摘要
本课题为创伤后深静脉血栓形成(Deep Venous Thrombosis, DVT)系列研究的一部分,在基于课题组前期动物建模方法的探索及基因芯片技术对相关基因筛选工作的基础上,我们发现炎性因素与DVT之间关系密切。结合经典文献关于DVT的研究,本课题就巨噬细胞炎性蛋白-1α (Macrophage Inflammatory Protein-1α, MIP-1α)及其调控的单核/巨噬细胞、基质金属蛋白酶2/9(MatrixMetalloproteinase-2/9, MMP-2/9)在DVT形成及溶解中的作用,对经结扎下腔静脉造模的淤滞型小鼠DVT模型血液、静脉壁和血栓栓子以及人血液中与DVT形成及溶解关系密切的MIP-1α、F4/80、MMP-2、-9的表达变化和其作用作一初步研究。
目的:
1.以结扎下腔静脉法建立淤滞型小鼠DVT模型,参照经典文献的报导,设立不同的时间点,获取相应时间点造模小鼠心脏血液、下腔静脉和血栓组织,HE染色观察造模小鼠建模后DVT形成的具体时间点,RT-PCR、 ELISA法检测其血液中MIP-1α在DVT形成前后的表达变化;
2.应用MIP-1α中和抗体(rh-MIP-1α Ab)腹腔注射建模后DVT形成的小鼠,明确其是否具有促进DVT溶解的作用;同时以RT-PCR、ELISA法检测不同组别造模后小鼠在预设时间点MIP-1α、 MMP-2、-9的表达变化,免疫组化法检测静脉壁和血栓栓子中巨噬细胞表面特异性抗原F4/80的表达,初步明确MIP-1α与DVT形成及溶解的关系,进而初步探讨其与血栓形成及溶解的相关机制;
3.对不同分组的人群血液样本,提取全血中MIP-1α、 F4/80、MMP-2、-9的mRNA,检测其在人血中的表达变化,探讨不同血栓形成状态下所关注的MIP-1α、 F4/80、 MMP-2、-9基因的表达变化及其与DVT的相关性。
方法:
1.96只昆明种小鼠参照随机数字表分为对照组(n=8)和模型组(n=88),模型组采用结扎下腔静脉法建立淤滞型小鼠DVT模型,参照经典文献报道,在建模后设立2h、4h、6h、8h、12h、1d、2d、4d、8d、14d、21d等11个时间点,在每个时间点将8只造模后小鼠麻醉后行心脏采血,同时获取造模段长约1.0cm的下腔静脉组织。将获取的小鼠血液样本以RT-PCR, ELISA法检测MIP-1α的表达,获取的下腔静脉组织以石蜡包埋后行静脉组织冠状面组织切片,观察血栓形成情况。
2.再取152只昆明种小鼠参照随机数字表分为对照组(n=8)和模型组(n=144),模型组采用结扎下腔静脉法建立淤滞型小鼠DVT模型,参照经典文献报道,造模后将模型组小鼠随机分为DVT组(n=72,小鼠不加任何干预措施)及DVT+MIP-1α-Ab组(n=72,小鼠造模后予以MIP-1α中和抗体0.5m1持续腹腔注射7d);建模后设立2d、4d、8d、12d、14d、21d等6个时间点,在每个时间点将12只造模后小鼠过量麻醉后处死,获取造模段的下腔静脉组织。质量/长度比值评估血栓的溶解程度,应用RT-PCR及ELISA法检测造模段静脉及血栓组织中MIP-1α、MMP-2、-9的表达量;同时免疫组化法检测相应时间点静脉壁及血栓组织中巨噬细胞表面特异抗原F4/80的表达。
3.采集人血并依据诊断(详见附表2)分为血栓组、血栓未形成组和健康对照组,用Paxgene blood RNA kit试剂盒提取mRNA,胶回收测序证实PCR所扩增基因准确,后以real-time PCR定量检测、统计学分析不同组间MIP-1α、F4/80、MMP-2、-9mRNA的表达。对于临床一般资料、血细胞检验、血生化检验、凝血指标和彩色多普勒超声检查等的结果进行搜集、统计,结合mRNA的表达结果进行综合分析。
结果:
1.结扎下腔静脉法建立小鼠淤滞型DVT模型,HE组织染色显示在6h时间点小鼠下腔静脉中有部分血栓形成,8h时间点有完全性血栓形成,与血管壁粘连;血栓主要为均匀分布的红细胞,其间可见纤维素网状结构及散在的血小板及白细胞;在14d时间点可见血栓明显机化缩小。
2.模型组小鼠MIP-1α mRNA、蛋白在血液中的表达呈造模后8h、12h、1d、2d、4d、8d逐渐升高的趋势(P<0.05),8d达到峰值(P<0.01),21d降至正常水平(P>0.05)。
3.DVT组及DVT+MIP-1α-Ab组血栓质量/长度比值随时间的延长呈逐渐缩小的趋势(P<0.05),相同时间点DVT组血栓栓子质量/长度比值减小更为明显(P<0.05)。
4.相同时间点DVT组F4/8、MMP-9的表达明显高于DVT+MIP-1α-Ab组(P<0.05), MMP-2的表达相同时间点组间无差异(P>0.05)。
5.PCR电泳检测半定量分析及Real-time PCR定量检测人血MIP-1α、 F4/80、MMP-2、-9mRNA在血栓组中的表达显著高于血栓未形成组和健康对照组,血栓未形成组的表达量与健康对照组无差别。
结论:
1.结扎下腔静脉法建立淤滞型小鼠DVT模型在8h左右可形成完全性血栓,14d左右血栓可发生明显机化。
2.小鼠DVT模型血液中MIP-1α的表达在建模后8d达到最高值,21d降至正常水平。
3. MIP-1α与深静脉血栓溶解密切相关,其中和抗体可减弱MIP-1α促进血栓溶解的效应。
4. MIP-1α促进血栓溶解的作用与单核/巨噬细胞聚集、MMP-9的表达密切相关,MMP-2与血栓溶解的关系可能是独立于MIP-1α之外存在的。
5. MIP-1α、F4/80、MMP-2、-9mRNA在人群中DVT形成后的血液中呈高表达,结合MIP-1α较强的诱导单核/巨噬细胞向损伤部位聚集、及诱导MMP-9表达的作用,提示MIP-1α可能参与了DVT的形成、溶解过程。
This study was one part of the research of post-traumatic deep venous thrombosis(DVT), based on the research of animal model and gene chip technology, we found that the inflammation and DVT had close relationship. Therefore, through experiments on DVT mouse model and human blood, initial research was made about macrophage inflammatory protein-1alpha (MIP-1α) and matrix metalloproteinase2/9(MMP-2/9) who have close relationship with DVT.
