【作者】 戴青原；

【导师】 郭涛；

【作者基本信息】 昆明医科大学 ， 外科学， 2014， 博士

【摘要】 目的1.建立稳定有效的脐静脉内皮细胞(HUVECs)体外培养方法,比较原代人脐静脉内皮细胞与人脐静脉内皮细胞株HUVEC-CS及EA.hy926的VWF、CD31、CD34表面抗原表达情况。2.采用过氧化氢(H202)体外诱导培养法,构建HUVECs氧化应激模型。观察体外正常培养HUVECs Visfatin的表达及氧化应激损伤对其表达的影响。3.观察Visfatin对体外正常培养及氧化应激损伤HUVECs的凋亡及多种细胞因子表达的影响,探讨其发挥效应的可能信号通路。4.探讨血浆Visfatin的影响因素及其与冠心病、糖代谢异常的相关性。应用eZscan系统和实验室方法对冠心病患者进行糖代谢异常的筛查,探讨eZscan系统的临床应用价值和前景。方法1.用0.1%Ⅱ型胶原酶消化分离原代人HUVECs,加入含内皮细胞生长因子的M199完全培养基内培养,]HUVEC-CS及EA.hy926细胞株用DMEM完全培养基培养。0.25%胰蛋白酶-0.02%EDTA消化传代,三种细胞在倒置相差显微镜下观察形态学,免疫荧光染色法测定VWF表达,流式细胞仪检测CD31、CD34表面抗原表达。2.①体外培养的人脐静脉内皮细胞株传代后进行实验,将实验细胞分为对照组、模型组,在剂量效应干预中,模型组分为6个不同浓度梯度(H202终浓度分别为100、200、400、600、800、1OOOμmol/L),筛选H202最佳作用浓度；在时间效应干预中,以筛选出的H202最佳作用浓度分别培养细胞6、12、24h,筛选最佳作用时间。收集各组细胞进行相关检测：倒置显微镜及透射电镜观察细胞形态学变化；采用细胞计数试剂盒(CCK-8法)检测细胞存活率；DCFH-DA荧光探针和流式细胞技术(FCM)检测细胞内活性氧水平；流式细胞技术TUNEL法检测细胞凋亡率和增殖指数。②体外培养的人脐静脉内皮细胞株传代后进行实验,将实验细胞随机分为正常对照组和H202处理组(H202终浓度分别为50、100、200、400、1000μmol/L),培养1、2、4、24h分别进行检测。Vestern blot检测visfatin蛋白水平的变化,Real time PCR测定Vistafin基因表达。3.体外培养的人脐静脉内皮细胞株传代后进行实验,将实验细胞随机分组后予Visfatin、SB203580、LY29004相应干预处理,流式细胞仪TUNEL法检测细胞凋亡率和细胞周期,ELISA定量检测IL-6、MCP-1、VCAM、VEGF、iNOS浓度。4.选取160例冠心病患者,根据患者是否合并糖代谢异常,将其分为冠心病合并糖代谢异常组及单纯性冠心病组；同期选择无器质性心脏病的糖代谢异常患者50例；同期选取50例健康人群作为正常对照组。空腹测定所有受试者实验室指标及血浆Visfatin水平,并进行baPWV及ABI检测,应用eZscan系统(糖尿病及并发症风险早期检测系统)对经冠脉造影确诊的冠心病患者进行糖代谢异常筛查,并将检测结果与实验室检查结果进行比较,评价其灵敏度和特异度。结果1.①原代HUVECs在接种24h后完全贴壁生长,呈多角形,单个或成团存在,4-5天后融合呈铺路石样镶嵌排列。HUVEC-CS和EA.hy926细胞为圆形或扁平形,生长速度快,2天左右即可铺满,融合后呈典型的“鹅卵石”状排列。②原代HUVECs及EA.hy926细胞的VWF免疫荧光染色呈阳性反应,大部分细胞内可见绿色荧光,HUVEC-CS细胞则染色呈阴性。HUVEC-CS细胞CD31阳性表达率(%)(4.70±0.46)显著低于原代HUVECs (77.93±0.25)或EA.hy926细胞(78.23±0.40),差异均有统计学意义(P均<0.001),原代HUVECs和EA.hy926细胞间无统计学差异(P>0.05)。原代HUVECs CD34阳性表达率(%)(43.1±0.20)显著高于HUVEC-CS细胞(1.20±0.40)或EA.hy926细胞(0.97±0.40),差异均有统计学意义(P均<0.001),HUVEC-CS和EA.hy926细胞间无统计学差异(P>0.05)。2.①与正常组比较,H202模型组HUVECs细胞形态学明显改变,细胞体积缩小,胞膜皱缩,细胞轮廓不清,形状不规则,细胞排列紊乱,部分细胞上浮,典型单层铺路石样细胞排列受到不同程度的破坏。梭型细胞减少,出现球形或椭圆形细胞,球形或椭圆形细胞数量随作用时间延长和剂量增加而增多,表明H202对HUVECs具有损伤作用。CCK-8显示随着H202作用浓度的增加和时间的延长,HUVECs存活率逐渐下降,呈现一定的量效和时效关系。DCFH-DA荧光探针和流式细胞技术(FCM)检测H202模型组细胞内有较高的活性氧水平。流式细胞技术TUNEL法观察证实H202能够诱导HUVECs发生凋亡,随着H202作用浓度的增加细胞凋亡率明显增加。②体外正常培养的HUVECs表达Visfatin。予不同浓度H202(50、100、200、400、1000μmol/L)作用HUVECs不同时间(1、2、4、24h),结果发现,在低浓度H202(50、100μmol/L)作用时,Visfatin的表达随H202作用时间延长而增加(P<0.05),但H202浓度≥200μmol/L时,Visfatin的表达随H202作用时间延长而减少；在H202短时间(1、2h)作用时,Visfatin的表达随H202作用浓度增加而增加(P<0.05),但H202作用时间延长后(≥4h),随H202作用浓度增加Visfatin的表达减少(P<0.05)。由、Vestern blot蛋白印迹灰度分析及Real time PCR检测Visfatin mRNA表达量得出的变化趋势是一致的。3.对体外正常培养的HUVECs, Visfatin100ng/ml、400ng/ml、800ng/m1培养细胞24h/48h细胞凋亡率及细胞增殖指数与正常对照组比较无统计学差异(P>0.05)。不同浓度Visfatin处理正常培养细胞24h/48h后,IL-6,MCP-1, VCAM, VEGF, iNOS表达较正常对照组均增加(P均<0.05),且与Visfatin呈浓度和时间依赖性。对氧化应激损伤的HUVECs, Visfatin100ng/ml、400ng/ml、800ng/ml预处理细胞24h/48h的细胞凋亡率较H202对照组明显下降(P<0.001),但3种浓度Visfatin处理组间两两比较,随着Visfatin浓度递增,细胞凋亡率反而逐渐增加,任意两组间均有统计学差异(P<0.05)。Visfatin预处理细胞24h后,氧化应激损伤HUVECs的增殖指数较H202对照组上升,差异有统计学意义(P<0.001)。