【作者】 吴永港；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 干细胞工程学， 2009， 博士

【摘要】 内皮祖细胞(EPC)是一类能循环、增殖并分化为血管内皮细胞,但尚未表达成熟血管内皮细胞表型特征的前体细胞。研究发现,EPC不仅参与人胚胎血管生成,同时也参与出生后血管新生和内皮损伤后的修复过程。干细胞因子(SCF)、血管内皮细胞生长因子(VEGF),HMG-coA还原酶抑制剂、粒系巨噬系集落刺激因子(GM-CSF)等都能白骨髓动员EPC,并促进其增殖、分化、粘附和迁移能力.氧化性低密度脂蛋白是冠心病独立危险因素,其生物学作用主要是影响内皮细胞功能,并进一步导致动脉粥样硬化。研究证明EPC对维持内皮结构和功能的完整性具有重要作用,并且参与了机体多种生理、病理性血管重建过程,在新形成的血管中,EPC来源的内皮细胞约占总内皮细胞的25%。EPC促进血管生成和内皮再生是因为EPC能够从骨髓动员到外周循环中,并归巢到血管损伤和生成的部位。EPC归巢需要多步骤协调作用,包括趋化、粘附、跨内皮迁移及最终分化为内皮细胞。最近,临床试验表明冠状动脉疾病患者培养的EPC数量及迁移能力明显下降。氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)为心血管疾病的重要独立危险因素,而且冠状动脉疾病及糖尿病患者血浆oxLDL生成明显增加。以前的研究表明oxLDL能够诱导内皮细胞凋亡、增加内皮细胞粘附分子的表达、抑制内皮细胞迁移而抑制血管新生。基于以上研究背景,我们推测。oxLDL可能为影响EPC数量和功能的因素。本研究的目的是观察oxLDL是否影响外周血EPC的数量;是否改变了EPC的增殖功能、迁移功能及粘附能力;oxLDL对EPC的体外血管生成能力的影响程度;同时观察oxLDL是否影响EPC的P38信号表达,来影响EPC的数量、功能。本研究分为二个部分,主要研究方法和结果如下:第一章氧化型低密度脂蛋白对内皮祖细胞存活和功能的影响目的:研究氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)对EPC存活及功能的影响方法:1.EPC的分离、培养:取健康成人空腹外周静脉血,用密度梯度离心法获取单个核细胞,培养7天,收集贴壁细胞。贴壁细胞随机分成5组:①对照组;②oxLDL各浓度组(共3组):在培养液中分别加入25,50,100,200μg/ml后培养24小时;③LDL组:含100μg/ml的条件培养液培养24h。2.细胞染色与鉴定:分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。用acLDL-Dil和FITC-UEA-I对细胞染色后,通过荧光显微镜鉴定,FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs,在荧光显微镜下(~*200)对每孔细胞进行计数3.细胞表型检测:将2×10~5贴壁细胞分别与FITC标记的VEGFR-2,CD31和CD34单克隆杭体以及PE标记的KDR,在4℃孵育30 min后,用300μl PBS悬浮细胞后上机检测。4.EPC粘附能力检测:收集贴壁细胞,悬浮在500μl培养液并计数,然后将等量EPC接种到包被有人纤维连接蛋白培养板,在37℃培养30 min,计数贴壁细胞。5.EPC迁移能力检测:收集贴壁细胞并计数。将600μl培养液和VEGF(50 ng/mL)加入改良的Boyden室的下室,将2×10~4 EPC悬浮在100μl培养液注入上室,培养24 h,刮去滤膜上面的未移动细胞,计数迁移到低层的细胞。6.EPC增殖能力检测:采用promega公司的Non-Radioactive CellProliferation Assay试剂盒MTS/PMS比色法检测细胞增殖率,按操作程序执行,简化如下:将消化的各组EPC细胞溶于含0.5%BSA的EBM-2中(含50ng/ml的VEGF)其中细胞浓度是1×10~5/ml,取各组细胞液50μl加入96孔,设三孔对照,培养72小时,各孔加入配备的染液15μl,再培养4小时,加停止液,过夜。置酶标仪于波长570nm记录各孔值,三孔对照均值作为各组本次试验值,7.体外血管生成能力检测:采用体外血管生成试剂盒检测EPC的血管生成能力.将ECM成胶。胰蛋白酶消化贴壁细胞获取EPC,并重新悬浮于培养液,调整细胞数为5×10~4/ml。将EPC接种于ECM胶上。37℃培养24h,在200倍倒置显微镜下观察小(血)管生成情况,随机选择6个显微镜视野(x200),计数小管数。结果:1.EPC的鉴定:分离获得的单个核细胞培养3天即可看到贴壁生长,7天后形成梭形的内皮样细胞。用acLDL-DiI和FITC-UEA-I对细胞染色后,通过荧光显微镜鉴定UEA-I和DiLDL双染色阳性细胞被认为是正在分化的EPC占贴壁细胞的90%。流式细胞仪检测贴壁细胞表面标志,结果显示:表达KDR(VECFR-2)(68.8±7.5)%,CD34(25.4±9.1%),CD31(71.2±7.2%)和CD144:(73.9±6.3)%,进一步明确为EPC。2.oxLDL对外周血EPC数量的影响:不同浓度的OxLDL干预EPC后24小时,结果显示:oxLDL显著减少EPC数量,并且EPC数量随着oxLDL的浓度的增加而减少。而LDL对EPC数量无影响。50μg/ml 100μg/ml,200μg/ml的oxLDL组导致EPC数量分别为46.3±4.85,34.2±3.59,22.7±2.38都较对照组EPC的70.7±7.41低,差异有显著性,P＜0.05。而oxLDL25μg/ml及LDL导致EPC数量为63.8±6.69,68.3±7.16,差异无显著性,P＞0.053.OxLDL对外周血EPC粘附能力的影响:oxLDL显著减少EPC的贴壁数,并且贴壁数随着其浓度的增加而减少,而LDL对EPC的贴壁无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC粘附数分别为21.7±2.28,16.3±1.71,10.2±1.07都较对照组EPC的30.2±3.17低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC粘附数为25.8±2.71,28.3±2.97,差异无显著性,P＞0.05。4.OxLDL对外周血EPC迁移功能的影响:OxLDL各浓度组明显减少EPC的迁移能力,而LDL对EPC的迁移无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC迁移数分别为16.5±1.73,10.8±1.13,6.2±0.65都较对照组EPC的25.5±2.67低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC迁移数为22.3±2.34,23.8±2.50,差异无显著性,P＞0.05。5.OxLDL对外周血EPC增殖功能的影响:EPC的增殖能力在OxLDL各浓度组抑制了EPC的增殖,并且随着其浓度的增加而加剧。LDL对EPC的增殖无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC增值率分别为0.73±0.762,0.45±0.048,0.31±0.033都较对照组EPC的1.29±0.135低,差异有显著性,P＜0.05。而oxLDL25μg/ml及LDL导致EPC增值率为1.12±0.118,1.198±0.126,差异无显著性,P＞0.05。6.OxLDL对外周血EPC体外血管生成能力的影响:体外血管生成实验模拟体内血管生成,检测EPC参与血管新生的能力.结果显示,OxLDL各浓度组显著减弱EPC的体外血管生成能力,在浓度为200μg/ml时最为显著。