初探内皮细胞膜微粒（EMPs）于体外对内皮细胞功能及凋亡的影响
|
摘要
研究背景:
股骨头微循环障碍、血液高凝倾向和小血管阻塞是激素性股骨头坏死诸多发病机制中比较重要的机制。内皮细胞膜微粒(Endothelial Microparticles,EMPs)是由内皮细胞分泌,磷脂膜包被的微小囊泡结构,具有影响血液凝血系统、血管舒张功能,且与多种内皮损伤性血栓及炎症疾病相关。同时EMPs参与细胞凋亡、活化、炎症细胞募集等多种病生理过程。
研究目的:
探讨体外试验中,激素对于内皮细胞EMPs释放的影响.
探讨EMPs对内皮细胞自身的影响,包括内皮功能和凋亡两方面,进而初探EMPs在激素相关性股骨头缺血坏死机制中的作用.
研究方法:
实验对象:实验中选择体外培养的人脐静脉内皮细胞株(EAhy926)。
实验刺激物:激素对内皮细胞实验中使用DEX(dexamethasone)激素,EMPs对内皮细胞影响实验则选择内皮细胞自分泌的EMPs。
检测方法:EMPs和内皮细胞共培养一段时间后倒置相差显微镜观察细胞形态学变化,用流式细胞仪检测EMPs数量,随后用Bradford法测BSA(BovineSerum Albumin)浓度值,判断内皮细胞通透性变化。以Caspase-3试剂盒和Annexin-V FITC试剂盒测定细胞凋亡状况,RT-PCR检测内皮细胞凋亡基因的表达。
研究结果:
1.流式细胞仪分析:10mM浓度激素刺激24h,内皮细胞释放EMPs达到最大。
2.中、高浓度EMPs对内皮细胞通透性产生损伤,低浓度无明显差异。
3.试剂盒检测结果:高中低浓度EMPs内皮细胞Caspase-3、PS(Phosphatidylserine)活性表达均高于对照。
4.Realtime-PCR、RT-PCR分析凋亡基因:高、中浓度EMPs组APAF-1、AIF基因表达较对照增高,低浓度组没有明显差异。
结论:
1.DEX在10mM,24h条件下刺激内皮细胞产生最大量的EMPs
2.7.66*10~4和7.66*10~3个/ml浓度EMPs对内皮细胞通透性可引起损伤,但7.66~*10~2个/mlEMPs则不会对内皮产生明显损伤。
3.7.66*10~4、7.66*10~3、7.66*10~2个/ml浓度EMPs均促内皮细胞凋亡发生;Caspase-3、PS活性表达均增高。
4.7.66*10~*和7.66*10~3个/ml浓度EMPs可使内皮细胞APAF-1、AIF基因表达增高,7.66*10~2个/ml浓度EMPs则没有明显作用。
Background:
microcirculation disorder,hypercoagulation state and microvascular occlusion are important pathways in the pathogenesis of Avascular Necrosis of Femoral Head(ANFH).Endothelial microparticles(EMPs) are phospholipid vesicles derived from endothelial cells.It has been reported that EMPs can influence plasma coagulation system and vasodilatation.Also,they are implicated in inflammation and vascular thrombosis associated with endothelial injury.Moreover,it has been shown that EMPs are involved in cell signaling,apoptosis and recruitment of inflammatory cells.
Object:
To investigate the effect of glucocortoid on EMPs releasing in vitro. To investigate the effect of EMPs on HUVECs,including endothelial function and apoptosis and to study the contribution of EMPs on ANFH.
Material and methods:
Object:Human Umbilical Vein Endothelial Cells(HUVECs) in vitro(EAhy926).
Stimulator:dexamethasone(DEX) and EMPs released by HUVECs.
Method:After cultured with EMps,HUVECs were examined under microscope, and then Mps were counted using flowcytometry.The value of bovine serum albumin was determined by Bradford method to evaluate the permeability of HUVECs. Cellular apoptosis were examined using Caspase-3 and Annexin-V FITC kit.The expression of genes associated with apoptosis were detected by reverses transcription-PCR.