经两两比较,Visfatin浓度由100ng/ml增加至400ng/ml或800ng/ml时,细胞增殖指数逐渐下降,差异有统计学意义(P<0.05), Visfatin400ng/ml预处理组与Visfatin800ng/ml预处理组间无统计学差异(P>0.05)。Visfatin100ng/ml、400ng/ml、800ng/ml预处理细胞48h不同处理组的细胞增殖指数较H202对照组上升,差异有统计学意义(P<0.001)。经两两比较,随着Visfatin浓度增加,增殖指数逐渐降低,当Visfatin浓度为800ng/ml时,细胞增殖指数较对照组下降,差异有统计学意义(P<0.01)。Visfatin预处理细胞24h后,IL-6, MCP-1, VCAM, VEGF, iNOS五个细胞因子表达增加,不同处理组间有统计学差异(P<0.05),并经两两比较,除了VCAM Visfatin100ng/ml组与H202对照组间差异无统计学意义外(P>0.05),各细胞因子表达随Visfatin浓度升高而增加,差异均有统计学意义(P<0.05)。Visfatin预处理细胞48h后,IL-6, MCP-1, VCAM, VEGF, iNOS五个细胞因子表达增加,不同处理组间有统计学差异(P<0.05),经两两比较,各细胞因子表达随Visfatn浓度升高而增加,任意两组间差异均有统计学意义(P<0.05)。p38MAPK抑制剂SB203580及PI3K抑制剂LY29004预处理后,可以部分阻断Visfatin抗细胞凋亡、促细胞增殖、促炎症因子表达的效应,各组间细胞凋亡率、细胞增殖指数、细胞因子IL-6、MCP-1、VCAM、iNOS水平的差异有统计学意义(P<均0.05),除MCP-1外,这2种不同的通路抑制剂的作用差异具有统计学意义,p38MAPK抑制剂的作用更为显著(P<0.05)。4.血浆Visfatin水平从高到低依次为冠心病组、糖代谢异常组、正常对照组(P<0.05)。冠心病不同临床类型(慢性稳定性心绞痛、不稳定性心绞痛、急性心肌梗死)血浆Visfatin水平递增性升高(P<0.05),冠心病合并糖代谢异常患者Visfatin显著高于未合并糖代谢异常冠心病患者(P<0.05)。Visfatin水平与BMI、SBP、TC、LDL-C、FPG、Fins、HOMA-IR正相关(P均<0.05), BMI、HOMA-IR对血浆Visfatin水平的影响尤为显著(P均<0.05)。eZscan系统筛查糖代谢异常的敏感度88.89%,特异度64.95%。受试者工作特征曲线表征EZSCAN筛查糖代谢异常的准确性有意义(曲线下面积0.769,P=0.000)。baPWV测值随冠脉狭窄程度有递增趋势,ABI测值则与冠脉狭窄程度负相关(P均<0.05)。结论1.人脐静脉灌注Ⅱ型胶原酶消化、加入含内皮生长因子的M199完全培养基培养,可获得高纯度、可传代的存活内皮细胞,改进消化培养方法能提高细胞数量,其表面抗原表达优于HUVEC-CS和EA.hy926细胞株,但细胞产出量不高,传代5-6次后细胞形态变化较大,在复杂基础研究中应用受限。EA.hy926永生细胞株培养方法简单,成活率高,生长迅速,细胞形态稳定,其表型与原代HUVECs相似度高,短时间内可获得足够数量细胞满足相关实验研究。两者均是组织工程及相关医学基础研究理想的细胞来源。2.①用含800gmol/L过氧化氢的培养基培养24h可成功建立HUVECs氧化应激模型。②体外正常培养的HUVECs表达Visfatin, H2O2促进Visfatin表达增加有量效和时效性,短时间内或低浓度作用能促进Visfatin的表达,而浓度增高或时间延长,反而抑制Visfatin的表达。3.①对体外正常培养的HUVECs, Visfatin100ng/ml、400ng/m;、800ng/ml培养细胞24h/48h未对细胞的凋亡及增殖未造成明显影响。Visfatin呈浓度及时间依赖性促进体外正常培养的HUVECs表达IL-6、MCP-1、CAM、 VEGF、iNOS。②对氧化应激损伤HUVECs, Visfatin100ng/ml、400ng/ml、800ng/ml培养细胞24h/48h细胞凋亡率较H202对照组明显下降、增殖指数上升,但Visfatin抗内皮细胞凋亡促增殖效应不随着浓度增加时间延长而递增。Visfatin呈浓度及时间依赖性引起氧化应激损伤HUVECs的IL-6、MCP-1、VCAM、VEGF、iNOS表达显著增加。③予p38MAPK抑制剂SB203580及P13K抑制剂LY29004预处理后,可以部分阻断visfatin抗细胞凋亡、促细胞增殖、促炎性细胞因子表达的效应,提示visfatin抗细胞凋亡、促细胞增殖、促炎性细胞因子表达的效应可能与p38MAPK信号通路及见P13K信号通路有关,其中以P38MAPK信号通路的作用更为显著。4. BMI、HOMA-IR对血浆Visfatin水平的影响有统计学意义。Visfatin可能在糖代谢异常、AS的发生发展过程中起到了一定作用。应用eZscan系统进行糖代谢异常的早期筛查简单省时、无创安全、不受空腹限制,具有较高的灵敏度和中等特异度。baPWV及ABI检测无创、简单、价廉、重复性好,对及早发现冠心病高危患者和评估冠脉病变的严重程度发挥着一定的作用。更多还原

【Abstract】 Objective:1. To explore a stable way of the culture and characterization of human umbilical vein endothelial cells (HUVECs) and compare the surface antigen expressions among primary HUVECs, HUVEC-CS and EA.hy926cell lines;2.①Hydrogen peroxide was used to induce the oxidative stress injury in HUVECs cultured in vitro;②To study the expression of visfatin in HUVECs under normal or oxidative stress injury situation.3. To study the effect of visfatin on the apoptosis of HUVECs and expression of various cytokines under normal and oxidative stress injury condition in vitro;4. To discuss the influencing factor of plasma visfatin and its correlation to coronary heart disease (CAD) and