Objective:
1. Established DVT mouse model by ligating inferior vena cava, set up different time points with reference to the classical literature reports, harvested the blood and inferior vena cava organization, observed the specific time point of DVT formation by HE dyeing, checked MIP-1α expression of blood before and after the formation of DVT.
2. Application of MIP-1α neutralizing antibody (rhMIP-1α Ab) with the DVT mouse model by intraperitoneal injection, to further clarify the role of promoting DVT resolution; at the same time, to detected MIP-1α and MMP-2,-9expression in different groups by RT-PCR and ELISA methods, immunohistochemical method to detect surface specific antigen F4/80expressio of macrophages in venous wall and thromboembolus, preliminary cleared the relationship of MIP-1α and DVT.
3. Gather blood samples and detect the expression changes of MIP-1, MMP-2,-9and F4/80in DVT group, non-thrombosis group and healthy control, then explor the relationship of gene expression changes and thrombosis states.
Method:
1.100KunMing mice were randemely divided into control group (n=8) and model group (n=92). Model was built by ligating inferior vena cava. After established model, set up2h,4h,6h,8h,12h,1d,2d,4d,8d,14d and21d etc.11time points with reference to the classical literature. Kill model mice in different time, harvested the blood and about1.0cm inferior vena cava organization. detected MIP-1α expression of the blood samples with RT-PCR and ELISA methods, observed thrombosis by paraffining the inferior vena cava organization.
2.200KunMing mice were randemely divided into control group (n=8) and model group (n=192). Model was built by ligating inferior vena cava. Model mice were randomly divided into DVT group (n=96, the mice were without any intervention measures) and DVT+MIP-1α-Ab group (n=96, mice had been intraperitoneal injection MIP-1α neutralizing antibody0.5mL for7days).Set up2d,4d,8d,12d,14d and21d etc. six time points after the model established, and in every time point killed12mice of different groups, harvested about1.0cm long inferior vena cava organization. Evaluated the degree of thrombus resolution by quality/length ratio; detected MIP-1α, MMP-2, and-9expression of thrombi and vein wall by RT-PCR and ELISA method; immunohistochemical method to detect the F4/80expression of vein wall and thrombi organization in corresponding time points.
3. Gather human blood samples and divided into DVT group, non-thrombosis group and healthy control group according to diagnosis. Extracte mRNA with Paxgene blood RNA kit, detected MIP-1a, MMP-2,-9and F4/80expression in human blood by RT-PCR, then verify PCR result by sequencing, and quantitatively detected by real-time PCR, then analyze the differences among the groups. Gather clinical data including the results of complete blood count, blood biochemistry, blood coagulation, color Doppler ultrasonography and etc, and then make statistical analysis.
Result:
1. The method to establish the stasis DVT mouse by ligating inferior vena cava, HE tissue staining displayed that at6h after modeling the inferior vena cava had partial thrombi intravascular filling and at8h it had complete thrombi; Thrombus mainly had uniform distribution of red blood cells, which visible cellulose reticular structure and scattered platelet and leukocyte; At14d the thrombi obviously reduce and organization.
2. MIP-1α mRNA and protein expression in the blood of the model mice showed rising trend from2h,4h,6h,8h,12h,1d,2d,4d to8d, and at8d reached peak, then at21d drop to normal levels.
3. The quality/length ratio of thrombi showed that was diminishing trend with the extension of time (P<0.05) in both DVT group and DVT+MIP-1α-Ab group; at the same time point, DVT group thromboembolus quality/length ratio is more obvious decrease (P<0.05).
4. AT the same time point, F4/80and MMP-9expression in DVT group is obviously higher than that of the DVT+MIP-1α-Ab group (P<0.05), MMP-2expression between the two groups had no difference (P>0.05).
5. Gene expression changes of MIP-1a, MMP-2,-9and F4/80were detected by PCR amplificate and electrophoresis, as well as real-time PCR. The gene expressions in DVT group were higher than non-thrombosis group and control group, and there is no difference between non-thrombosis and control group.
