本文選題：內(nèi)皮微粒 + 內(nèi)皮細(xì)胞　； 參考：《河北醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:研究血管內(nèi)皮細(xì)胞脫落的內(nèi)皮微粒對(duì)體外培養(yǎng)的T淋巴細(xì)胞活化增殖、產(chǎn)生細(xì)胞因子及膜蛋白表達(dá)的影響,探討內(nèi)皮微粒對(duì)在急性排斥反應(yīng)中的作用及部分機(jī)制。 方法:1.內(nèi)皮細(xì)胞的培養(yǎng):①人內(nèi)皮細(xì)胞株(Eahy926),用含10 %胎牛血清的高糖DMEM培養(yǎng)基在37℃、5 % CO2、飽和濕度下培養(yǎng),至Eahy926細(xì)胞生長成單層后備用;②大鼠肺微血管內(nèi)皮細(xì)胞(PMVEC)的培養(yǎng)及鑒定:取健康Sprague Dawley (SD)大鼠的外周肺組織,采用組織塊貼壁法建立大鼠PMVEC的原代培養(yǎng)技術(shù)。采用形態(tài)學(xué)觀察及第Ⅷ因子相關(guān)抗原免疫組織化學(xué)染色法對(duì)所培養(yǎng)的大鼠PMVEC進(jìn)行鑒定。2.誘導(dǎo)內(nèi)皮細(xì)胞活化產(chǎn)生內(nèi)皮微粒:以TNF-α(100 ng/ml)刺激Eahy926細(xì)胞或第二代大鼠PMVEC,于48 h后收集培養(yǎng)液上清分離細(xì)胞微粒。3.內(nèi)皮微粒的分離及定量測(cè)定:采用高速離心法分離所收集培養(yǎng)液上清中的細(xì)胞微粒。AnnexinⅤ-FITC熒光染色,激光共聚焦顯微鏡下觀察所分離微粒形態(tài),并采用Bradford法進(jìn)行定量測(cè)定。4.大鼠脾臟淋巴細(xì)胞的分離培養(yǎng):分離健康成年Brown -Norway(BN)大鼠脾臟淋巴細(xì)胞并隨機(jī)分為正常對(duì)照組、不同濃度(10、20、40μg/ml)Eahy926細(xì)胞來源EMP干預(yù)組、ConA刺激組及PMVEC來源EMP(40μg/ml)干預(yù)組。5.檢測(cè)評(píng)價(jià)EMPs對(duì)T淋巴細(xì)胞的影響:①采用噻唑蘭比色法(MTT法)檢測(cè)淋巴細(xì)胞增值程度;②采用流式細(xì)胞術(shù)檢測(cè)T細(xì)胞(CD3+淋巴細(xì)胞)早期活化分子CD69的表達(dá);③采用ELISA法檢測(cè)各組細(xì)胞培養(yǎng)液上清中相關(guān)細(xì)胞因子(IFN-γ,IL-2)水平的表達(dá)。④采用RT-PCR技術(shù)檢測(cè)各組細(xì)胞FasL mRNA的表達(dá)。 結(jié)果:1.成功培養(yǎng)出狀態(tài)良好的PMVEC。細(xì)胞在倒置顯微鏡下呈現(xiàn)明顯的上皮樣細(xì)胞形態(tài),細(xì)胞聚集成一片,外觀呈鵝卵石樣或鋪路石樣鑲嵌狀排列生長。經(jīng)免疫組織化學(xué)對(duì)其內(nèi)皮特異性第Ⅷ因子相關(guān)抗原呈陽性表達(dá),證明成功培養(yǎng)出了高純度的PMVEC。2.EMP的誘生及其分離鑒定結(jié)果:經(jīng)TNF-α刺激,內(nèi)皮細(xì)胞因活化而產(chǎn)生大量內(nèi)皮微粒。激光共聚焦顯微鏡觀察,微粒因與AnnexinⅤ-FITC結(jié)合而呈綠色囊泡狀外觀。我們采用Bradford法定量所分離EMP,以蛋白量計(jì),EMP濃度在0.5~1μg/μl之間。3.淋巴細(xì)胞活化增殖測(cè)定:倒置顯微鏡直接觀察,各EMP干預(yù)組及ConA組細(xì)胞都有較高程度的聚集生長,較對(duì)照組增殖旺盛;MTT結(jié)果顯示:與對(duì)照組相比較,各Eahy926細(xì)胞來源EMP干預(yù)組、ConA組、PMVEC來源EMP干預(yù)組細(xì)胞代謝活力均顯著提高(P0.05);且與ConA組比較,EMP高濃度組、PMVEC來源EMP干預(yù)組細(xì)胞代謝活力的增高無顯著差異(P0.05)。4.T細(xì)胞早期活化標(biāo)志分子CD69的表達(dá):流式細(xì)胞術(shù)的檢測(cè)結(jié)果顯示,EMP各濃度組、ConA組、PMVEC來源EMP干預(yù)組CD3+細(xì)胞CD69分子的表達(dá)較對(duì)照組均明顯增高(P0.01);ConA組CD3+細(xì)胞CD69分子表達(dá)顯著高于其他各組(P0.01)。5. ELISA結(jié)果顯示:與對(duì)照組相比較,EMP各濃度組、ConA組、PMVEC來源EMP干預(yù)組培養(yǎng)液上清IFN-γ水平較對(duì)照組有顯著增高(P0.05),且EMP各濃度組間具有明顯的效量關(guān)系;而與對(duì)照組相比較,EMP中濃度組、EMP高濃度組、ConA組及PMVEC來源EMP干預(yù)組培養(yǎng)液上清IL-2水平顯著增高(P0.05),EMP低濃度組較空白對(duì)照組IL-2增高則無顯著性差異(P0.05)。6.FasL mRNA水平的表達(dá):與對(duì)照組相比,EMP中濃度組、EMP高濃度組、ConA組及PMVEC來源EMP干預(yù)組FasL mRNA表達(dá)水平顯著增高(P0.01);ConA組FasL mRNA的表達(dá)增高較各EMP干預(yù)組具有顯著差異(P0.05);且EMP高濃度組與PMVEC來源EMP干預(yù)組細(xì)胞FasL mRNA表達(dá)無顯著性差異(P0.05)。 結(jié)論:研究發(fā)現(xiàn)Eahy926細(xì)胞來源EMP及原代PMVEC來源EMP均可誘導(dǎo)體外培養(yǎng)的淋巴細(xì)胞活化增殖,誘導(dǎo)T細(xì)胞早期活化分子CD69的迅速表達(dá),且細(xì)胞的活化程度與EMP干預(yù)濃度有關(guān)。另外,EMP誘導(dǎo)活化的T細(xì)胞分泌IL-2、IFN-γ等炎性細(xì)胞因子并高表達(dá)FasL。體內(nèi)這些炎性因子及重要分子作用于免疫細(xì)胞及靶細(xì)胞,在機(jī)體免疫應(yīng)答如排斥反應(yīng)中發(fā)揮著重要作用。EMP可能是免疫調(diào)節(jié)性治療的一個(gè)重要靶點(diǎn)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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