Results:
Flowcytometry analysis showed that EMPs were released at maximum concentration after 24 hours stimulated by 10mM DEX.
Moderate and high concentrations of EMPs caused injury to the permeability of HUVECs,while low concentration of EMP didn't.
All concentrations of EMPs induced higher activity of Caspase-3 and PS than control did.
Moderate and high concentration groups showed increased expression of APAF-1,AIF gene in comparison with control,while low concentration group didn't.
Conclusion:
DEX(10mM,24h) stimulated HUVECs to release EMPs at maximum concentration.
EMPs at the concentration of 7.66*104 and 7.66*103/ml caused injury to the permeability of HUVECs,while 7.66*102/ml did not show significant difference from control.
All three concentrations of EMPs induced HUVECs apoptosis with increased activity of Caspase-3 and PS.
EMPs at the concentration of 7.66*104 and 7.66*103/ml showed increased expression of APAF-1,AIF gene in comparison with control,while low concentration group didn't.
股骨头微循环障碍、血液高凝倾向和小血管阻塞是激素性股骨头坏死诸多发病机制中比较重要的机制。内皮细胞膜微粒(Endothelial Microparticles,EMPs)是由内皮细胞分泌,磷脂膜包被的微小囊泡结构,具有影响血液凝血系统、血管舒张功能,且与多种内皮损伤性血栓及炎症疾病相关。同时EMPs参与细胞凋亡、活化、炎症细胞募集等多种病生理过程。
研究目的:
探讨体外试验中,激素对于内皮细胞EMPs释放的影响.
探讨EMPs对内皮细胞自身的影响,包括内皮功能和凋亡两方面,进而初探EMPs在激素相关性股骨头缺血坏死机制中的作用.
研究方法:
实验对象:实验中选择体外培养的人脐静脉内皮细胞株(EAhy926)。
实验刺激物:激素对内皮细胞实验中使用DEX(dexamethasone)激素,EMPs对内皮细胞影响实验则选择内皮细胞自分泌的EMPs。
检测方法:EMPs和内皮细胞共培养一段时间后倒置相差显微镜观察细胞形态学变化,用流式细胞仪检测EMPs数量,随后用Bradford法测BSA(BovineSerum Albumin)浓度值,判断内皮细胞通透性变化。以Caspase-3试剂盒和Annexin-V FITC试剂盒测定细胞凋亡状况,RT-PCR检测内皮细胞凋亡基因的表达。
研究结果:
1.流式细胞仪分析:10mM浓度激素刺激24h,内皮细胞释放EMPs达到最大。
2.中、高浓度EMPs对内皮细胞通透性产生损伤,低浓度无明显差异。
3.试剂盒检测结果:高中低浓度EMPs内皮细胞Caspase-3、PS(Phosphatidylserine)活性表达均高于对照。
4.Realtime-PCR、RT-PCR分析凋亡基因:高、中浓度EMPs组APAF-1、AIF基因表达较对照增高,低浓度组没有明显差异。
结论:
1.DEX在10mM,24h条件下刺激内皮细胞产生最大量的EMPs
2.7.66*10~4和7.66*10~3个/ml浓度EMPs对内皮细胞通透性可引起损伤,但7.66~*10~2个/mlEMPs则不会对内皮产生明显损伤。
3.7.66*10~4、7.66*10~3、7.66*10~2个/ml浓度EMPs均促内皮细胞凋亡发生;Caspase-3、PS活性表达均增高。
4.7.66*10~*和7.66*10~3个/ml浓度EMPs可使内皮细胞APAF-1、AIF基因表达增高,7.66*10~2个/ml浓度EMPs则没有明显作用。
Background:
microcirculation disorder,hypercoagulation state and microvascular occlusion are important pathways in the pathogenesis of Avascular Necrosis of Femoral Head(ANFH).Endothelial microparticles(EMPs) are phospholipid vesicles derived from endothelial cells.It has been reported that EMPs can influence plasma coagulation system and vasodilatation.Also,they are implicated in inflammation and vascular thrombosis associated with endothelial injury.Moreover,it has been shown that EMPs are involved in cell signaling,apoptosis and recruitment of inflammatory cells.
