血管内皮细胞微粒对T淋巴细胞功能影响的实验研究
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摘要
目的:研究血管内皮细胞脱落的内皮微粒对体外培养的T淋巴细胞活化增殖、产生细胞因子及膜蛋白表达的影响,探讨内皮微粒对在急性排斥反应中的作用及部分机制。
方法:1.内皮细胞的培养:①人内皮细胞株(Eahy926),用含10 %胎牛血清的高糖DMEM培养基在37℃、5 % CO2、饱和湿度下培养,至Eahy926细胞生长成单层后备用;②大鼠肺微血管内皮细胞(PMVEC)的培养及鉴定:取健康Sprague Dawley (SD)大鼠的外周肺组织,采用组织块贴壁法建立大鼠PMVEC的原代培养技术。采用形态学观察及第Ⅷ因子相关抗原免疫组织化学染色法对所培养的大鼠PMVEC进行鉴定。2.诱导内皮细胞活化产生内皮微粒:以TNF-α(100 ng/ml)刺激Eahy926细胞或第二代大鼠PMVEC,于48 h后收集培养液上清分离细胞微粒。3.内皮微粒的分离及定量测定:采用高速离心法分离所收集培养液上清中的细胞微粒。AnnexinⅤ-FITC荧光染色,激光共聚焦显微镜下观察所分离微粒形态,并采用Bradford法进行定量测定。4.大鼠脾脏淋巴细胞的分离培养:分离健康成年Brown -Norway(BN)大鼠脾脏淋巴细胞并随机分为正常对照组、不同浓度(10、20、40μg/ml)Eahy926细胞来源EMP干预组、ConA刺激组及PMVEC来源EMP(40μg/ml)干预组。5.检测评价EMPs对T淋巴细胞的影响:①采用噻唑兰比色法(MTT法)检测淋巴细胞增值程度;②采用流式细胞术检测T细胞(CD3+淋巴细胞)早期活化分子CD69的表达;③采用ELISA法检测各组细胞培养液上清中相关细胞因子(IFN-γ,IL-2)水平的表达。④采用RT-PCR技术检测各组细胞FasL mRNA的表达。
结果:1.成功培养出状态良好的PMVEC。细胞在倒置显微镜下呈现明显的上皮样细胞形态,细胞聚集成一片,外观呈鹅卵石样或铺路石样镶嵌状排列生长。经免疫组织化学对其内皮特异性第Ⅷ因子相关抗原呈阳性表达,证明成功培养出了高纯度的PMVEC。2.EMP的诱生及其分离鉴定结果:经TNF-α刺激,内皮细胞因活化而产生大量内皮微粒。激光共聚焦显微镜观察,微粒因与AnnexinⅤ-FITC结合而呈绿色囊泡状外观。我们采用Bradford法定量所分离EMP,以蛋白量计,EMP浓度在0.5~1μg/μl之间。3.淋巴细胞活化增殖测定:倒置显微镜直接观察,各EMP干预组及ConA组细胞都有较高程度的聚集生长,较对照组增殖旺盛;MTT结果显示:与对照组相比较,各Eahy926细胞来源EMP干预组、ConA组、PMVEC来源EMP干预组细胞代谢活力均显著提高(P<0.05);且与ConA组比较,EMP高浓度组、PMVEC来源EMP干预组细胞代谢活力的增高无显著差异(P>0.05)。4.T细胞早期活化标志分子CD69的表达:流式细胞术的检测结果显示,EMP各浓度组、ConA组、PMVEC来源EMP干预组CD3+细胞CD69分子的表达较对照组均明显增高(P<0.01);ConA组CD3+细胞CD69分子表达显著高于其他各组(P<0.01)。5. ELISA结果显示:与对照组相比较,EMP各浓度组、ConA组、PMVEC来源EMP干预组培养液上清IFN-γ水平较对照组有显著增高(P<0.05),且EMP各浓度组间具有明显的效量关系;而与对照组相比较,EMP中浓度组、EMP高浓度组、ConA组及PMVEC来源EMP干预组培养液上清IL-2水平显著增高(P<0.05),EMP低浓度组较空白对照组IL-2增高则无显著性差异(P>0.05)。6.FasL mRNA水平的表达:与对照组相比,EMP中浓度组、EMP高浓度组、ConA组及PMVEC来源EMP干预组FasL mRNA表达水平显著增高(P<0.01);ConA组FasL mRNA的表达增高较各EMP干预组具有显著差异(P<0.05);且EMP高浓度组与PMVEC来源EMP干预组细胞FasL mRNA表达无显著性差异(P>0.05)。
结论:研究发现Eahy926细胞来源EMP及原代PMVEC来源EMP均可诱导体外培养的淋巴细胞活化增殖,诱导T细胞早期活化分子CD69的迅速表达,且细胞的活化程度与EMP干预浓度有关。另外,EMP诱导活化的T细胞分泌IL-2、IFN-γ等炎性细胞因子并高表达FasL。体内这些炎性因子及重要分子作用于免疫细胞及靶细胞,在机体免疫应答如排斥反应中发挥着重要作用。EMP可能是免疫调节性治疗的一个重要靶点。
Objective: To study the effects of vascular endothelial microparticles (EMP) on the activation and proliferation of spleen lymphocytes, to analyze the alterations of its phenotype and released cytokines. And to study the effects of EMP on acute rejection.