Conclusion:
1. By ligating inferior vena cava method to establish stasis DVT mouse model, at8h there were complete thrombosis in vein, and at14d the thrombi can occur obvious organization.
2. MIP-1α expression in model group showed that it rised to the peak after8days, and at21d it droped to the normal levels.
3. MIP-1α and deep vein thrombosis, which is closely related to the resolution, and its antibody could weaken the effect of thrombolysis.
4. The effect of MIP-1a promoting thrombi resolution were related with monocyte/macrophage gathered and MMP-9expression; the relationship between MMP-2and thrombi resolution may be independent of the MIP-1 a effect.
5. MIP-1α, MMP-2,-9and F4/80may play important role in recruit inflammatory cells and thrombosis recanalization.
引文
[I]Tenna AM, Kappadath S, Stansby G. Diagnostic tests and strategies in venous thromboembolism.Phlebology.2012;27 Suppl 2:43-52.
[2]中华医学会心血管病学分会肺血管病学组.急性肺血栓栓塞症诊断治疗中国专家共识.中华内科杂志,2010,2(4):10-57
[3]Kahn SR, Ginsberg JS. Relationship between deep venous thrombosis and the postthrombotic syndrome. Archives of internal medicine.2004,164(1):17-26.
[4]Vazquez SR, Kahn SR. Advances in the diagnosis and management of postthrombotic syndrome.Best Pract Res Clin Haematol.2012 Sep;25(3):391-402.
[5]Armstrong awadegah TE. Principles of pharmacology:the pathophysiologic basis of drug therapy. Philadelphia:Wolters Kluwer Health/Lippincott Williams & Wilkins.2008:396.
[6]中华医学会骨科学分会.中国骨科大手术静脉血栓栓塞症预防指南.中华骨科杂志,2009,29(6):600-603.
[7]邱贵兴.骨科领域静脉血栓栓塞症的预防.中国血管外科杂志(电子版).2011;03(01).
[8]Liew NC, Chang YH, Choi G, et al. Asian venous thromboembolism guidelines:prevention of venous thromboembolism.Int Angiol.2012 Dec;31(6):501-16.
[9]Zhao JM, He ML, Xiao ZM, et al. Different types of intermittent pneumatic compression devices for preventing venous thromboembolism in patients after total hip replacement.Cochrane Database Syst Rev.2012 Nov 14;11:CD009543.
[10]Januel JM, Chen G, Ruffieux C, et al. Symptomatic in-hospital deep vein thrombosis and pulmonary embolism following hip and kneearthroplasty among patients receiving recommended prophylaxis:a systematic review. JAMA.2012 Jan 18;307(3):294-303.
[11]Jawa RS, Warren K, Young D, et al. Venous Thromboembolic Disease in Trauma and Surveillance. Journal of Surgical Research.2011; 167(1):24-31.
[12]Al-Dujaili TM, Majer CN, Madhoun TE, et al. Deep venous thrombosis in spine surgery patients:incidence and hematoma formation. Int Surg.2012 Apr;97(2):150-4.
[13]孙志坚.关于药物预防骨科大手术后静脉血栓形成的争论.中国骨与关节外科.2011;04(02):67-70.
[14]Majoor CJ, Kamphuisen PW, Zwinderman AH, et al. Risk of deep-vein thrombosis and pulmonary embolism in asthma. Eur Respir J.2012 Dec 20.
[15]Guanella R, Kahn SR. Post-thrombotic syndrome:current prevention and management strategies.Expert Rev Cardiovasc Ther.2012 Dec;10(12):1555-66.
[16]O'Donnell MJ, McRae S, Kahn SR, et al. Evaluation of a venous-return assist device to treat severe post-thrombotic syndrome (VENOPTS). A randomized controlled trial.Thromb Haemost.2008 Mar;99(3):623-9.
[17]Turkoski B. Preventing DVT in orthopaedic patients.Orthop Nurs,2000,19:93-99.
[18]Moheimani F, Jackson DE. Venous thromboembolism:classification, risk factors, diagnosis, and management. ISRN hematology.2011:124610-124610.
[19]Holbrook A, Schulman S, Witt DM, et al. Evidence-based management of anticoagulant therapy:Antithrombotic Therapy and Prevention of Thrombosis,9th ed:American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest.2012; 141(2 Suppl): e152S-184S.
[20]Haut ER, Noll K, Efron DT, et al. Can increased incidence of deep vein thrombosis (DVT) be used as a marker of quality of care in the absence of standardized screening? The potential effect of surveillance bias on reported DVT rates after trauma.2007; 63:1132-1135; discussion 1135-1137.
[21]Flinterman LE, van Hylckama VA, Cannegieter SC,et al. Long-term survival in a large cohort of patients with venous thrombosis:incidence and predictors. PLoS Med.2012; 9(1): 1100-1155.
[22]SooHoo NF, Eagan M, Krenek L, et al. Incidence and factors predicting pulmonary embolism and deep venous thrombosis following surgical treatment of ankle fractures. Foot Ankle Surg. 2011,17(4):259-262.
[23]Ramzi DW, Leeper KV. DVT and pulmonary embolism:Part Ⅰ. Diagnosis. American family physician.2004,69(12):2829-2836.