Object:
To investigate the effect of glucocortoid on EMPs releasing in vitro. To investigate the effect of EMPs on HUVECs,including endothelial function and apoptosis and to study the contribution of EMPs on ANFH.
Material and methods:
Object:Human Umbilical Vein Endothelial Cells(HUVECs) in vitro(EAhy926).
Stimulator:dexamethasone(DEX) and EMPs released by HUVECs.
Method:After cultured with EMps,HUVECs were examined under microscope, and then Mps were counted using flowcytometry.The value of bovine serum albumin was determined by Bradford method to evaluate the permeability of HUVECs. Cellular apoptosis were examined using Caspase-3 and Annexin-V FITC kit.The expression of genes associated with apoptosis were detected by reverses transcription-PCR.
Results:
Flowcytometry analysis showed that EMPs were released at maximum concentration after 24 hours stimulated by 10mM DEX.
Moderate and high concentrations of EMPs caused injury to the permeability of HUVECs,while low concentration of EMP didn't.
All concentrations of EMPs induced higher activity of Caspase-3 and PS than control did.
Moderate and high concentration groups showed increased expression of APAF-1,AIF gene in comparison with control,while low concentration group didn't.
Conclusion:
DEX(10mM,24h) stimulated HUVECs to release EMPs at maximum concentration.
EMPs at the concentration of 7.66*104 and 7.66*103/ml caused injury to the permeability of HUVECs,while 7.66*102/ml did not show significant difference from control.
All three concentrations of EMPs induced HUVECs apoptosis with increased activity of Caspase-3 and PS.
EMPs at the concentration of 7.66*104 and 7.66*103/ml showed increased expression of APAF-1,AIF gene in comparison with control,while low concentration group didn't.
引文
1．孙伟：股骨头坏死的病因、病理和发病机制。中华全科医师杂志,2006,5(2)：75-77
2. Jones JR Intravascular coagulatiow and osteoncrosis. Clin Orthop, 1992, 277 : 41-53
3. Glueck CJ , Freiberg R, Glueck H I, et al. Hypofibrinolysis: a common, major caus of Osteonecrosis. AM J Hematol, 1994, 45:156-166
4. Glueck CJ , Freiberg R, Tracy T, et al. Thrombopilia and hypofibrinolysis. Pathophysioligies of osteonerosis. Clin Orthop, 1997, 334:43-56
5. Wei-li Chen et al. In-Vitro Effects of Dexamethasone on Cellular Proliferation, Apoptosis, and Na+-K+-ATPase Activity of Bovine Corneal Endothelial Cells. Ocular Immunology and Inflammation,2006,14;215-223
6. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tisues. Science, 1997, 276 : 71-74
7. Bersford JN , Bennett JH, Devlin C, et al. Evidence for an inverse ralation ship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci, 1992, 102 : 341-351
8. Thompson D 1, lum KD, N ygaard SC, et al. The derivation and characteri zatvon of stromal cell lines from the bone marrow of p53212 mice: new insights into osteoblast and adipocyte differen tiation. J Bone M iner Res, 1998, 13: 195- 204
9. Cui Q , Wang GJ , Balin G. Stcroid2induccd adipogcncsis ina Pluripotential cell line from bonemarrow. J Bone Joint Surg (Am) , 1997, 79 : 1054-1063
10. Oreffo RO, Virdi A S, Triffitt JT. Modulatior of osteogenesis and adipogenesis by human serum in human bone marrow cultues. Eur J Gell Biol, 1997, 74 : 251 - 261
11. ang GJ. Fat-cell changes as a mechanism of avascular necrosis on the femoral head in cortison - treated rabbits. J Bone Joint Surg (Am) , 1977, 59: 729
12. Martin S, Tesse A, Hugel B, et al: Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation 109:1653- 1659, 2004
13. Brodsky SV, Zhang F, Nasjletti A, et al: Endotheliumderived microparticles impair endothelial function in vitro. Am J Physiol Heart Circ Physiol 286:H1910-H1915, 2004
14. Boulanger CM, Scoazec A, Ebrahimian T, et al: Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 104:2649- 2652, 2001