Methods: 1 The culture of endothelial cells(ECs):①Eahy926 cells, a human endothelial hybrid cell line,were cultured in a modified Eagle’s Minimum Essential Medium (acc. to Dulbecco) containing with 10 % Fetal bovine serum (FBS). Cells were maintained in 5 % CO2 atmosphere and saturated humidity at 37℃, for reaching subconfluent monolayers.②The culture and identification of rat pulmonary microvascular endothelial cells(PMVEC):PMVECs were obtained from lung tissue of male Sprague Dawley (SD) rats by tissue block pasted culture method. The cultured cells were identified by cell morphology observation and immuneohistochemistical staining. 2 Generation of EMPs from endothelial cells: Confluent Eahy926 cells or PMVECs were incubated for 48 h with 100 ng/ml TNF-α, then the culture supernatants were collected. 3 The isolation and quantification of EMPs: The collected supernatants were then centrifuged at different speeds to get EMPs. After fluorescence staining, EMPs were observed by confocal microscopy. Pelleted EMPs were determined by Bradford method, and used immediately. 4 The culture of rat lymphocytes: lymphocytes were isolated from spleen of Brown-Norway (BN) rats.The cultured lymphocytes were divided randomly into control group, intervention groups of EMPs derived from Eahy926 cells at different concentrations (10, 20, 40μg/ml), ConA group and intervention group of EMPs derived from PMVECs (40μg/ml). 5 To evaluate the effect of EMPs on T lymphocytes:①The proliferation and metabolic activity assay of lymphocytes was measured by MTT.②The activation marker CD69 of CD3+ lymphocytes was measured by flow cytometer.③Cytokines such as IL-2 and IFN-γwere measured by enzyme linked immunosorbent assay (ELISA).④The expression of FasL mRNA in T lymphocytes was measured by RT-PCR.
Results: 1 The culture of endothelial cells:①The normal morphology of Eahy926 cells were observed by convert microscope, the cells showed productive growth condition, polygonal or spindle epithelial-like shape adhered to the plate.②PMVECs were isolated by tissue block pasted culture method. The cultured PMVECs of rats in vitro displayed a typical cobble stone or paving stone like morphology, and the PMVECs expressed factorⅧassociated antigen. 2 Generation and identification of EMPs: EMPs were released from ECs after stimulating with TNF-αfor 48 hours. EMPs were stained with AnnexinⅤ-FITC, and green distributed spots were observed by confocal microscopy. EMPs were quantified by Bradford method, and the concentration was 0.5~1μg/μl. 3 The proliferation and metabolic activity assay of lymphocytes: After incubation,the lymphocytes of all EMPs intervention groups and ConA group show more productive growth condition observed under convert microscope than that of control group. MTT assay showed: Compared with control group, the lymphocytes vitality of each EMPs intervention group and ConA group was increased remarkably(P<0.05); No significant difference of the lymphocytes vitality was observed in high EMPs concentration group,EMPs derived from PMVECs intervention group and ConA group (P>0.05). 4 The expression of early activation marker CD69: Compared with control group, the expression of CD69 on CD3+ cell in each EMPs intervention group and ConA Group was increased remarkably(P<0.01); The expression of CD69 in ConA group was higher than other groups significantly(P<0.01). 5 ELISA assay: Compared with control group, the level of IFN-γin culture supernatant of each EMPs intervention group and ConA group was increased remarkably(P<0.05), significant difference of IFN-γexpression was observed in each EMPs intervention group; Compared with control group, the level of IL-2 was increased in culture supernatant of the high and intermediate EMPs concentration group, ConA group, EMP derived from PMVEC intervention group (P<0.05); But compared with control group, no significant increase of IL-2 was observed in low concentration group(P>0.05). 6 The expression of FasL mRNA:Compared with control group, the express levels of FasL mRNA was increased remarkably in the high and intermediate EMPs concentration group,ConA group,the EMP derived from PMVEC intervention group(P<0.01); The level of FasL mRNA in ConA group was increased significantly compared with each EMP intervention group (P<0.05); No significant difference of FasL mRNA expression was observed between the high EMPs concentration group and EMP derived from PMVEC intervention group(P>0.05).