[24]Herrera S, Comerota AJ. Embolization during treatment of deep venous thrombosis: incidence, importance, and prevention. Tech Vasc Interv Radiol,2011,14(2):58-64.
[25]Engbers MJ, van Hylckama Vlieg A, et al. Venous thrombosis in the elderly:incidence, risk factors and risk groups. J Thromb Haemost,2010,8(10):2105-2112.
[26]Muntz JE. Deep vein thrombosis and pulmonary embolism in the perioperative patient. The American journal of managed care.2000; 6(20 Suppl):S1045-1052.
[27]Jacquemin M, Saint-Remy JM. The use of antibodies to coagulation factors for anticoagulant therapy. Current medicinal chemistry.2004,11(17):2291-2296.
[28]Cushman M, Callas PW, Denenberg JO, et al. Risk factors for peripheral venous disease resemble those for venous thrombosis:the San Diego Population Study. J Thromb Haemost. 2010,8(8):1730-1735.
[29]Esmon CT. Basic mechanisms and pathogenesis of venous thrombosis. Blood Rev.2009, 23(5):225-229.
[30]Rosendaal FR, Reitsma PH. Genetics of venous thrombosis. J Thromb Haemost.2009,7 Suppl 1:301-304.
[31]Bezemer ID, Bare La, Doggen CJM,et al. Gene variants associated with deep vein thrombosis. JAMA:the journal of the American Medical Association.2008,299(11):1306-1314.
[32]van Schie MC, van Loon JE, de Maat MPM, et al. Genetic determinants of von Willebrand factor levels and activity in relation to the risk of cardiovascular disease:a review. Journal of thrombosis and haemostasis:JTH.2011,9(5):899-908.
[33]Lopez-Beret P, Pinto JM, Romero A, et al. Systematic study of occult pulmonary thromboembolism in patients with deep venous thrombosis. Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter.2001,33(3):515-521.
[34]Solis G, Saxby T. Incidence of DVT following surgery of the foot and ankle.2002,23: 411-414.
[35]Hippisley-cox J, Coupland C. Development and validation of risk prediction algorithm (QThrombosis) to estimate future risk of venous thromboembolism:prospective cohort study.2011:1-12.
[36]Ogeng'o JA, Obimbo MM, Olabu BO,et al. Pulmonary thromboembolism in an East African tertiary referral hospital. J Thromb Thrombolysis.2011 Oct;32(3):386-91.
[37]Press D. Deep vein thrombosis:a clinical review. Access.2011:59-69.
[38]Christie S, Thibault-Halman G, Casha S. Acute pharmacological DVT prophylaxis after spinal cord injury. Journal of neurotrauma.2011,28(8):1509-1514.
[39]Decousus H, Frappe P, Accassat S,et al. Epidemiology, diagnosis, treatment and management of superficial-vein thrombosis of the legs. Best Pract Res Clin Haematol.2012 Sep;25(3):275-84.
[40]Bauersachs RM. Clinical presentation of deep vein thrombosis and pulmonary embolism. Best Pract Res Clin Haematol.2012 Sep;25(3):243-51.
[41]Riopel C, Bounameaux H. Doppler ultrasound and D-dimer:friend or foe?Hamostaseologie. 2012;32(1):28-36.
[42]McLintock C, Brighton T, Chunilal S, et al. Recommendations for the diagnosis and treatment of deep venous thrombosis and pulmonary embolism in pregnancy and the postpartum period.Aust N Z J Obstet Gynaecol.2012 Feb;52(1):14-22.
[43]Vedantham S. Interventional approaches to deep vein thrombosis. Am J Hematol.2012 May;87 Suppl 1:S113-8.
[44]Nyamekye I, Merker L. Management of proximal deep vein thrombosis. Phlebology.2012;27 Suppl 2:61-72.
[45]Bambrah RK, Pham DC, Zaiden R, et al. Heparin-induced thrombocytopenia. Clin Adv Hematol Oncol.2011 Aug;9(8):594-9.
[46]Dasari TW, Pappy R, Hennebry TA. Pharmacomechanical thrombolysis of acute and chronic symptomatic deep vein thrombosis:a systematic review of literature. Angiology.2012 Feb;63(2):138-45.
[47]Agnelli G, Becattini C. Treatment of DVT:how long is enough and how do you predict recurrence.J Thromb Thrombolysis.2008 Feb;25(1):37-44.
[48]Romualdi E, Ageno W. Venous thrombosis at unusual site in women. Thromb Res.2011 Feb;127 Suppl 3:S86-8.
[49]Brooks EG, Trotman W, Wadsworth MP, et al. Valves of the deep venous system:an overlooked risk factor. Blood.2009;114:1276-1279.
[50]Charkes ND, Dugan MA, Malmud LS, et al. Letter:labelled leucocytes in thrombi. Lancet. 1974;2:600.
[51]Wakefield TW, Strieter RM, Prince MR, Downing LJ, Greenfield LJ. Pathogenesis of venous thrombosis:a new insight. Cardiovasc Surg.1997;5:6-15.
[52]Wang MF, Yang LH, Yang XL, et al. [1059 g/c gene polymorphism of C-reactive protein in patients with deep vein thrombosis]. Zhongguo Shi Yan Xue Ye Xue Za Zhi.2011 Jun;19(3):769-71. Chinese.