而LDL对EPC的成血管能力无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC成血管数数分别为19.2±2.01,13.2±1.38,9.5±1.00都较对照组EPC的26.2±2.75低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC迁移数为23.0±2.41,25.8±2.70,差异无显著性,P＞0.05。结论:1.OxLDL在体外能减少EPC数量,作用呈浓度依赖性;2.OxLDL在体外能抑制EPC的增殖、迁移、粘附能力,作用呈浓度和依赖性;3.OxLDL能降低EPC的体外血管生成能力:提示OxLDL是导致EPC数量及功能减弱的因素之一。第二章P38信号通路参与oxLDL对内皮祖细胞数量和功能的影响目的:研究oxLDL是否导致EPC细胞内P38及P-P38的表达异常,以及oxLDL对EPC的作用是否与细胞内P38的信号有关。方法:1.EPC分离,培养,及分组:EPC的分离及培养见第一章,收集贴壁细胞。贴壁细胞随机三组:①对照组:②oxLDL组:在培养液中加入100μg/ml的oxLDL③SB203580组:在培养液中加入100μg/ml的oxLDL前,预先半小时加入0.5μM的SB203580。2.Western检测p38及p-p38的表达:oxLDL(100μg/ml)处理EPC后0,5,15,25,35分钟及SB203580预处理后细胞内p38,及p-p38蛋白的表达;以及oxLDL 25,50,100,200μg/ml以及LDL100μg/ml处理30分钟后细胞内p38,及p-p38蛋白的表达。3.EPC凋亡分析:按BD Biosciences公司的说明进行操作:各组EPC处理结束后,用0.1%胰酶消化,4°C PBS洗两次后,重悬于结合缓冲液中,调整细胞浓度为1 X 106/ml。取100μl细胞悬液至5 ml试管中,加5μlAnnexin V-FITC标记液和5μl PI,轻轻混匀。并设立无Annexin V及PI的空白对照、仅Annexin V的对照、仅PI的对照。室温避光孵育15min后,加400μl结合缓冲液,1小时内上流式细胞仪检测EPC双染色鉴定EPC数量,EPC粘附能力检测,迁移能力检测,EPC增殖能力检测,体外血管生成能力检测实验方法见第一章结果:1.western检测结果:100μg/ml的oxLDL处理EPC后,细胞内p-p38成时间依赖性表达增强,大约25min时达顶峰,5,15,25,25,35分钟细胞内p-p38的表达是对照组的1.4±0.15倍,2.1±0.22倍,3.2±0.34倍,1.5±0.13倍,且p38拮抗剂sb203580可抑制其表达,是对照组的1.2±0.13倍,与各时间点比较有显著性差异,P＜0.05。不同浓度的OxLDL处理25min后观察,细胞内p-p38成剂量依赖性表达增强,25μg/ml,50μg/ml,100μg/ml,200μg/ml处理EPC后,其细胞内p-p38的表达是分别对照组的1.2±0.13,1.7±0.18,3.2±0.35,3.4±0.35。而LDL对其没影响。细胞内p38表达不受时间和浓度的影响。2.100μg/ml的oxLDL处理EPC后,导致EPC数量减少,增殖减低,凋亡增加,粘附及迁移能力减低,体外成血管能力减弱。而sb203580能减弱100μg/ml的oxLDL对EPC的作用。其中对照组,oxLDL组和SB+oxLDL组的EPC数量分别为71.8±7.52,32.5±3.40,52.8±5.85;粘附细胞数分别为31.7±3.32,16.3±1.71,24.5±2.57;迁移细胞数分别为24.2±2.53,10.5±1.10,18.2±1.91;体外成血管细胞数分别为25.2±2.64,12.3±1.29,17.8±1.87。各组增殖率分别为1.32±0.139,0.45±0.048,1.133±0.119。各组的凋亡率分别10.2±1.07%,21.3±2.23%,13.2±1.4%。SB+oxLDL与oxLDL组比较,在细胞数量,增殖,凋亡及黏附,迁移,体外成血管能力方面有差异显著性,P＜0.05。结论:oxLDL对EPC的作用与细胞内磷酸化的p38表达及活性有关。P38的抑制剂sb203580能抑制oxLDL对EPC的毒性作用。提示oxLDL对EPC的作用是通过细胞p38信号作用的。更多还原

【Abstract】 To generate the precursor cell which has the phenotypic characteristics of mature vascular endothelial cell with the endothelial progenitor cell(EPC),which can circulate,proliferate and differentiate into vascular endothelial cell.The previous studies found that EPC is involved not only in the human embryonic angiogenesis but also in the vascular neogenesis after birth and the reparative process after endothelial injuries.The stem cell factor(SCF),vascular endothelial cell growth factor(VEGF),HMG-CoA reductase inhibitor,granulocyte macrophage colony stimulating factor(GM-CSF),etc can mobilize the EPC in marrow and stimulate its abilities of proliferation,differentiation, adhesion and migration.The oxidized low density lipoprotein is an independent risk factor of coronary artery disease and its biological activity is mainly to affect the functions of endothelial cell to further result in artherosclerosis.It has been proved that EPC plays an important role in the maintenance of endothelial structure and the functional integrity and it is also involved in multiple physiological and pathological reconstruction of blood vessel of the organism,meanwhile,the proportion of endothelial cells from EPC is 25%of the total endothelial cells in the new vessel.The function of EPC to stimulate angiogenesis and endothelium regeneration is because that it can be mobilized from the marrow into the peripheral circulation and home to the areas with injury of blood vessel and angiogenesis.The homing