Conclusions: Our data establishes that EMPs derived from both Eahy926 cells and rat pulmonary microvascular endothelial cells induce T lymphocyte activation and expression of early activation marker CD69, and the effect is related to the intervention concentration of EMPs. The activated T cells produce inflammatory cytokines such as IL-2 and IFN-γ, and express FasL highly. These cytokines and molecules play an important part in immune response in vivo.Endothelial microparticles may be an important immunomodulatory therapeutic target.
方法:1.内皮细胞的培养:①人内皮细胞株(Eahy926),用含10 %胎牛血清的高糖DMEM培养基在37℃、5 % CO2、饱和湿度下培养,至Eahy926细胞生长成单层后备用;②大鼠肺微血管内皮细胞(PMVEC)的培养及鉴定:取健康Sprague Dawley (SD)大鼠的外周肺组织,采用组织块贴壁法建立大鼠PMVEC的原代培养技术。采用形态学观察及第Ⅷ因子相关抗原免疫组织化学染色法对所培养的大鼠PMVEC进行鉴定。2.诱导内皮细胞活化产生内皮微粒:以TNF-α(100 ng/ml)刺激Eahy926细胞或第二代大鼠PMVEC,于48 h后收集培养液上清分离细胞微粒。3.内皮微粒的分离及定量测定:采用高速离心法分离所收集培养液上清中的细胞微粒。AnnexinⅤ-FITC荧光染色,激光共聚焦显微镜下观察所分离微粒形态,并采用Bradford法进行定量测定。4.大鼠脾脏淋巴细胞的分离培养:分离健康成年Brown -Norway(BN)大鼠脾脏淋巴细胞并随机分为正常对照组、不同浓度(10、20、40μg/ml)Eahy926细胞来源EMP干预组、ConA刺激组及PMVEC来源EMP(40μg/ml)干预组。5.检测评价EMPs对T淋巴细胞的影响:①采用噻唑兰比色法(MTT法)检测淋巴细胞增值程度;②采用流式细胞术检测T细胞(CD3+淋巴细胞)早期活化分子CD69的表达;③采用ELISA法检测各组细胞培养液上清中相关细胞因子(IFN-γ,IL-2)水平的表达。④采用RT-PCR技术检测各组细胞FasL mRNA的表达。
结果:1.成功培养出状态良好的PMVEC。细胞在倒置显微镜下呈现明显的上皮样细胞形态,细胞聚集成一片,外观呈鹅卵石样或铺路石样镶嵌状排列生长。经免疫组织化学对其内皮特异性第Ⅷ因子相关抗原呈阳性表达,证明成功培养出了高纯度的PMVEC。2.EMP的诱生及其分离鉴定结果:经TNF-α刺激,内皮细胞因活化而产生大量内皮微粒。激光共聚焦显微镜观察,微粒因与AnnexinⅤ-FITC结合而呈绿色囊泡状外观。我们采用Bradford法定量所分离EMP,以蛋白量计,EMP浓度在0.5~1μg/μl之间。3.淋巴细胞活化增殖测定:倒置显微镜直接观察,各EMP干预组及ConA组细胞都有较高程度的聚集生长,较对照组增殖旺盛;MTT结果显示:与对照组相比较,各Eahy926细胞来源EMP干预组、ConA组、PMVEC来源EMP干预组细胞代谢活力均显著提高(P<0.05);且与ConA组比较,EMP高浓度组、PMVEC来源EMP干预组细胞代谢活力的增高无显著差异(P>0.05)。4.T细胞早期活化标志分子CD69的表达:流式细胞术的检测结果显示,EMP各浓度组、ConA组、PMVEC来源EMP干预组CD3+细胞CD69分子的表达较对照组均明显增高(P<0.01);ConA组CD3+细胞CD69分子表达显著高于其他各组(P<0.01)。5. ELISA结果显示:与对照组相比较,EMP各浓度组、ConA组、PMVEC来源EMP干预组培养液上清IFN-γ水平较对照组有显著增高(P<0.05),且EMP各浓度组间具有明显的效量关系;而与对照组相比较,EMP中浓度组、EMP高浓度组、ConA组及PMVEC来源EMP干预组培养液上清IL-2水平显著增高(P<0.05),EMP低浓度组较空白对照组IL-2增高则无显著性差异(P>0.05)。6.FasL mRNA水平的表达:与对照组相比,EMP中浓度组、EMP高浓度组、ConA组及PMVEC来源EMP干预组FasL mRNA表达水平显著增高(P<0.01);ConA组FasL mRNA的表达增高较各EMP干预组具有显著差异(P<0.05);且EMP高浓度组与PMVEC来源EMP干预组细胞FasL mRNA表达无显著性差异(P>0.05)。
结论:研究发现Eahy926细胞来源EMP及原代PMVEC来源EMP均可诱导体外培养的淋巴细胞活化增殖,诱导T细胞早期活化分子CD69的迅速表达,且细胞的活化程度与EMP干预浓度有关。另外,EMP诱导活化的T细胞分泌IL-2、IFN-γ等炎性细胞因子并高表达FasL。体内这些炎性因子及重要分子作用于免疫细胞及靶细胞,在机体免疫应答如排斥反应中发挥着重要作用。EMP可能是免疫调节性治疗的一个重要靶点。
Objective: To study the effects of vascular endothelial microparticles (EMP) on the activation and proliferation of spleen lymphocytes, to analyze the alterations of its phenotype and released cytokines. And to study the effects of EMP on acute rejection.
Methods: 1 The culture of endothelial cells(ECs):①Eahy926 cells, a human endothelial hybrid cell line,were cultured in a modified Eagle’s Minimum Essential Medium (acc. to Dulbecco) containing with 10 % Fetal bovine serum (FBS). Cells were maintained in 5 % CO2 atmosphere and saturated humidity at 37℃, for reaching subconfluent monolayers.②The culture and identification of rat pulmonary microvascular endothelial cells(PMVEC):PMVECs were obtained from lung tissue of male Sprague Dawley (SD) rats by tissue block pasted culture method. The cultured cells were identified by cell morphology observation and immuneohistochemistical staining. 2 Generation of EMPs from endothelial cells: Confluent Eahy926 cells or PMVECs were incubated for 48 h with 100 ng/ml TNF-α, then the culture supernatants were collected. 