[53]Ripplinger CM, Kessinger CW, Li C,et al. Inflammation modulates murine venous thrombosis resolution in vivo:assessment by multimodal fluorescence molecular imaging.Arterioscler Thromb Vasc Biol.2012 Nov;32(11):2616-24.
[54]Jezovnik MK, Poredos P. Factors influencing the recanalisation rate of deep venous thrombosis.Int Angiol.2012 Apr;31(2):169-75.
[55]Henke PK, Wakefield T. Thrombus resolution and vein wall injury:dependence on chemokines and leukocytes.Thromb Res.2009; 123 Suppl 4:S72-8.
[56]Shbaklo H, Holcroft CA, Kahn SR. Levels of inflammatory markers and the development of the post-thrombotic syndrome.Thromb Haemost.2009 Mar;101(3):505-12.
[57]Wakefield TW, Myers DD, Henke PK. Mechanisms of venous thrombosis and resolution. Arterioscler Thromb Vasc Biol.2008 Mar;28(3):387-91.
[58]Smits HA, Rijsmus A, vanLoon JH, et al. Amyloid- beta-induced chemokine Production in Primary human macrophages and astroeytes.J Neurolrnrnunol.2002;127:160 - 168
[59]Chen YK, Jiang XM, Gong JP. Recombinant human granulocyte colony-stimulating factor enhanced the resolution of venous thrombi. J Vasc Surg.2008 May;47(5):1058-65.
[60]Stoll G, Bendszus M. Inflammation and atherosclerosis:novel insights into plaque formation and destabilization. Stroke.2006 Jul;37(7):1923-32.
[61]Ishikawa K, Yoshida S, Nakao S, et al. Bone marrow-derived monocyte lineage cells recruited by MIP-1β promote physiological revascularization in mouse model of oxygen-induced retinopathy. Lab Invest.2012 Jan;92(1):91-101.
[62]Marcel Levi, Janine Dorffler—Melly, Gerhard J.Johnson, et al.Usefulness and Limitations of Animal Models of Venous Thrombosis [J].Thromb Haemost,2001,86:1331.
[63]顾毅,时德,赵渝,等.不同时相球囊导管取栓的实验研究[J].中华普通外科杂志,2003,18(6):370-372.
[64]赛力克·马高维亚,王护国,罗军,等.一种适合动态研究的大鼠深静脉血栓动物模型[J].中国普外基础与临床杂志,2008,15(6):408-411.
[65]高峰,胡建国,关薇薇.SD大鼠深静脉血栓模型的建立及其静脉壁形态学研究[J].江西医学院学报,2008,48(2):21-23.
[66]戎建杰,伍波,何礼荣,等.导管直接溶栓治疗犬股静脉急性深静脉血栓后静脉壁形态学的变化[J].苏州大学学报(医学版),2004,24(2):169-172.
[67]周为民,李晓强,余朝文,等.血栓消融、溶栓和取栓对犬股静脉壁形态学影响的研究[J].中国普通外科杂志,2003,18(4):214-216.
[68]何飞,胡敏,王兵,等.构建创伤性肢体深静脉血栓动物模型及其应用评价[J].中国临床康复,2005,9(22):112-113.
[69]张春强,黄河,赵学凌,等.两种创伤性深静脉血栓大鼠模型中炎症相关基因的表达研究[J].生物医学工程研究,2008,26(1):66-70.
[70]Tran A, Taylor DM. Medical model for hospital in the home:effects on patient management. Aust Health Rev.2009 Aug;33(3):494-501
[71]Didisheim P. Animal models useful in the study of thrombosis and antithrombotic agents. ProgHemost Thromb.1972; 1:165-197.
[72]Jeske WP, Iqbal O, Fareed J, Kaiser B. A survey of venous thrombosis models. Methods Mol Med.2004; 93:221-237.
[73]Myers DD, Hawley AE, Farris DM, et al. P-selectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg.2003; 38:1075-1089.
[74]Wojcik BM, Wrobleski SK, Hawley AE, et al. Interleukin-6:A potential target for post-thrombotic syndrome. Ann Vasc Surg.2011; 25:229-239..
[75]Brill A, Fuchs TA, Chauhan AK, et al. Von willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models. Blood.2011; 117:1400-1407.
[76]Nazario R, DeLorenzo LJ, Maguire GP, et al. Treatment of venous thromboembolism. Heart Dis.2000 Nov-Dec;2(6):438-45.
[77]Kearon C. Natural history of venous thromboembolism. Circulation.2003 Jun 17;107(23 Suppl 1):I22-30.
[78]Dewyer NA, Sood V, Lynch EM, et al. Plasmin inhibition increases MMP-9 activity and decreases vein wall stiffness during venous thrombosis resolution. J Surg Res.2007 Oct;142(2):357-63.
[79]Wakefield TW, Henke PK. The role of inflammation in early and late venous thrombosis:Are there clinical implications? Semin Vasc Surg.2005 Sep;18(3):118-29.
[80]Myers DD, Wrobleski SK, Longo C, et al. Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation. Thromb Haemost.2007 Mar;97(3):400-7.
[81]Henke PK. Plasmin and matrix metalloproteinase system in deep venous thrombosis resolution. Vascular.2007 Nov-Dec;15(6):366-71.
[82]Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing J Leukoc Biol 2001;69:513-21.