abnormal glucose metabolism. EZscan system and laboratory method were used to screen sugar metabolic abnormalities of patients with CAD to study the clinical application value and prospect of eZscan system.Methods:1. HUVECs were obtained from human umbilical vein digested with0.1%collagenase type Ⅱ and cultivated in M199complete medium containing20μg/mL ECGF. HUVEC-CS and EA.hy926were cultured in DMEM complete medium. Subculture was attained with trypsin/EDTA digestion when cells were90%confluent. Cell morphology was observed under inverted phsase contrast microscope, immunofluorescent staining and flow cytometry (FCM) were used to test the expression of VWF, CD31and CD34of these three cells;2.①EA.hy926cell lines subcultured in vitro were divided into two groups:model group and control group. Six concentration gradients (H2O2final concentration was100,200,400,600,800or100μmol/L) were tested to find out the optimal effect concentration in dose effect of intervention in model group. HUVECs were treated with the optimal concentration of H2O2for6hours,12hours or24hours to find out the optimal effect time in time effect of intervention. HUVECs were collected and tested:cells morphology change were observed under inverted phase contrast microscope, cell counting kit (CCK-8) was applied to test cell viability rate; DCFH-DA fluorescence probe and FCM was applied to test intracellular reactive oxygen levels, FCM TUNEL method was used to test cell apoptosis rate and proliferation rate;②EA.hy926cell line passages sbucultured in vitro was divided into two groups randomly:control group and H2O2treatment group (H2O2final concentration was50,100,200,400or1000μmol/L) and testing was conducted after1hour,2,4or24hours of cell culture. Visfatin protein and gene expression were tested via western blot and real time PCR.3. EA.hy926cell line subcultured in vitro were divided randomly and given corresponding treatment. Cells apoptosis rate and cell cycle were tested by flow cytometry, IL-6, MCP-1, VCAM, VEGF and iNOS concentration were tested quantitatively by ELISA.4. One hundred and sixty patients with CAD were recruited and divided into two groups:CAD combined sugar metabolic abnormalities group and CAD group. Fifty sugar metabolism disorder patients without any organic heart disease were recruited as sugar metabolism disorder group,50healthy persons were recruited as control group at the same time. Fasting laboratory index and plasma visfatin levels were tested and conducted with baPWN and ABI examination in all subjects. Then eZscan system (a system for early detection of diabetes and its complication risk) was applied to screen sugar metabolic abnormalities of established CAD patients via coronary angiography. The results were compared with that of laboratory examination to test the sensitivity and specificity of eZscan system.Results:1. The primary HUVECs were polygon with the