of EPC is accomplished through the coordination of multiple steps,which include chemotaxis, adhesion,migration through the endothelim and the final differentiation into endothelial cell.Recently,the clinical studies indicated that the number and the ability of migrated EPC from the patients with coronary disease are significantly decreased.The oxidized low density lipoprotein (oxLDL) is an important and independent risk factor of cardiovascular disease,and the plasma level of oxLDL in the patients with coronary disease and diabetes is significantly increased.The previous studies indicated that oxLDL can induce the endothelial cellular apoptosis, increase the expression of endothelial cell adhesion molecules and inhibit the epithelial cell migration to inhibit the angiogenesis.Thus,we presumed that oxLDL might be the factor which affects the number and function of EPC.The purpose of this study is to observe that whether oxLDL affect the number of EPC in the peripheral blood or change the EPC abilities of proliferation,migration and adhesion,to detect the impact degree of oxLDL to the EPC ability of angiogenesis and to observe that whether oxLDL affect the expression of the P38 signal in EPC to affect the number and function of EPC.This study includes two parts and the main methods and results are as the following:ChapterⅠ.The effects of the oxidized low density lipoprotein to the survival and function of epithelial progenitor cellObject:to investigate the effects of the oxidized low density lipoprotein (oxLDL) to the survival and function of epithelial progenitor cell.Methods:1.The separation and culture of EPC:harvest the peripheral venous blood from the healthy adults at the state of empty stomach, obtain the mononucleated cells through density gradient centrifugation, culture for 7 days and collect the adherent cells.The adherent cells are randomly assigned into 5 groups:1) the control group;2) the groups treated with oxLDL of different concentrations(3 groups):add 25,50, 100 and 200μg/ml oxLDL into the medium,respectively,and culture for 24 h;and 3) LDL group:culture in the conditioned medium containing 100μg/ml LDL for 24 h.2.Staining and identification of the cells:the separated mononucleated cells transformed into spindle-shaped endothelioid cells after 7 d of culture.The cells were identified by the luorescence microscope after the staining by acLDL-Dil and FITC-UEA-I,in which, the positive cells of double staining are proliferating EPCs.Count the cells of each well under the fluorescence microscope(×200).3.Examination of the cellular phenotype:incubate 2×10~5 cells with VEGFR-2,CD31 and monoclonal antibody of CD34 labeled by FITC and KDR labeled by PE for 30 min at 4℃,respectively,and transfer 300μl PBS suspension cells for examination.4.Examination of the adhesion ability of EPC:collect the adherent cells and suspended in 500μl medium for counting,and then inoculate the same amount of cells into the culture plate covered by human fibronectin and count the adherent cells after 30 min of culture.5.Examination of the migration ability of EPC:collect the adherent cells and count.Transferred 25μl medium with VEGF(50 ng/mL) into the lower chamber of the modified Boyden chamber,inject 2×10~4 EPC suspended in 200μl medium into the upper chamber,culture for 24 h,scrape off the cells without migration on the filter membrane