3 The isolation and quantification of EMPs: The collected supernatants were then centrifuged at different speeds to get EMPs. After fluorescence staining, EMPs were observed by confocal microscopy. Pelleted EMPs were determined by Bradford method, and used immediately. 4 The culture of rat lymphocytes: lymphocytes were isolated from spleen of Brown-Norway (BN) rats.The cultured lymphocytes were divided randomly into control group, intervention groups of EMPs derived from Eahy926 cells at different concentrations (10, 20, 40μg/ml), ConA group and intervention group of EMPs derived from PMVECs (40μg/ml). 5 To evaluate the effect of EMPs on T lymphocytes:①The proliferation and metabolic activity assay of lymphocytes was measured by MTT.②The activation marker CD69 of CD3+ lymphocytes was measured by flow cytometer.③Cytokines such as IL-2 and IFN-γwere measured by enzyme linked immunosorbent assay (ELISA).④The expression of FasL mRNA in T lymphocytes was measured by RT-PCR.
Results: 1 The culture of endothelial cells:①The normal morphology of Eahy926 cells were observed by convert microscope, the cells showed productive growth condition, polygonal or spindle epithelial-like shape adhered to the plate.②PMVECs were isolated by tissue block pasted culture method. The cultured PMVECs of rats in vitro displayed a typical cobble stone or paving stone like morphology, and the PMVECs expressed factorⅧassociated antigen. 2 Generation and identification of EMPs: EMPs were released from ECs after stimulating with TNF-αfor 48 hours. EMPs were stained with AnnexinⅤ-FITC, and green distributed spots were observed by confocal microscopy. EMPs were quantified by Bradford method, and the concentration was 0.5~1μg/μl. 3 The proliferation and metabolic activity assay of lymphocytes: After incubation,the lymphocytes of all EMPs intervention groups and ConA group show more productive growth condition observed under convert microscope than that of control group. MTT assay showed: Compared with control group, the lymphocytes vitality of each EMPs intervention group and ConA group was increased remarkably(P<0.05); No significant difference of the lymphocytes vitality was observed in high EMPs concentration group,EMPs derived from PMVECs intervention group and ConA group (P>0.05). 4 The expression of early activation marker CD69: Compared with control group, the expression of CD69 on CD3+ cell in each EMPs intervention group and ConA Group was increased remarkably(P<0.01); The expression of CD69 in ConA group was higher than other groups significantly(P<0.01). 5 ELISA assay: Compared with control group, the level of IFN-γin culture supernatant of each EMPs intervention group and ConA group was increased remarkably(P<0.05), significant difference of IFN-γexpression was observed in each EMPs intervention group; Compared with control group, the level of IL-2 was increased in culture supernatant of the high and intermediate EMPs concentration group, ConA group, EMP derived from PMVEC intervention group (P<0.05); But compared with control group, no significant increase of IL-2 was observed in low concentration group(P>0.05). 6 The expression of FasL mRNA:Compared with control group, the express levels of FasL mRNA was increased remarkably in the high and intermediate EMPs concentration group,ConA group,the EMP derived from PMVEC intervention group(P<0.01); The level of FasL mRNA in ConA group was increased significantly compared with each EMP intervention group (P<0.05); No significant difference of FasL mRNA expression was observed between the high EMPs concentration group and EMP derived from PMVEC intervention group(P>0.05).
