.79±138.46,血栓未消退组309.95±131.19,血栓消退组511.32±215.65。血栓消退组与正常组及血栓未消退组相比Plat、Plau升高,差异具有统计学意义(P0.05),血栓未消退组Plat、Plau表达与正常组无明显差异(P0.05)。第三部分:基因信息学寻找Plat、Plau与内皮细胞在血栓消退中的联系 1.在动、静脉血栓和肺栓中,Plat、Plau可溶解纤维蛋白,促进血栓溶解,血栓形成后可刺激内皮细胞释放Plat、Plau,发挥溶解血栓的作用,上述研究多探讨临床药物疗效,针对蛋白水平检测较少。 2.血管内皮细胞的刺激释放Plat、Plau,激活内皮细胞表面的纤溶酶原,产生纤溶酶,纤维蛋白水解的肽链和胞外基质的溶解,血管退化。 3. Plat、Plau结合内皮细胞膜上受体,可激活纤溶酶原,纤溶酶生成增加,使纤溶蛋白多肽链水解,Plat经ERK及p38信号通路(ERK也可激活p38),促使内皮细胞生成(血管内皮细胞生长因子)VEGF,抑制血管平滑肌增生、抑制血小板聚集、促使血管新生,促进血管腔再通、血栓机化；Plau经ERK、p38信号通路表达MIP-1a,刺激单核/巨噬细胞分泌MMP-9、MMP-12、MMP-13降解细胞外基质中Ⅰ、Ⅲ型胶原蛋白,促进血栓溶解。 4.内皮细胞受刺激,可经Complement and cogulation cascades Pathway,即补体与凝血途径信号通路,并在其中起重要作用,使Plat、Plau表达升高,促进血栓溶解；Plau调节升高,经Heparan sulfate proteoglycans(HSPGs) Pathway即硫酸类肝素蛋白聚酶信号通路,激活MMP-2、MMP-9分泌增加,参与血管新生和细胞外基质降解促进血栓溶解。 [结论] 1. Plat、Plau在兔、人DVT消退中表达均上调,趋势基本与血栓生物学过程相符合,可反映机体纤溶的状态,有效指导治疗。 2. Plat、Plau主要通过补体和凝血途径与内皮细胞膜上受体结合,激活纤溶酶水解纤维蛋白多聚体；Plat经ERK及p38信号通路,促使内皮细胞生成VEGF,抑制血管平滑肌增生、抑制血小板聚集、促使血管新生,促进血管腔再通、血栓机化；Plau通过HSPGs途径激活MMPs系统降解细胞外基质Ⅰ、Ⅲ型胶原蛋白致血栓消退。为进一步在静脉内皮细胞中通过基因干扰技术研究DVT消退的分子机制提供理论基础。
[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.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R619

【参考文献】