15. Vanwijk MJ, Svedas E, Boer K, et al: Isolated microparticles,but not whole plasma, from women with preeclampsia impair endothelium-dependent relaxation in isolated myometrialarteries from healthy pregnant women. Am J Obstet Gynecol 187:1686 -1693, 2002
16. Gilbert GE, Sims PJ, Wiedmer T, et al: Platelet-derived microparticles express high affinity receptors for factor VIII. J.Biol Chem 266:17261 - 17268, 1991
17. Hoffman M, Monroe DM, Roberts HR: Coagulation factor IXa binding to activated platelets and platelet-derived microparticles: A flow cytometric study. Thromb Haemost 68:74 - 78, 1992
18. So AK, Varisco PA, Kemkes-Matthes B, et al: Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways. J Thromb Haemost 1:2510-2515, 2003
19. Bogdanov VY, Balasubramanian V, Hathcock J, et al: Alternatively spliced human tissue factor: A circulating, soluble,thrombogenic protein. Nat Med 9:458 - 462, 2003
20. Butenas S, Bouchard BA, Brummel-Ziedins KE, et al:Tissue factor activity in whole blood. Blood 105:2764- 2770,2005
21. Osterud B, Bjorklid E: The production and availability of tissue thromboplastin in cellular populations of whole blood exposed to various concentrations of endotoxin. An assay for detection of endotoxin. Scand J Haematol29:175 - 184, 1982
22. Egorina EM, Sovershaev MA, Bjorkoy G, et al:Intracellular and surface distribution of monocyte tissue factor: Application to intersubject variability. Arterioscler Thromb Vasc.Biol 25:1493- 1498, 2005
23. Mallat Z, Hugel B, Ohan J, et al: Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: A role for apoptosis in plaque thrombogenicity. Circulation 99:348 - 353, 1999
24. Satta N, Toti F, Feugeas O, et al: Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 153:3245- 3255, 1994
25. Combes V, Simon AC, Grau GE, et al: In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest 104:93-102,1999
26. Wang GT. The effect of core decompression on femoral headblood flow in steroid2induced avascular necrosis of the femoral head. J BoneJoint Surg (Am), 1985,67(1): 121
27．李子荣,孙伟,屈辉,等．皮质类固醇与骨坏死关系的临床研究．中华外科杂志,2005．43：104821053
28. Camille,J et al.Microvascular Endothelial Cell Death and Rarefaction in the Glucocortoid-Induced Hypertensive Rat Microcirculation 2001,8 129-139
29. Denise B et al,Endothelium-Drived Microparticles Inhibt Human Cardiac Valve Endothelial Cell Function SHCOKVol 25.No 6 .575-580 .2006
30. Berckmans RJ, Neiuwland R, Boing AN, et al: Cellderived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 85:639 - 646,2001
31. Joop K, Berckmans RJ, Nieuwland R, et al: Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms. Thromb Haemost 85:810 - 820, 2001
32. Simak J, Holada K, Risitano AM, et al: Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria. Br J Haematol 125:804- 813,2004
33. Shet AS, Aras O, Gupta K, et al: Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 102:2678-2683, 2003
34. Diamant M, Nieuwland R, Pablo RF, et al: Elevated numbers of tissue-factor exposing microparticles correlate with components of the metabolic syndrome in uncompli-CELL MICROPARTICLES IN BLOOD 23 cated type 2 diabetes mellitus. Circulation 106:2442- 2447,2002
35. Sabatier F, Darmon P, Hugel B, et al: Type 1 and type 2 diabetic patients display different patterns of cellular microparticles.Diabetes 51:2840-2845,2002
36. Bretelle F, Sabatier F, Desprez D, et al: Circulating microparticles: A marker of procoagulant state in normal pregnancy and pregnancy complicated by preeclampsia or intrauterine growth restriction. Thromb Haemost 89:486 - 492, 2003
37. Matsumoto N, Nomura S, Kamihata H, et al: Increased level of oxidized LDL-dependent monocyte-derived microparticles in acute coronary syndrome. Thromb Haemost 91:146-154, 2004
38. Omoto S, Nomura S, Shouzu A, et al: Detection of monocyte-derived microparticles in patients with type II diabetes mellitus. Diabetologia 45:550 - 555, 2002