characteristic "cobblestone" morphology, while HUVEC-CS and EA.hy926cell lines exhibited rounded or flat shape, also with "cobblestone" morphology. Primary HUVECs and EA.hy926were positive for von Willebrand factor (vWF) immunostaining, whereas HUVEC-CS as negative. The expression of CD31in primary HUVECs (77.93±0.25) or EA.hy926(78.23±0.40) was much higher than that of HUVEC-CS(4.70±0.46), while the expression of CD34in primary HUVECs (43.1±0.20) was much higher than that of HUVEC-CS or EA.hy926(1.20±0.40or0.97±0.40, respectively). The differences were statistically significant (P<0.001).2.①Our results indicated that cell morphology of HUVECs changed a lot in model group:cell outline was ambiguous, cell shape was irregular, cell volume and membrane shrinked, cell arrangement was confused, some cells floated and the characteristic "cobblestone" structure was destroyed to varying degree. Spindle cells reduced, sphere or oval cells appeared and these cells became more and more with the extension of action time and dose increase, which meant that H2O2had harmful effect on HUVECs. CCK-8results indicated the survival rate of HUVECs decreased gradually with the extension of dose increase and action time of H2O2. presenting a certain relationship of dose-effect and time-effect. Results of DCFH-DA fluorescence probe and flow cytometry indicated higher level of intracellular active oxygen. Flow cytometry technique TUNEL method results verified that H2O2did induce the apoptosis of HUVECs and the apoptosis rate increased distinctly with the increase of H2O2concentration;②HUVECs could express visfatin when culturing in vitro. At different H2O2concentration (50,100,200,400or1000μmol/L) for different period of time (1hour,2,4or24hours), significant increase of visfatin levels were detected when treated with low concentration of H2O2(50or100μmol/L), while significant decrease was detected if the concentration of H2O2was higher than200μmol/L with the extension of time (P<0.05); Visfatin levels increased when treating in short time of H2O2(1hour or2hours), while decreased when treating no less than4hours of H2O2with the increase of its concentration (P<0.05). Western blot gray analysis and real time PCR results showed no difference of variation trend of visfatin expression.3. After treating HUVECs with different concentration of visfatin (100,400or800ng/ml) for24/48hours in normal culture, the effect of visfatin on apoptosis rate and proliferation index of cells showed no difference between treating group and control group, while the expression of IL-6, MCP-1, VCAM, VEGF and iNOS showed significantly difference between each group (visfatin100,400,800ng/ml and normal control).After treating HUVECs in oxidative stress with100,400or800ng/ml visfatin for24/48hours, cells apoptosis rate decreased significantly compared with H2O2group, while its apoptosis rate gradually was upregulated with the