and count the cells migrated into the lower part.6.Examination of the proliferation ability of EPC:apply the Non-Radioactive Cell Proliferation Assay(Promega company) kit to detecte the cell proliferation rate by MTS/PMS chromatometry and operate according to the procedures which were simplified as the following:suspended the digested cells of various EPC groups in the EBM-2 containing 0.5%BSA(containing 50 ng/ml VEGF) with the concentration of 1×10~5 cells/ml,respectively,transfer 50μl cell suspension from each group into the 96-well plate in triplication as control,culture for 72 h,add the 15μl prepared staining solution into each well and culture for another 4 h,add the stop solution and incubate overnight.Examine each well by the enzyme-labeled instrument at the wavelength of 570 nm and take the mean value of the triplication as the test value of each group.7.Examination of the in vitro angiogenesis ability:use the in vitro angiogenesis assay kit to detect the angiogenesis ability of EPC. Mix the ECMa t r i x" glum-solution and 10×dilution of ECM after freeze thawing at the proper ratio to form the gum.Digest the adherent cells with 0.250%trypsin to obtain the EPC,re-suspend in the medium and adjust the concentration to 5 x 104cells/ml.Inoculate the EPC on the ECMa t r i x" glum.Culture for 24 h,observe the formation of tubules under the 200×inverted microscope,choose 5 fields(×200) randomly and count the number of tubules.Results:1.Identification of the EPC:the separated mononucleated cells turned to the adherent growth after 3 d of culture and transformed into the spindle-shaped endothelioid cells after 7 d.The cells,which with double staining of UEA-I and DiLDL under the fluorescence microscope after the staining by acLDL-DiI and FITC-UEA-I,were recognized as the proliferating ECP which composed 90%of the adherent cells.The surface markers of the adherent cells were detected by the flow cytometer and the results indicated that the cells expressed KDR(VECFR-2)(68.8±7.5)%, CD34(25.4±9.1%),CD31((77.1±7.2%) and CD144:(73.9±6.3)%, which further confirmed that the cells were EPC.2.The effect of oxLDL to the number of EPC in peripheral blood:after 24 h-exposure of EPC to different concentrations of oxLDL, the results indicated that oxLDL significantly decreased the number of EPC and the number of EPC decreased with the concentration increase of oxLDL.However,the LDL had no effect to the number of EPC.The numbers of EPC in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 46.3±4.85,34.2±3.59 and 22.7±2.38, respectively,which were all significantly lower than the number of EPC in the control group which was 70.7±7.41(P＜0.05).The numbers of EPC in the groups treated by 25μg/ml oxLDL and LDL were 63.8±6.69 and 68.3±7.16,respectively,and there was not significant difference between them(P＞0.05).3.The effect of oxLDL to the adherent ability of the EPC in peripheral blood:oxLDL significantly decreased the number of adherent cells and the number of adherent cells decreased with the concentration increase of oxLDL.However,the LDL had no effect to the adherence. The numbers of adherent EPC in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 21.7±2.28,16.3±1.71 and 10.2±1.07,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 30.2±3.17(P＜0.05).The numbers of adherent EPC in the groups treated by 25μg/ml oxLDL and LDL were 25.8±2.71 