39. Jimenez JJ, Jy W, Mauro LM, et al: Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation. Br J Haematol 123:896- 902, 2003
40. Jimenez JJ, Jy W, Mauro LM, et al: Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: Findings from brain and renal microvascular cell culture and patients with active disease. Br J Haematol 112:81 -90,2001
41. Minagar A, Jy W, Jimenez JJ, et al: Elevated plasma endothelial microparticles in multiple sclerosis. Neurology 56:1319- 1324, 2001
42. Shet AS, Aras O, Gupta K, et al: Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 102:2678-2683,2003
43. Horstman LL, Ahn YS: Platelet microparticles: A wideangle perspective. Crit Rev Oncol Hematol 30:111 - 142, 1999
44. Day SM, Reeve JL, Pedersen B, et al: Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood 105:192- 198, 2005
45. Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, et al: Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant. Circulation96:3534- 3541, 1997
46. Biro E, Sturk-Maquelin KN, Vogel GM, et al: Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner. J Thromb Haemost 1:2561 - 2568, 2003
47. Mallat Z, Hugel B, Ohan J, et al: Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: A role for apoptosis in plaque thrombogenicity. Circulation 99:348 - 353, 1999
48. Jy W, Mao WW, Horstman L, et al: Platelet microparticles bind, activate and aggregate neutrophils in vitro. Blood Cells Mol Dis 21:217 - 231, 1995 (discussion 231a)
49. Barry OP, Pratico D, Savani RC, et al: Modulation of monocyte-endothelial cell interactions by platelet microparticles.J Clin Invest 102:136- 144, 1998
50. Barry OP, Pratico D, Lawson JA, et al: Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest 99:2118-2127,1997
51. Brill A, Dashevsky O, Rivo J, et al: Platelet-derived microparticles induce angiogenesis and stimulate post-ischemic revascularization. Cardiovasc Res 67:30- 38, 2005
52. Taraboletti G, D'Ascenzo S, Borsotti P, et al: Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1- MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol 160:673- 680, 2002
53. Mezentsev A, Merks RM, O'Riordan E, et al: Endothelial microparticles affect angiogenesis in vitro: The role of oxidative stress. Am J Physiol Heart Circ Physiol 289:H1106-H1114,2005
54. Mallat Z, Benamer H, Hugel B, et al: Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 101:841- 843, 2000
55. Bernal-Mizrachi L, Jy W, Jimenez JJ, et al: High levels of circulating endothelial microparticles in patients with acute coronary syndromes. Am Heart J 145:962-970,2003
56. Nomura S, Suzuki M, Katsura K, et al: Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus. Atherosclerosis 116:235 - 240, 1995
57. More O, Hugel B, Jesel L, et al: Sustained elevated amounts of circulating procoagulant membrane-microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus. Thromb Haemost 91:345 - 353, 2004
2. Jones JR Intravascular coagulatiow and osteoncrosis. Clin Orthop, 1992, 277 : 41-53
3. Glueck CJ , Freiberg R, Glueck H I, et al. Hypofibrinolysis: a common, major caus of Osteonecrosis. AM J Hematol, 1994, 45:156-166
4. Glueck CJ , Freiberg R, Tracy T, et al. Thrombopilia and hypofibrinolysis. Pathophysioligies of osteonerosis. Clin Orthop, 1997, 334:43-56
5. Wei-li Chen et al. In-Vitro Effects of Dexamethasone on Cellular Proliferation, Apoptosis, and Na+-K+-ATPase Activity of Bovine Corneal Endothelial Cells. Ocular Immunology and Inflammation,2006,14;215-223
6. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tisues. Science, 1997, 276 : 71-74
7. Bersford JN , Bennett JH, Devlin C, et al. Evidence for an inverse ralation ship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci, 1992, 102 : 341-351
8. Thompson D 1, lum KD, N ygaard SC, et al. The derivation and characteri zatvon of stromal cell lines from the bone marrow of p53212 mice: new insights into osteoblast and adipocyte differen tiation. J Bone M iner Res, 1998, 13: 195- 204