Conclusions: Our data establishes that EMPs derived from both Eahy926 cells and rat pulmonary microvascular endothelial cells induce T lymphocyte activation and expression of early activation marker CD69, and the effect is related to the intervention concentration of EMPs. The activated T cells produce inflammatory cytokines such as IL-2 and IFN-γ, and express FasL highly. These cytokines and molecules play an important part in immune response in vivo.Endothelial microparticles may be an important immunomodulatory therapeutic target.
引文
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24 Yousef GM, Diamandis EP. An overview of the kallikrein gene families in humans and other species: emerging candidate tumour markers[J]. Clin biochem, 2003, 36(6): 443-452
25王惠明,王沂芹,阮志华,等. IFN-γ上调的单核细胞表面MHC-I类链相关分子促进NK细胞活化[J].细胞与分子免疫学杂志, 2008, 24(4): 413-418
26 Kelliher MA, Grimm S, Ishida Y, Kuo F, et al. The death domain kinase RIP mediates the TNF induced NF-kappaB signal[J]. Immunity, 1998, 8(3): 297-303
27 Tian L, Qu X, Wang ME, et al. Selective inhibition of IL-2 mRNA blocks allograft rejection by limiting T cell clonal expansion[J]. Transplant Proc, 2001, 33 (1-2):330
28 Bernard A, Lamy, Alberti I. The two signal model of T-cel1 activation after
30 years[J]. Transplantation, 2002, 73(1 supply): S31-35
29 Dugre FJ, Gaudregu S, Belles-Isles M, et al. Cytokine and cytotoxic molecule gene expression determined in peripheral blood mononuclear cells in the diagnosis of acute renal rejection[J]. Transplantation, 2000, 70(7): 1074-1080
30 Matsuno T, Sasaki H, Nakagawa K, et al. Expression of Fas/Fas ligand and apoptosis induction during renal allograft rejection[J]. Transplantation Proceedings, 1998, 30(7): 2947-2949
31姜英,王业华,贾筱琴,等.外周血淋巴细胞FasL在大鼠肾移植急性排斥的表达及意义[J].实用临床医药杂志, 2005, 9(9): 71-82
32 Richardson BC, Lalwani ND, Johnson KJ, et al. Fas ligation triggers apoptosis in macrophages but not endothelial cells[J]. Eur J Immunol, 1994, 24(11): 2640-2645
1 Enjeti AK, Lincz LF, Seldon M. Detection and measurement of microparticles: An evolving research tool for vascular biology[J]. Semin Thromb Hemost, 2007, 33(8): 771-779
2 Kereiakes DJ, Michelson AD. Platelet activation and progression to complications[J]. Rev Cardiovasc Med, 2006, 7(2): 75-81
3 Freyssinet JM. Cellular microparticles: what are they bad or good for?[J]. Thromb Haemost, 2003, 1(7): 1655-1662
4 Horstman LL, Jy W, Jimenez JJ, et al. Endothelial microparticles as markers of endothelial dysfunction[J]. Front Biosci, 2004, 9: 1118-1135
5 Mackman N. Role of tissue factor in hemostasis and thrombosis [J]. Blood Cells Mol Dis, 2006, 36(2): 104-107
6 Raybagkar DA, Patchipulusu S, Mast AE, et al. In vitro ?ow evaluation of recombinant tissue factor pathway inhibitor immobilized on collagen impregnated Dacron[J]. ASAIO J, 2004, 50(4): 301-305
7 Martinez MC, Tesse A, Zobairi F, et al. Shed membrane microparticles from circulating and vascular cells in regulating vascular function[J]. Am J Physiol Heart Circ Physiol, 2005, 288(3): H1004-H1009
8 Biro E, Sturk-Maquelin KN, Voqel GM, et al. Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor -dependent manner[J]. Thromb Haemost, 2003, 1(12): 2561-2568
9 Koga H, Sugiyama S, Kugiyama K, et al. Elevated levels of remnant lipoproteins are associated with plasma platelet microparticles in patients with type-2 diabetes mellitus without obstructive coronary artery disease[J]. Eur Heart, 2006, 27(7): 817-823