increasing concentration of visfatin. Any two of the groups showed significantly difference by pairwise comparison (P<0.05). After treating cells with visfatin for24hours, proliferation index of HUVECs in oxidative stress went up than H2O2group, different groups showed significant differences. Between different groups showed no statistical difference (P>0.05) after comparing one to the other, except for statistical difference between visfatin400ng/ml plus H2O2800μmol/L for24hours group and visfatin800ng/ml plus H2O2800μmol/L for24hours group (P>0.05). When the concentration of visfatin rose from100ng/ml to400or800ng/ml, cells proliferation index decreased gradually (P<0.05). After treating cells with visfatin100,400or800ng/ml for48hours, there was statistical difference between any two treatment groups. IL-6, MCP-1, VCAM, VEGF and iNOS expression all showed statistical differences between4treatment groups after treating cells with visfatin for24hours. For VCAM, each group showed statistical differences except between visfatin100ng/ml group and oxidative stress group. IL-6, MCP-1, VCAM, VEGF and iNOS expression all showed statistical differences between any2of4treatment groups after comparing one to the other when treating cells with visfatin for48hours (P<0.05).When treating cells with SB203580(p38MAPK inhibitor) and LY29004(PI3K inhibitor), apoptosis rate and proliferation index of different groups showed statistical differences (P<0.001):H2O2800μmol/L for24h, visfatin400ng/ml+H2O2800μmol/L for24h, SB20358020μmol/L90min+Visfatin400ng/ml for24h+H2O2800μmol/L for24h, LY29400220μmol/L90min+Visfatin400ng/ml for24h+H2O2800μmol/L for24h, any2groups also showed statistical differences (P<0.01) by pairwise comparison. IL-6, MCP-1, VCAM, VEGF and iNOS expression all showed statistical differences between4treatment groups. For MCP-1, each group showed statistical differences (P<0.05) except between SB20358090min treatment group and LY2900490min treatment group. For IL-6, VCAM, VEGF and iNOS, each group showed statistical difference (P<0.05).4. Plasma visfatin level was CAD group, sugar metabolic abnormalities group and control group in turns from high to low. Subgroup analysis indicated that plasma visfatin levels increased progressively among different clinical types of CAD (chronic stable angina pectoris, unstable angina and acute myocardial infarction). The visfatin level of CAD combined sugar metabolic abnormalities group was obviously higher than that of simple CAD patients (P<0.05). Linear dependence analysis results indicated that visfatin was positively correlated to BMI, SBP, TC, LDL-C, FPG, Fins or HOMA-IR (P<0.05). Multiple linear regression analysis results indicated that independence variable BMI and HOMA-IR had significantly effect on plasma visfatin levels (P<0.05). The sensitivity and specificity of eZscan system screening sugar metabolic abnormalities