9. Cui Q , Wang GJ , Balin G. Stcroid2induccd adipogcncsis ina Pluripotential cell line from bonemarrow. J Bone Joint Surg (Am) , 1997, 79 : 1054-1063
10. Oreffo RO, Virdi A S, Triffitt JT. Modulatior of osteogenesis and adipogenesis by human serum in human bone marrow cultues. Eur J Gell Biol, 1997, 74 : 251 - 261
11. ang GJ. Fat-cell changes as a mechanism of avascular necrosis on the femoral head in cortison - treated rabbits. J Bone Joint Surg (Am) , 1977, 59: 729
12. Martin S, Tesse A, Hugel B, et al: Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation 109:1653- 1659, 2004
13. Brodsky SV, Zhang F, Nasjletti A, et al: Endotheliumderived microparticles impair endothelial function in vitro. Am J Physiol Heart Circ Physiol 286:H1910-H1915, 2004
14. Boulanger CM, Scoazec A, Ebrahimian T, et al: Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 104:2649- 2652, 2001
15. Vanwijk MJ, Svedas E, Boer K, et al: Isolated microparticles,but not whole plasma, from women with preeclampsia impair endothelium-dependent relaxation in isolated myometrialarteries from healthy pregnant women. Am J Obstet Gynecol 187:1686 -1693, 2002
16. Gilbert GE, Sims PJ, Wiedmer T, et al: Platelet-derived microparticles express high affinity receptors for factor VIII. J.Biol Chem 266:17261 - 17268, 1991
17. Hoffman M, Monroe DM, Roberts HR: Coagulation factor IXa binding to activated platelets and platelet-derived microparticles: A flow cytometric study. Thromb Haemost 68:74 - 78, 1992
18. So AK, Varisco PA, Kemkes-Matthes B, et al: Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways. J Thromb Haemost 1:2510-2515, 2003
19. Bogdanov VY, Balasubramanian V, Hathcock J, et al: Alternatively spliced human tissue factor: A circulating, soluble,thrombogenic protein. Nat Med 9:458 - 462, 2003
20. Butenas S, Bouchard BA, Brummel-Ziedins KE, et al:Tissue factor activity in whole blood. Blood 105:2764- 2770,2005
21. Osterud B, Bjorklid E: The production and availability of tissue thromboplastin in cellular populations of whole blood exposed to various concentrations of endotoxin. An assay for detection of endotoxin. Scand J Haematol29:175 - 184, 1982
22. Egorina EM, Sovershaev MA, Bjorkoy G, et al:Intracellular and surface distribution of monocyte tissue factor: Application to intersubject variability. Arterioscler Thromb Vasc.Biol 25:1493- 1498, 2005
23. Mallat Z, Hugel B, Ohan J, et al: Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: A role for apoptosis in plaque thrombogenicity. Circulation 99:348 - 353, 1999
24. Satta N, Toti F, Feugeas O, et al: Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 153:3245- 3255, 1994
25. Combes V, Simon AC, Grau GE, et al: In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest 104:93-102,1999
26. Wang GT. The effect of core decompression on femoral headblood flow in steroid2induced avascular necrosis of the femoral head. J BoneJoint Surg (Am), 1985,67(1): 121
27．李子荣,孙伟,屈辉,等．皮质类固醇与骨坏死关系的临床研究．中华外科杂志,2005．43：104821053
28. Camille,J et al.Microvascular Endothelial Cell Death and Rarefaction in the Glucocortoid-Induced Hypertensive Rat Microcirculation 2001,8 129-139
29. Denise B et al,Endothelium-Drived Microparticles Inhibt Human Cardiac Valve Endothelial Cell Function SHCOKVol 25.No 6 .575-580 .2006
30. Berckmans RJ, Neiuwland R, Boing AN, et al: Cellderived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 85:639 - 646,2001
31. Joop K, Berckmans RJ, Nieuwland R, et al: Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms. Thromb Haemost 85:810 - 820, 2001
32. Simak J, Holada K, Risitano AM, et al: Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria. Br J Haematol 125:804- 813,2004
33. Shet AS, Aras O, Gupta K, et al: Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 102:2678-2683, 2003
