内皮细胞源性HMGB-1、KLF-2在DVT形成初期作用的研究

发布时间：2018-05-16 19:48

本文选题：Kr(u|)ppel样转录因子-2 + 高迁移率族蛋白-B1　；参考：《昆明医科大学》2015年硕士论文

【摘要】：深静脉血栓因其形成原因复杂多变,应用传统诊断方法难以进行早期确诊,早期确诊困难的现状严重影响了治疗的及时性,如不能得到及时有效的早期诊治,可引发肢体进一步病理性损害,并可能会遗留肢体静脉功能不全；严重者血栓脱落随血液循环进入肺组织引起肺动脉栓塞,甚至可危及生命。因此,与DVT早期诊断和预防相关的标志物的研究一直是国内外研究者的探讨热点。近年来,众多可引起DVT的复杂因素中,围绕内皮细胞损伤方面的讨论逐渐成为DVT发病原因的研究热点。内皮细胞损伤释放出的各种炎症因子可激活白细胞,活化血小板,导致或加速了血栓的形成。KLF-2、HMGB-1与DVT形成的关键角色内皮细胞损伤关系密切,通过保护内皮细胞膜稳定,负向调控炎症因子等功能,发挥了抑制血栓的作用。本研究围绕KLF-2、HMGB-1进行研究,实验通过DVT动物模型构建,结合分子生物学技术检测手段,观察上述标志物在DVT形成过程中的表达变化,初步讨论两种细胞因子的内在联系及其在深静脉血栓形成中的作用。目的1.下腔静脉狭窄法建立C57小鼠DVT模型,病理切片观察血栓形成情况,获取各组小鼠下腔静脉组织；2. Real-time PCR检测下腔静脉和血栓组织中,KLF-2、HMGB-1在转录层面的表达变化,初步探讨其与DVT的联系及作用,为进一步分析其与DVT的关系提供基础实验证据；3. ELISA法检测血浆中t-PA蛋白的表达变化,探讨其与DVT的联系及作用,为进一步分析其与DVT的关系提供基础实验证据。方法1.建立DVT小鼠模型并取材行相关检测：将120只C57小鼠采用随机分组原则,分为正常对照组(n=40)、假手术(n=40)和DVT组(n=40)。造模后24小时,正常组、假手术组取肾静脉下方1.5cm下腔静脉组织；DVT组取血栓形成范围内下腔静脉组织及其内容物；并记录下腔静脉直径等数据。2.下腔静脉组织样本行病理切片,HE染色后观察血栓形成情况；Real-time PCR检测各组静脉组织中KLF-2、HMGB-1基因mRNA表达水平,分析其与DVT形成关系。3. ELISA法检测各组血浆中t-PA蛋白的表达变化；分析其与DVT形成关系。结果1.DVT组小鼠模型静脉组织HE染色：DVT组可见完全血栓形成、血管壁内炎细胞侵润明显；正常组、假手术组未见形成血栓。2. Real-time PCR检测静脉组织KLF-2 mRNA表达：DVT组较其它两组表达上调,具统计学意义(P0.05)；正常对照组与假手术组相比(P0.05),没有统计学差异。HMGB-1 mRNA表达：DVT组较正常对照组及假手术组表达下调,具统计学意义(P0.05)；正常对照组与假手术组相比(P0.05),没有统计学差异。3. ELISA法检测血浆中t-PA蛋白表达：DVT组较正常对照组及假手术组表达上调,具统计学意义(P0.05)；正常对照组与假手术组相比(P0.05),没有统计学差异。结论1.小鼠DVT模型中KLF-2、HMGB-1、t-PA表达变化提示,它们与DVT形成有关；在DVT形成初期它们对血栓形成可能起到一定作用。2. KLF-2、HMGB-1可能通过影响t-PA蛋白含量,从而打破纤溶-抗纤系统动态平衡,起到影响血栓形成的作用。
[Abstract]:The cause of deep venous thrombosis is complicated and changeable, and it is difficult to make early diagnosis with traditional diagnostic method. The difficult situation of early diagnosis seriously affects the timeliness of treatment. If it can't get timely and effective diagnosis and treatment, it can cause further pathological damage of limbs, and can leave limb venous insufficiency; serious thrombosis The study of the markers associated with early diagnosis and prevention of DVT has been a hot spot for researchers both at home and abroad. In recent years, many of the complex factors that can cause DVT have gradually become the cause of the pathogenesis of DVT. The various inflammatory factors released by endothelial cell damage can activate leukocytes, activate platelets, lead to or accelerate the formation of.KLF-2, and HMGB-1 is closely related to the key role of endothelial cells in the formation of DVT, and the inhibition of thrombus is played by protecting endothelial cell membrane stability and negatively regulating inflammatory factors. This study was conducted around KLF-2 and HMGB-1. The experiment was constructed by DVT animal model and combined with molecular biological techniques to observe the changes in the expression of the above markers in the formation of DVT. The internal relations of the two cytokines and their role in the formation of deep venous thrombosis were preliminarily discussed. Objective 1. inferior vena cava stenosis method was established. C57 mice DVT model, pathological sections to observe the formation of thrombosis, to obtain the inferior vena cava tissue in each group; 2. Real-time PCR detection of the inferior vena cava and thrombus tissue, KLF-2, HMGB-1 at the transcriptional level changes, preliminarily explore its relationship with DVT and the role of the further analysis of the relationship with DVT to provide basic experimental evidence; 3. EL ISA method was used to detect the changes in the expression of t-PA protein in plasma and to explore its relationship with DVT and to provide basic experimental evidence for further analysis of its relationship with DVT. Method 1. the model of DVT mice was established and the correlation detection was taken. 120 C57 mice were randomly divided into normal control group (n=40), sham operation (n=40) and DVT group (n=40). 24 hours after the model, the normal group and the sham group took the inferior vena cava inferior vena cava below the renal vein in the sham operation group; the DVT group took the inferior vena cava tissue and its contents within the range of thrombus formation, and recorded the lower vena cava diameter and other data of the inferior vena cava tissue in.2., and observed the formation of thrombus after HE staining; Real-time PCR was used to detect the veins in.2.. The expression level of mRNA, HMGB-1 gene mRNA in tissue, and analysis of the relationship between the expression of t-PA protein in the plasma with.3. ELISA method and DVT formation, and the relationship with DVT formation. Results the 1.DVT group mice model venous tissue HE staining: DVT group can see complete thrombosis, inflammatory cells in the vascular wall are invaded obviously; normal group, sham operation group. The expression of KLF-2 mRNA in venous tissue was not detected by the formation of thrombus.2. Real-time PCR: the expression of DVT in the group of the other two groups was up, with statistical significance (P0.05); there was no statistical difference of.HMGB-1 mRNA expression between the normal control group and the sham operation group (P0.05), and the expression of the DVT group was lower than that of the normal control group and the sham operation group, with a statistical significance (P0.05). Compared with the sham operation group (P0.05), there was no statistical difference between the normal control group and the sham group (P0.05) to detect the expression of t-PA protein in the plasma: the expression of t-PA in the DVT group was higher than that in the normal control group and the sham operation group (P0.05), and there was no statistical difference between the normal control group and the sham operation group (P0.05). Conclusion the 1. mice DVT model was found to be in KLF-2 and HMGB-1, The changes in the expression of t-PA suggest that they are related to the formation of DVT; they may play a role in the formation of.2. KLF-2 in the early stage of the formation of DVT, and HMGB-1 may affect the dynamic balance of fibrinolytic and fibrinolytic system by affecting the content of t-PA protein, thus affecting the formation of thrombus.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R543.6

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