[83]Henke PK, Pearce CG, Moaveni DM, et al. Targeted deletion of CCR2 impairs deep vein thombosis resolution in a mouse model J Immunol,2006;177:3388-97.
[84]Varma MR, Varga AJ, Knipp BS, et al. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg 2003;38:1090-8.
[85]Varma MR, Moaveni DM, Dewyer NA, et al. Deep vein thrombosis resolution is not accelerated with increased neovascularization J Vasc Surg,2004;40:536-42.
[86]Hogaboam CM, Steinhauser ML, Chensue SW, et al.Novel roles for chemokines and fibroblasts in interstitial fibrosis. Kidney Int,1998;54:2152-9.
[87]Stewart GJ. Neutrophils and deep venous thrombosis. Haemostasis 1993;23(Suppl 1):127-40.
[88]Humphries J, McGuinness CL, Smith A, et al. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi.J Vasc Surg 1999;30:894-9.
[89]Grainger DJ, Wakefield L, Bethell HW, et al. Release and activation of platelet latent TGFb in blood clots during dissolution with plasmin.Nat Med,1995; 1:932-7.
[90]Deatrick KB, Eliason JL, Lynch EM, et al. Vein wall remodeling after deep vein thrombosis involves matrix metalloproteinases and late fibrosis in a mouse model J Vasc Surg 2005;42:140-8.
[91]Streetly M, Hunt BJ, Parmar K, et al. Markers of endothelial and haemostatic function in the treatment of relapsed myeloma with the immunomodulatory agent Actimid (CC-4047) and their relationship with venous thrombosis. Eur J Haematol.2005 Apr;74(4):293-6
[92]Flannery CR. MMPs and ADAMTSs:functional studies. Front Biosci,2006,11:544-569.
[93]Zhang XM, Li YK, Shen CY, et al. [Preliminary study on etiology of Budd-Chiari syndrome through clinic and experiment]. Zhonghua Wai Ke Za Zhi.2010 Apr 15;48(8):569-72.
[94]Siefert SA, Sarkar R. Matrix metalloproteinases in vascular physiology and disease. Vascular. 2012 Aug;20(4):210-6.
[95]Sood V, Luke CE, Deatrick KB, et al. Urokinase plasminogen activator independent early experimental thrombus resolution:MMP2 as an alternative mechanism. Thromb Haemost. 2010 Dec;104(6):1174-83. doi:10.1160/TH10-03-0184. Epub 2010 Sep 30.
[96]Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system J Clin Invest 1991;88:1067-72.
[97]Thanaporn P, Myers DD, Wrobleski SK, et al. P-selectin inhibition decreases post-thrombotic vein wall fibrosis in a rat model. Surgery.2003 Aug;134(2):365-71.
[98]Singh I, Burnand KG, Collins M, et al. Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice:rescue by normal bone marrow-derived cells Circulation 2003;107:869-75.
[99]Kohler HP, Grant PJ. Plasminogen-activator inhibitor type 1 and coronary artery disease. N Engl J Med 2000;342:1792-801.
[100]Oda T, Jung YO, Kim HS, et al. PAI-a deficiency attenuates the fibrogenic response to ureteral obstruction. Kidney Int 2001;30:587-96.
[101]Moses MA, Harper J, Folkman J. Doxycycline Treatment for Lymphangioleiomyo-matosis with Urinary Monitoring for MMPs. N. Engl. J. Med,2006,354:2621-2622.
[102]Luttun A, Lupu F, Storkebaum E, et al. Lack of plasminogen activator inhibitor-1 promotes growth and abnormal matrix remodeling of advanced atherosclerotic plaques in apolipoprotein E-deficient mice Arterioscler Thromb Vasc Biol 2002;22:499-505.
[103]Baldwin JF, Sood V, Elfline MA, et al. The role of urokinase plasminogen activator and plasmin activator inhibitor-1 on vein wall remodeling in experimental deep vein thrombosis. J Vasc Surg.2012 Oct;56(4):1089-97.
[104]Modarai B, Burnand KG, Humphries J, et al.. The role of neovascularisation in the resolution of venous thrombus. Thromb Haemost.2005 May;93(5):801-9.
[105]Nosaka M, Ishida Y, Kimura A, et al. Absence of IFN-y accelerates thrombus resolution through enhanced MMP-9 and VEGF expression in mice. J Clin Invest. 2011 Jul;121(7):2911-20.
[106]Henke PK, Varga A, De S, et al. Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol.2004 Jun;24(6):1130-7.
[107]Wilbur J, Shian B. Diagnosis of deep venous thrombosis and pulmonary embolism. Am Fam Physician.2012 Nov 15;86(10):913-9.
[108]Krishan S, Panditaratne N, Verma R, et al. Incremental value of CT venography combined with pulmonary CT angiography for the detection of thromboembolic disease:systematic review and meta-analysis. AJR Am J Roentgenol.2011 May; 196(5):1065-72.
[109]Shiver SA, Lyon M, Blaivas M,et al. Prospective comparison of emergency physician-performed venous ultrasound and CT venography for deep venous thrombosis. Am J Emerg Med.2010 Mar;28(3):354-8.
[110]Cassou-Birckholz MF, Engelhorn CA, Salles-Cunha SX, et al. Assessment of deep venous thrombosis by grayscale median analysis of ultrasound images. Ultrasound Q.2011 Mar;27(1):55-61.