10 Gupta AK, Hasler P, Holzgreve W, et al. Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia[J]. Hum Immunol, 2005, 66(11): 1146-1154
11 Gross PL, Furie BC, Merrill-Skoloff G, et al. Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombusdevelopment[J]. Leukoc Biol, 2005, 78(6): 1318-1326
12 Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis[J]. Scand J Immunol, 2007, 66(2-3): 159-165
13 Faye RS, Aamdal S, Hoifodt HK, et al. Immunomagnetic detection and clinical significance of micrometastatic tumor cells in malignant melanoma patients[J]. Clin Cancer Res, 2004, 10(12 pt 1): 4134-4139
14 Bruland OS, Hoifodt H, Saeter G, et al. Hematogenous micrometastases in osteosarcoma patients[J]. Clin Cancer Res, 2005, 11(13): 4666-4673
15 Gilles N, Chironi, Chantal M, et al. Endothelial microparticles in diseases[J]. Cell Tissue Res, 2009, 335(1): 143-151
16 Dale GL, Remenyi G, Friese P. Quantitation of microparticles released from coated-platelets[J]. Thromb Haemost, 2005, 3(9): 2081-2088
17 Horstman LL, Jy W, Jimenez JJ, et al. New horizons in the analysis of circulating cell derived microparticles[J]. Keio J Med, 2004, 53(4): 210-230
18 Jy W, Horstman LL, Jimenez JJ, et al. Measuring circulating cell-derived microparticles[J]. Thromb Haemost, 2004, 2(10): 1842-1851
19 Exner T, Joseph J, Low J, et al. A new activated factor X-based clotting method with improved specificity for procoagulant phospholipids [J]. Blood Coagul Fibrinolysis, 2003, 14(8): 773-779
20 Enjeti AK, Lisa L, Seldon M. Bio-Maleimide as a generic stain for detection and quantitation of microparticles[J]. Int J Lab Haematol , 2008, 30(3): 196-199
21 Banfi C, Brioschi M, Wait R, et al. Proteome of endothelial cell-derived procoagulant microparticles[J]. Proteomics, 2005, 5(17): 4443-4455
22 Mona D, Angela L, Jing-Fei Dong, et al. Flow cytometric measurement of microparticles: Pitfalls and protocol modifications[J]. Platelets, 2008, 19(5): 365-372
23 Kim HK, Song KS, Lee ES, et al. Optimized ?ow cytometric assay for the measurement of platelet microparticles inplasma: pre-analytic and analyticconsiderations[J]. Blood Coagul Fibrinolysis, 2002, 13(5):393-397
24 Robert S, Poncelet P, Camoin L, et al. Standardization of microparticle counting using ?ow cytometry: new options and tools for the control of criticalinstrument settings[J]. Thromb Haemost 2007, 5(Suppl 2): P-M-442
2 Fanny A, Estelle S, Sabeha B, et al. Endothelial cell -derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases[J]. Haematologica, 2009, 94(11), 1502-1512
3柏桦,海春旭,张晓迪,等.组织块法培养大鼠肺微血管内皮细胞的方法研究[J].科学技术与工程, 2008, 8(5): 1160-1164
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5 Banfi C, Brioschi M, Wait R, et al. Proteome of endothelial cell-derived procoagulant microparticles[J]. Proteomics, 2005, 5(17): 4443-4455
6 Basse F, Gaffet P, Bienvenue A. Correlation between inhibition of cytoskeleton proteolysis and anti-vesiculation effect of calpeptin during A238177 induced activation of human platelets: are vesicles shed by filopod fragmentation?[J]. Biochim Biophys Acta, 1994, 1190(2): 217-224
7王昌富,李琳芸.血液中微粒的形成及生物学活性[J].循环学杂志, 2006, 16(1): 59-61
8 Kereiakes DJ, Michelson AD. Platelet activation and progression to complications[J]. Rev Cardiovasc Med, 2006(7): 75-81
9 Freyssinet JM. Cellular microparticles: what are they bad or good for?[J]. Thromb Haemost, 2003(1): 655-662
10 Preston RA, JYW, Jimenez JJ, et a1. Effects of severe hypertension on endothelial and platelet microparticles[J]. Hypertension, 2003, 41(2): 211-217
