【作者】 刘长营；

【导师】 孙奕；

【作者基本信息】 河北医科大学 ， 免疫学， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原