[111]Giannini M, Rollo HA, Maffei FH, et al. Advances in ultrasound techniques improve early detection of deep vein thrombosis. Int Angiol.2008 Dec;27(6):466-74
[112]Schellong SM. Complete compression ultrasound for the diagnosis of venous thromboembolism. Curr Opin Pulm Med.2004 Sep;10(5):350-5.
[113]Theodoro D, Blaivas M, Duggal S,et al. Real-time B-mode ultrasound in the ED saves time in the diagnosis of deep vein thrombosis (DVT). Am J Emerg Med.2004 May;22(3):197-200
[114]Olowoyeye OA, Awosanya GO, Soyebi KO. Duplex ultrasonographic findings in patients with suspected DVT. Niger Postgrad Med J.2010,17(2):128-132.
[115]Zierler BK. Screening for acute DVT:optimal utilization of the vascular diagnostic laboratory. Semin Vasc Surg.2001 Sep;14(3):206-14.
[116]Crowther MA, Cook DJ, Griffith LE, et al. Neither baseline tests of molecular hypercoagulability nor D-dimer levels predict deep venous thrombosis in critically ill medical-surgical patients. Intensive Care Med.2005 Jan;31(1):48-55.
[117]Srinivasan D, Watzak B. Anticoagulant Use in Real Time. J Pharm Pract.2012 Nov 25.
[118]Wadajkar AS, Santimano S, Rahimi M, et al. Deep vein thrombosis:Current status and nanotechnology advances. Biotechnol Adv.2012 Aug 23.
[119]Albertsen IE, Larsen TB, Rasmussen LH, et al. Prevention of venous thromboembolism with new oral anticoagulants versus standard pharmacological treatment in acute medically ill patients:a systematic review and meta-analysis. Drugs.2012 Sep 10;72(13):1755-64.
[120]Garcia D, Libb YE, Crowther MA. The new oral anticoagulants [J]. Blood,2010, 115 (1):15-20.
[121]Comerota A J, Aldridge SC. Thrombolytic therapy for deep venous thrombosis:a clinical review [J]. Can J Surg,1993,36 (4):359-364.
[122]Watson LI, Armon MP. Thrombolysis for acute deep vein thrombosis [J]. Cochrane Database Syst Rev,2004,18 (4):CD002783.
[123]Baekgaard N, Broholm R, Just S, et al. Long-term results using catheter-directed thrombolysis in 103 lower limbs with acute iliofemoral venous thrombosis [J]. Eur J Vasc Endovasc Surg,2010,39 (1):112-117.
[124]Kanphausen M, Barbera L, Mumme A, et al.Clinical and functional result after transfemoral thrombectomy for iliofemoral deep venous thrombosis:A 5-year-follow-up [J]. Zentralbl Chir,2005,130 (5):454-461
[125]张福先,金英姬,马佐田,等.肢体静脉血栓形成与肺动脉栓塞的关系探讨[J].中华结核和呼吸杂志,2009,23(9):531-533
[126]Hann CL,Streiff MB.The role of vena caval filter in the management of venous thromboembolism[J]. Blood Rev,2005,19(4):179-202
[127]Shibuya N, Frost CH, Campbell JD, et al. Incidence of acute deep vein thrombosis and pulmonary embolism in foot and ankle trauma:analysis of the National Trauma Data Bank. J Foot Ankle Surg.2012 Jan-Feb;51(1):63-8.
[128]Toker S, Hak DJ, Morgan SJ. Deep vein thrombosis prophylaxis in trauma patients. Thrombosis.2011;2011:505373.
[129]Frits RR. Venous thrombosis:the role of genes, environment, and behavior. J Hematology Am Soc Hematol Educ Program,2005:1-12.
[130]DeDea L. DVT prophylaxis with aspirin in orthopedic surgery patients. JAAPA.2012 Apr;25(4):21.
[131]Sobieraj DM, Lee S, Coleman CI, et al. Prolonged versus standard-duration venous thromboprophylaxis in major orthopedic surgery:a systematic review.
1. Majoor CJ, Kamphuisen PW, Zwinderman AH, et al. Risk of deep-vein thrombosis and pulmonary embolism in asthma.Eur Respir J.2012 Dec 20.
2. Guanella R, Kahn SR. Post-thrombotic syndrome:current prevention and management strategies. Expert Rev Cardiovasc Ther.2012 Dec;10(12):1555-66.
3. Liew NC, Chang YH, Choi G, et al. Asian venous thromboembolism guidelines:prevention of venous thromboembolism.Int Angiol.2012 Dec;31(6):501-16.
4. Streiff MB, Lau BD. Thromboprophylaxis in nonsurgical patients. Hematology Am Soc Hematol Educ Program.2012;2012:631-7.
5. Wakefield TW, Myers DD, Henke PK. Mechanisms of venous thrombosis and resolution. Arterioscler Thromb Vasc Biol.2008;28:387-391.
6. Brooks EG, Trotman W, Wadsworth MP, et al. Valves of the deep venous system:an overlooked risk factor. Blood.2009;114:1276-1279.
7. Charkes ND, Dugan MA, Malmud LS, et al. Letter:labelled leucocytes in thrombi. Lancet, 1974;2:600.
8. Wakefield TW, Strieter RM, Prince MR, et al. Pathogenesis of venous thrombosis:a new insight. Cardiovasc Surg.1997;5:6-15.