11 Gonzalez-Quintero VH, Jimenez JJ, Jy W, et a1. Elevated plasma endothelial microparticles in preeclampsia[J]. Obstet Gynecol, 2003,189(2): 589-593
12 Jimenez JJ, Jy W, Mauro LM, et al. Endothelial microparticles(EMP) as vascular disease markers[J]. Adv Clin Chem, 2005, 39: 131-157
13 Arteaga RB, Chirinos JA, Soriano AO, et al. Endothelial microparticles and platelet and leukocyte activation in patients with the metabolic syndrome. Am J Cardiol, 2006, 98(1): 70-74
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15武焕玲.内皮细胞微颗粒的研究进展[J].国际检验医学杂志, 2006, 27(7):644-647
16徐艳,郑永唐,何黎,等.昆明山海棠片对小鼠淋巴细胞体外增殖活化影响的研究[J].中药材, 2008, 31(4): 557-561
17 Craston R, Koh M, Dermott AM, et al. Temporal dynamics of CD69 expression only mphoid cells. Immuno Methods[J], 1997, 10(209): 37-45
18 Testi R, Ambrosio D, Maria R, et al. The CD69 receptor: a multipurpose cell surface trigger for hematopoitic cells[J]. Immunol Today, 1994, 15(10): 479-483
19 Poosselt AM, Vincenti F, Bedolli M, et al. CD69 espression on peripheral CD8 T cells correlates with acute rejection in renal transplant recipients[J]. Transplantation, 2003, 15:76(1): 190-195
20 Schowengerdt KO, Fricker FJ, Bahjat KS, et al. Increased expression of the lymphocyte early activation marker CD69 in peripheral blood correlates with histologic evidenced of cardiac allograft rejection[J]. Transplantation, 2000, 27:69(10): 2102-2107
21钟敬祥,徐锦堂,郭萌,等.角膜移植患者外周血T细胞及其亚群CD69分子表达研究[J].广东医学, 2005, 26(5): 620-621
22 Vincent-Schneider H, Stumptner-Cuvelette P, Lankar D, et al. Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells[J]. Int Immunol, 2002, 14(7): 713-722
23 Nolte-'t Hoen EN, Wagenaar-Hilbers JP, Peters PJ, et al. Uptake of membrane molecules from T cells endows antigen-presenting cells with novel functional properties[J]. Eur J Immunol, 2004, 34(11): 3115-3125
24 Yousef GM, Diamandis EP. An overview of the kallikrein gene families in humans and other species: emerging candidate tumour markers[J]. Clin biochem, 2003, 36(6): 443-452
25王惠明,王沂芹,阮志华,等. IFN-γ上调的单核细胞表面MHC-I类链相关分子促进NK细胞活化[J].细胞与分子免疫学杂志, 2008, 24(4): 413-418
26 Kelliher MA, Grimm S, Ishida Y, Kuo F, et al. The death domain kinase RIP mediates the TNF induced NF-kappaB signal[J]. Immunity, 1998, 8(3): 297-303
27 Tian L, Qu X, Wang ME, et al. Selective inhibition of IL-2 mRNA blocks allograft rejection by limiting T cell clonal expansion[J]. Transplant Proc, 2001, 33 (1-2):330
28 Bernard A, Lamy, Alberti I. The two signal model of T-cel1 activation after
30 years[J]. Transplantation, 2002, 73(1 supply): S31-35
29 Dugre FJ, Gaudregu S, Belles-Isles M, et al. Cytokine and cytotoxic molecule gene expression determined in peripheral blood mononuclear cells in the diagnosis of acute renal rejection[J]. Transplantation, 2000, 70(7): 1074-1080
30 Matsuno T, Sasaki H, Nakagawa K, et al. Expression of Fas/Fas ligand and apoptosis induction during renal allograft rejection[J]. Transplantation Proceedings, 1998, 30(7): 2947-2949
31姜英,王业华,贾筱琴,等.外周血淋巴细胞FasL在大鼠肾移植急性排斥的表达及意义[J].实用临床医药杂志, 2005, 9(9): 71-82
32 Richardson BC, Lalwani ND, Johnson KJ, et al. Fas ligation triggers apoptosis in macrophages but not endothelial cells[J]. Eur J Immunol, 1994, 24(11): 2640-2645
1 Enjeti AK, Lincz LF, Seldon M. Detection and measurement of microparticles: An evolving research tool for vascular biology[J]. Semin Thromb Hemost, 2007, 33(8): 771-779
2 Kereiakes DJ, Michelson AD. Platelet activation and progression to complications[J]. Rev Cardiovasc Med, 2006, 7(2): 75-81