and 28.3±2.97,respectively,and there was not significant difference between them(P＞0.05).4.The effect of oxLDL to the migration ability of EPC in peripheral blood:oxLDL of different concentrations all decreased the migration ability of the EPC significantly,but the LDL had no effect to the migration of the EPC.The number of migrated cells in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 16.5±1.73, 10.8±1.13 and 6.2±0.65,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 25.5±2.67(P＜0.05).The numbers of migrated EPC in the groups treated by 25μg/ml oxLDL and LDL were 22.3±2.34 and 23.8±2.50, respectively,and there was not significant difference between them (P＞0.05).5.The effect of oxLDL to the proliferation ability of the EPC in peripheral blood:the proliferation ability of EPC in the groups treated by oxLDL of different concentrations were all inhibited and the inhibitory effect increased with the increase of the concentration.The LDL had no effect to the proliferation of the EPC.The EPC proliferation rates of the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 0.73±0.762,0.45±0.048 and 0.31±0.033,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 1.29±0.135(P＜0.05).The EPC proliferation rates in the groups treated by 25μg/ml oxLDL and LDL were 1.12±0.118 and 1.198±0.126,respectively,and there was not significant difference between them(P＞0.05). 6.The effect of oxLDL to the in vitro angiogenesis ability of EPC in peripheral blood:the in vitro angiogenesis experiment was applied to simulate the in vivo angiogenesis in order to detect the angiogenesis ability of EPC.The results indicated that oxLDL of different concentrations all decreased the angiogenesis ability of the EPC significantly and the inhibitory effect was most significant in the group treated by 200μg/ml oxLDL.However,the LDL had no effect to the angiogenesis ability of the EPC.The numbers of the generated blood vessels in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 19.2±2.01,13.2±1.38 and 9.5±1.00,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 26.2±2.75(P＜0.05).The EPC proliferation rates in the groups treated by 25μg/ml oxLDL and LDL were 23.0±2.41 and 25.8±2.70,respectively,and there was not significant difference between them(P＞0.05).Conclusions:1.OxLDL can decrease the number of EPC in vitro and the effect is concentration dependent;2.OxLDL can impair the EPC abilities of proliferation,migration and adherence and the effect is concentration dependent.3.OxLDL can decrease the in vitro angiogenesis ability of EPC.These results indicated that oxLDL is one of the factors leading to the decreases of EPC amount and the function. ChapterⅡThe effect of p38 MAPK on the endothelial progenitor cell induced by oxLDLObjective:to study that whether oxLDL lead to the abnormal expressions of P38 and P-P38 in EPC and whether the effect of oxLDL to EPC be related to the P38 signal in the cells.Methods:1.The separation,culture and grouping of EPC:see chapterⅠfor the separation and culture of EPC and collect the adherent cells.The adherent cells are randomly assigned into 3 groups:1) the control group; 2) oxLDL group:the medium contains 100μg/ml oxLDL;and 3) SB203580 group:add SB203580 in the medium to 0.5μM at half an hour before adding oxLDL to 100μg/ml.2.The detection of p38 and p-p38 expression by Western blot: detect the p38 and p-p38 