9. Wang MF, Yang LH, Yang XL, et al. [1059 g/c gene polymorphism of C-reactive protein in patients with deep vein thrombosis]. Zhongguo Shi Yan Xue Ye Xue Za Zhi.2011 Jun;19(3):769-71. Chinese.
10. Ripplinger CM, Kessinger C W, Li C,et al. Inflammation modulates murine venous thrombosis resolution in vivo:assessment by multimodal fluorescence molecular imaging.Arterioscler Thromb Vasc Biol.2012 Nov;32(11):2616-24.
11. Jezovnik MK, Poredos P. Factors influencing the recanalisation rate of deep venous thrombosis.Int Angiol.2012 Apr;31(2):169-75.
12. Henke PK, Wakefield T. Thrombus resolution and vein wall injury:dependence on chemokines and leukocytes.Thromb Res.2009; 123 Suppl 4:S72-8.
13. Shbaklo H, Holcroft CA, Kahn SR. Levels of inflammatory markers and the development of the post-thrombotic syndrome.Thromb Haemost.2009 Mar;101(3):505-12.
14. Stewart GJ, Alburger PD Jr, Stone EA, Soszka TW. Total hip replacement induces injury to remote veins in a canine model. J Bone Joint Surg Am.1983;65:97-102.
15. Thomas D. Venous thrombogenesis. Br Med Bull.1994;50:803-812.
16. McGuinness CL, Humphries J, Waltham M, et al. Recruitment of labelled monocytes by experimental venous thrombi. Thromb Haemost.2001;85:1018-1024.
17. Ramacciotti E, Myers DD Jr, Wrobleski SK,et al. P-selectin/PSGL-1 inhibitors versus enoxaparin in the resolution of venous thrombosis:a meta-analysis.Thromb Res.2010 Apr;125(4):e138-42.
18. Manly DA, Boles J, Mackman N. Role of tissue factor in venous thrombosis. Annu Rev Physiol.2010.
19. Meier TR, Myers DD Jr, Wrobleski SK, et al. Prophylactic P-selectin inhibition with PSI-421 promotes resolution of venous thrombosis without anticoagulation.Thromb Haemost.2008 Feb;99(2):343-51.
20. Day SM, Reeve JL, Pedersen B, et al. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood.2005; 105:192-198.
21. Wakefield TW, Strieter RM, Wilke CA, et al. Venous thrombosisassociated inflammation and attenuation with neutralizing antibodies to cytokines and adhesion molecules. Arterioscler Thromb Vasc Biol.1995;15:258-268.
22. McDonald B, Pittman K, Menezes GB, et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science.2010;330:362-366.
23. Varma MR, Varga AJ, Knipp BS, et al. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg.2003;38:1090-1098.
24. Henke PK, Varga A, De S, et al.Deep vein thrombosis resolution is modulated by monocyte cxcr2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol.2004;24:1130-1137.
25. Pillay J, Ramakers BP, Kamp VM, et al. Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia. J Leukoc Biol. 2010;88:211-220.
26. Woollard KJ, Sturgeon S, Chin-Dusting JP,et al.Erythrocyte hemolysis and hemoglobin oxidation promote ferric chlorideinduced vascular injury. J Biol Chem.2009; 284:13110-13118.
27. Mustafa S, Weltermann A, Fritsche R,et al. Genetic variation in heme oxygenase 1 (HMOX1) and the risk of recurrent venous thromboembolism. J Vasc Surg.2008;47:566-570.
28. Saha P, Humphries J, Modarai B, et al. Leukocytes and the natural history of deep vein thrombosis:current concepts and future directions. Arterioscler Thromb Vasc Biol.2011 Mar;31(3):506-12.
29. Martin P, D'Souza D, Martin J, et al. Wound healing in the PU.1 null mouse-tissue repair is not dependent on inflammatory cells. Curr Biol.2003;13:1122-1128.
30. Humphries J, Gossage JA, Modarai B, et al. Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis. J Vasc Surg. 2009;50:1127-1134.
31. Flick MJ, LaJeunesse CM, Talmage KE, et al. Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin αmβ2 binding motif. J Clin Invest. 2007; 117:3224-3235.
32. Smiley ST, King JA, Hancock WW. Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4.J Immunol.2001;167:2887-2894.
33. Henke PK, Mitsuya M, Luke CE, et al. Toll-like receptor 9 signaling is critical for early experimental deep vein thrombosis resolution. Arterioscler Thromb Vasc Biol.2011;31: 43-49.
34. Varma MR, Moaveni DM, Dewyer NA, et al. Deep vein thrombosis resolution is not accelerated with increased neovascularization. J Vasc Surg.2004;40:536-542.
35. Humphries J, McGuinness CL, Smith A, et al. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg. 1999;30:894-899.
36. Henke PK, Pearce CG, Moaveni DM, et al. Targeted deletion of CCR2 impairs deep vein thombosis resolution in a mouse model. J Immunol.2006;177:3388-3397.
37. Henke PK, Varma MR, Moaveni DK, et al. Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis. Thromb Haemost. 2007;98:1045-1055.
38. Nahrendorf M, Swirski FK, Aikawa E, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007;204:3037-3047.
39. Arnold L, Henry A, Poron F, et al. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med. 2007;204:1057-1069.