3 Freyssinet JM. Cellular microparticles: what are they bad or good for?[J]. Thromb Haemost, 2003, 1(7): 1655-1662
4 Horstman LL, Jy W, Jimenez JJ, et al. Endothelial microparticles as markers of endothelial dysfunction[J]. Front Biosci, 2004, 9: 1118-1135
5 Mackman N. Role of tissue factor in hemostasis and thrombosis [J]. Blood Cells Mol Dis, 2006, 36(2): 104-107
6 Raybagkar DA, Patchipulusu S, Mast AE, et al. In vitro ?ow evaluation of recombinant tissue factor pathway inhibitor immobilized on collagen impregnated Dacron[J]. ASAIO J, 2004, 50(4): 301-305
7 Martinez MC, Tesse A, Zobairi F, et al. Shed membrane microparticles from circulating and vascular cells in regulating vascular function[J]. Am J Physiol Heart Circ Physiol, 2005, 288(3): H1004-H1009
8 Biro E, Sturk-Maquelin KN, Voqel GM, et al. Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor -dependent manner[J]. Thromb Haemost, 2003, 1(12): 2561-2568
9 Koga H, Sugiyama S, Kugiyama K, et al. Elevated levels of remnant lipoproteins are associated with plasma platelet microparticles in patients with type-2 diabetes mellitus without obstructive coronary artery disease[J]. Eur Heart, 2006, 27(7): 817-823
10 Gupta AK, Hasler P, Holzgreve W, et al. Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia[J]. Hum Immunol, 2005, 66(11): 1146-1154
11 Gross PL, Furie BC, Merrill-Skoloff G, et al. Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombusdevelopment[J]. Leukoc Biol, 2005, 78(6): 1318-1326
12 Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis[J]. Scand J Immunol, 2007, 66(2-3): 159-165
13 Faye RS, Aamdal S, Hoifodt HK, et al. Immunomagnetic detection and clinical significance of micrometastatic tumor cells in malignant melanoma patients[J]. Clin Cancer Res, 2004, 10(12 pt 1): 4134-4139
14 Bruland OS, Hoifodt H, Saeter G, et al. Hematogenous micrometastases in osteosarcoma patients[J]. Clin Cancer Res, 2005, 11(13): 4666-4673
15 Gilles N, Chironi, Chantal M, et al. Endothelial microparticles in diseases[J]. Cell Tissue Res, 2009, 335(1): 143-151
16 Dale GL, Remenyi G, Friese P. Quantitation of microparticles released from coated-platelets[J]. Thromb Haemost, 2005, 3(9): 2081-2088
17 Horstman LL, Jy W, Jimenez JJ, et al. New horizons in the analysis of circulating cell derived microparticles[J]. Keio J Med, 2004, 53(4): 210-230
18 Jy W, Horstman LL, Jimenez JJ, et al. Measuring circulating cell-derived microparticles[J]. Thromb Haemost, 2004, 2(10): 1842-1851
19 Exner T, Joseph J, Low J, et al. A new activated factor X-based clotting method with improved specificity for procoagulant phospholipids [J]. Blood Coagul Fibrinolysis, 2003, 14(8): 773-779
20 Enjeti AK, Lisa L, Seldon M. Bio-Maleimide as a generic stain for detection and quantitation of microparticles[J]. Int J Lab Haematol , 2008, 30(3): 196-199
21 Banfi C, Brioschi M, Wait R, et al. Proteome of endothelial cell-derived procoagulant microparticles[J]. Proteomics, 2005, 5(17): 4443-4455
22 Mona D, Angela L, Jing-Fei Dong, et al. Flow cytometric measurement of microparticles: Pitfalls and protocol modifications[J]. Platelets, 2008, 19(5): 365-372
23 Kim HK, Song KS, Lee ES, et al. Optimized ?ow cytometric assay for the measurement of platelet microparticles inplasma: pre-analytic and analyticconsiderations[J]. Blood Coagul Fibrinolysis, 2002, 13(5):393-397
24 Robert S, Poncelet P, Camoin L, et al. Standardization of microparticle counting using ?ow cytometry: new options and tools for the control of criticalinstrument settings[J]. Thromb Haemost 2007, 5(Suppl 2): P-M-442