were88.89%and64.95%, respectively. The accuracy of eZscan system screening sugar metabolic abnormalities was expressed by receiver operating characteristic (ROC) curve, with statistical significance (area under the curve was0.769, P=0.000). With the aggravation of coronary stenosis, baPWV showed an increasing trend and ABI was negatively correlated to the degree of coronary stenosis (P<0.05)Conclusion:1. Human umbilical vein digested with collagenase type Ⅱ and cultured in M199complete medium can quickly obtain highly purified endothelial cells. EA.Hy926cell line can be characterized by more amounts of cells with simple culture method and high survival rate. Both of them are good sources for tissue engineering and related basic research;2.①The oxidative stress model in human umbilical vein endothelial cells could be established by coculturing cells with culture medium containing800μmol/L hydrogen peroxide;②Our findings suggest that H2O2had dose-effect and time-effect in promoting the expression of visfatin. H2O2could promote the expression of visfatin in short time or low concentration, while suppress the visfatin expression in long time or high concentration;3.①Our results indicated that apoptosis and proliferation of HUVECs in normal culture in vitro were not affected when treating cells with100,400or800ng/ml visfatin. The expression of IL-6, MCP-1, VCAM, VEGF and iNOS increased with the increasing concentration of visfatin and extension of action time;②Apoptosis rate of HUVECs in oxidative stress injury decreased significantly while its proliferation index rose up when treating cells with100,400or800ng/ml visfatin for24/48hours, compared to H2O2control group. But its effect didn’t increase with the increasing concentration of visfatin. The expression of IL-6, MCP-1, VCAM, VEGF and iNOS increased remarkably with the increasing concentration of visfatin and extension of its action tim;③Pretreating HUVECs with SB203580(p38MAPK inhibitor) and LY29004(PI3K inhibitor) could partially block the effect of visfatin on cell apoptosis, proliferation and expression of inflammatory cytokines, which indicated that the effect of visfatin on apoptosis, proliferation and expression of inflammatory cytokines of HUVECs may be related to p38MAPK signal pathway and PI3K signal pathway, especially the p38MAPK signal pathway;4. Our findings suggest that visfatin may have some effects on the process of development of sugar metabolic abnormalities and atherosclerosis. BMI, HOMA-IR had significantly effect on the level of plasma visfatin. EZscan system was simple, time-saving and noninvasively safe, not subject to fasting restrictions in early screening of sugar metabolic abnormalities, with higher sensitivity and moderate specificity. BaPWV and ABI test was noninvasive, simple, cost-effective and repeatable, playing a role in early screening of high-risk CAD patients and assessing coronary lesions.更多还原