expressions in the EPC at 0,5,15,25 and 35 min when the cells were treated by oxLDL and preteated by SB203580, meanwhile,the p38 and p-p38 expressions were also detected in the cells treated by 25,50,100 and 200μg/ml oxLDL and 100μg/ml LDL for 30 min.3.EPC apoptosis analysis:Operate according to the instruction of BD Biosciences company:digest the EPC of each group by 0.1%trypsin after the treatments,wash twice by 4℃PBS and re-suspend in the binding buffer and adjust the concentration to 1×10~6 cells/ml.Transfer 100μl cell suspension into the 5 ml test tube and add 5μl Annexin V-FITC labeling sultion and 5μl PI,and then mix gently.Set the blank control without Annexin V and PI,the control only with Annexin V and the control only with PI.Incubate away from light and at temperature for 15 min,then add 400μl binding buffer and detect by the flow cytometer within 1 h.See chapterⅠfor the methods of the EPC double staining for EPC number and the determinations of the EPC abilities of adherence, migration,proliferation and in vitro angiogenesis.Results:1.The results of Western blot:the p-p38 expression increased in the EPC with time dependence when treated by 100μg/ml oxLDL and the expression level reached peak at 25 min,the ratios between the expression levels of p-p38 in the oxLDL group at 5,15,25,25 and 35 min and those of the control group were 1.4±0.15,2.1±0.22,3.2±0.34 and 1.5±0.13,respectively,meanwhile,the p38 inhibitor SB203580 could inhibit the expression and the ratio to the control group is 1.2±0.13,and there were significant differences at different times(P＜0.05).The observations at 25 min after the treatment of different concentrations indicated that the p-p38 expressions increased with dose dependence,and the ratios between the expression levels of p-p38 in the groups treated by 25μg /ml,50μg /ml,100μg /ml,200μg/ml oxLDL were 1.2±0.13, 1.7±0.18,3.2±0.35 and 3.4±0.35,respectively.LDL had no effect to p-p38 expression.The p38 expression in the EPC was not affected by the time and concentrations.2.The treatment of 100μg/ml oxLDL led to the decrease of EPC amount,reduce of proliferation,increase of apoptosis,the ability decreases of adherence,migration and in vitro angiogenesis.However, the SB203580 could decrease the effect of 100μg/ml oxLDL to EPC. The EPC numbers in the control group,oxLDL group and SB+oxLDL group were 71.8±7.52,32.5±3.40 and 52.8±5.85,respectively;and the adherent cell numbers were 31.7±3.32,16.3±1.71 and 24.5±2.57, respectively;and the migrated cell numbers were 24.2±2.53,10.5±1.10 and 18.2±1.91,respectively;and the numbers of the cells forming in vitro blood vessels were 25.2±2.64,12.3±1.29 and 17.8±1.87,respectively; and the proliferations were 1.32±0.139,0.45±0.048 and 1.133±0.119, respectively;and the apoptosis rates were 10.2±1.07%,21.3±2.23%and 13.2±1.4%respectively.The comparison between the SB+oxLDL group and oxLDL group indicated that there were significant differences on the cell numbers,proliferation,apoptosis and the abilities of adherence, migration and in vitro angiogenesis between these two groups(P＜0.05).Conclusions:the effect of oxLDL to EPC is related to the expression and activity of the intracellular phosphorylated p38.The p38 inhibitor SB203580 can inhibit the toxic actions of oxLDL to EPC.These results suggested that the effect of oxLDL to EPC is transduced by the p38 signal.更多还原