血管内皮细胞微粒对T淋巴细胞功能影响的实验研究

发布时间：2018-05-20 08:14

本文选题：内皮微粒 + 内皮细胞　；参考：《河北医科大学》2010年硕士论文

【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(P0.05);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(P0.05)。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(P0.01);ConA组CD3+细胞CD69分子表达显著高于其他各组(P0.01)。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(P0.05),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(P0.05),EMP低浓度组较空白对照组IL-2增高则无显著性差异(P0.05)。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(P0.01);ConA组FasL mRNA的表达增高较各EMP干预组具有显著差异(P0.05);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(P0.05)。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。
[Abstract]:Objective: To study the effect of endothelial microparticles off endothelial cells on the activation and proliferation of T lymphocytes in vitro, to produce cytokines and membrane protein expression, and to explore the role and mechanism of endothelial particles in the acute rejection.
Methods: 1. the culture of endothelial cells: 1 human endothelial cell line (Eahy926), with high glucose DMEM medium containing 10% fetal bovine serum at 37, 5% CO2, under saturated humidity, to Eahy926 cells to grow into single layer; (2) the culture and identification of rat lung microvascular endothelial cells (PMVEC): the peripheral lung tissues of healthy Sprague Dawley (SD) rats were taken. The primary culture technique of rat PMVEC was established by tissue block adherence method. The cultured rat PMVEC was identified by morphological observation and immuno histochemical staining of factor VIII related antigen..2. induced endothelial cells were activated by.2. induced endothelial cells: TNF- alpha (100 ng/ml) was used to stimulate Eahy926 cells or PMVEC in second generation rats and to be collected after 48 h. The separation and quantitative determination of.3. endothelial particles in the supernatant cell particles of the collection culture liquid supernatant: using high speed centrifugation to separate the cell particles from the supernatant of the cultured liquid,.Annexin V -FITC fluorescence staining, the morphology of the particles separated by the confocal laser scanning microscope, and the quantitative determination of the spleen lymphocytes of the.4. rats by Bradford method. Isolation and culture: isolation of spleen lymphocytes from healthy adult Brown -Norway (BN) rats and randomly divided into normal control group, EMP intervention group of different concentrations (10,20,40 mu g/ml) Eahy926 cells, ConA stimulation group and PMVEC source EMP (40 mu g/ml) intervention group. The degree of lymphocyte increment; (2) the expression of early activation molecule CD69 of T cells (CD3+ lymphocyte) was detected by flow cytometry; and ELISA was used to detect the expression of related cytokines (IFN-, IL-2) in the supernatant of cell culture liquid. (4) the expression of FasL mRNA in each group was detected by RT-PCR technique.
Results: 1. the successful cultured PMVEC. cells showed obvious epithelioid cell morphology under the inverted microscope. The cells aggregated a piece, and the appearance was cobblestone like or paved stone like arrangement. The immuno histochemistry showed positive expression of the specific factor VIII related antigen of its inner skin, which proved to be highly cultured. The purity of PMVEC.2.EMP induced and its isolation and identification results: the endothelial cells produced a large number of endothelial particles by TNF- alpha stimulation. The laser confocal microscope observed that the particles were green vesicles with Annexin V -FITC. We used the Bradford statutory quantity to separate EMP, the protein quantity, and the EMP concentration between 0.5~1 and g/ micron L. .3. lymphocyte activation proliferation assay: direct observation of inverted microscope, EMP intervention group and ConA group cells have a high degree of aggregation growth, more proliferation than the control group, MTT results showed that: compared with the control group, the Eahy926 cell source EMP intervention group, ConA group, PMVEC source EMP intervention group significantly increased cell metabolism activity (P0.05); And compared with the ConA group, there was no significant difference in the cell metabolism activity in the EMP high concentration group and the PMVEC source EMP intervention group (P0.05), the expression of CD69 in the early activation marker of.4.T cells: the results of flow cytometry showed that the expression of CD3+ cells in EMP concentration group, ConA group and PMVEC source EMP intervention group was significantly higher than that of the control group. .01); the expression of CD69 molecule in CD3+ cells in group ConA was significantly higher than that of other groups (P0.01).5. ELISA results: compared with the control group, the level of IFN- gamma in each concentration group of EMP, ConA group, PMVEC source of EMP intervention group was significantly higher than that of the control group, and there was a significant relationship between the concentration groups, and compared with the control group. The level of IL-2 in medium concentration group, EMP high concentration group, ConA group and PMVEC source EMP intervention group increased significantly (P0.05), and there was no significant difference (P0.05).6.FasL mRNA level in the EMP low concentration group than that in the blank control group (P0.05). The expression level was significantly higher (P0.01), and the increased expression of FasL mRNA in group ConA was significantly higher than that in the EMP intervention group (P0.05), and there was no significant difference between the EMP high concentration group and the FasL mRNA expression in the EMP intervention group of PMVEC source (P0.05).
Conclusion: it is found that Eahy926 cell source EMP and primary PMVEC source EMP can induce lymphocyte activation and proliferation in vitro, induce the rapid expression of early activated molecular CD69 in T cells, and the activation degree of the cells is related to the concentration of EMP intervention. In addition, EMP induces activated T cells to secrete IL-2, IFN- gamma and other inflammatory cytokines and highly express F. AsL. in vivo, these inflammatory factors and important molecules play an important role in immune cells and target cells, and play an important role in the immune response, such as rejection,.EMP may be an important target for immunoregulatory therapy.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【参考文献】