34. Diamant M, Nieuwland R, Pablo RF, et al: Elevated numbers of tissue-factor exposing microparticles correlate with components of the metabolic syndrome in uncompli-CELL MICROPARTICLES IN BLOOD 23 cated type 2 diabetes mellitus. Circulation 106:2442- 2447,2002
35. Sabatier F, Darmon P, Hugel B, et al: Type 1 and type 2 diabetic patients display different patterns of cellular microparticles.Diabetes 51:2840-2845,2002
36. Bretelle F, Sabatier F, Desprez D, et al: Circulating microparticles: A marker of procoagulant state in normal pregnancy and pregnancy complicated by preeclampsia or intrauterine growth restriction. Thromb Haemost 89:486 - 492, 2003
37. Matsumoto N, Nomura S, Kamihata H, et al: Increased level of oxidized LDL-dependent monocyte-derived microparticles in acute coronary syndrome. Thromb Haemost 91:146-154, 2004
38. Omoto S, Nomura S, Shouzu A, et al: Detection of monocyte-derived microparticles in patients with type II diabetes mellitus. Diabetologia 45:550 - 555, 2002
39. Jimenez JJ, Jy W, Mauro LM, et al: Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation. Br J Haematol 123:896- 902, 2003
40. Jimenez JJ, Jy W, Mauro LM, et al: Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: Findings from brain and renal microvascular cell culture and patients with active disease. Br J Haematol 112:81 -90,2001
41. Minagar A, Jy W, Jimenez JJ, et al: Elevated plasma endothelial microparticles in multiple sclerosis. Neurology 56:1319- 1324, 2001
42. Shet AS, Aras O, Gupta K, et al: Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 102:2678-2683,2003
43. Horstman LL, Ahn YS: Platelet microparticles: A wideangle perspective. Crit Rev Oncol Hematol 30:111 - 142, 1999
44. Day SM, Reeve JL, Pedersen B, et al: Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood 105:192- 198, 2005
45. Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, et al: Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant. Circulation96:3534- 3541, 1997
46. Biro E, Sturk-Maquelin KN, Vogel GM, et al: Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner. J Thromb Haemost 1:2561 - 2568, 2003
47. Mallat Z, Hugel B, Ohan J, et al: Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: A role for apoptosis in plaque thrombogenicity. Circulation 99:348 - 353, 1999
48. Jy W, Mao WW, Horstman L, et al: Platelet microparticles bind, activate and aggregate neutrophils in vitro. Blood Cells Mol Dis 21:217 - 231, 1995 (discussion 231a)
49. Barry OP, Pratico D, Savani RC, et al: Modulation of monocyte-endothelial cell interactions by platelet microparticles.J Clin Invest 102:136- 144, 1998
50. Barry OP, Pratico D, Lawson JA, et al: Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest 99:2118-2127,1997
51. Brill A, Dashevsky O, Rivo J, et al: Platelet-derived microparticles induce angiogenesis and stimulate post-ischemic revascularization. Cardiovasc Res 67:30- 38, 2005
52. Taraboletti G, D'Ascenzo S, Borsotti P, et al: Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1- MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol 160:673- 680, 2002
53. Mezentsev A, Merks RM, O'Riordan E, et al: Endothelial microparticles affect angiogenesis in vitro: The role of oxidative stress. Am J Physiol Heart Circ Physiol 289:H1106-H1114,2005
54. Mallat Z, Benamer H, Hugel B, et al: Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 101:841- 843, 2000
55. Bernal-Mizrachi L, Jy W, Jimenez JJ, et al: High levels of circulating endothelial microparticles in patients with acute coronary syndromes. Am Heart J 145:962-970,2003
56. Nomura S, Suzuki M, Katsura K, et al: Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus. Atherosclerosis 116:235 - 240, 1995
57. More O, Hugel B, Jesel L, et al: Sustained elevated amounts of circulating procoagulant membrane-microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus. Thromb Haemost 91:345 - 353, 2004