【作者】 朱颖；

【导师】 罗心平； 朱军； 马端； 李剑； 梁旺；

【作者基本信息】 复旦大学 ， 内科学心血管， 2008， 硕士

【摘要】 背景:动脉粥样硬化疾病已成为威胁国人健康的主要疾病之一,而动脉粥样硬化(atherosclerosis,AS)的发生与局部血栓形成密切相关。组织因子(tissue factor,TF)是血栓形成的起始因子,组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)作为TF的生理抑制剂,它有两种同族异形体,TFPI-1和TFPI-2。大量研究证实TFPI-1在抑制血栓形成和血管重塑等方面发挥重要作用;TFPI-2与粥样斑块的稳定性相关,而在动脉粥样斑块形成阶段的作用尚无相关研究,我们前期动物模型研究初步提示TFPI-2在其中具有一定作用。单核细胞源性的泡沫细胞是粥样斑块早期形成阶段的典型病变,贯穿AS疾病发生发展。我们希望以建立泡沫细胞模型为研究的切入点,初步探讨TFPI-2与AS斑块形成之间的联系。目的:1.研究TFPI-2蛋白在U937单核细胞和U937源性泡沫细胞的表达定位;2.观察氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导U937源性泡沫细胞形成过程中,TFPI-2蛋白表达和基因水平的变化规律;3.观察外源性hrTFPI-2蛋白干预,对U937源性泡沫细胞胞内胆固醇蓄积的影响,以确定TFPI-2与泡沫细胞形成是否存在联系,并探讨可能的机制和意义;4.观察泡沫细胞形成中,影响TFPI-2表达的因素。与人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)共同培养,U937源性泡沫细胞中TFPI-2蛋白表达和基因水平的变化规律,并探讨该变化可能存在机制及意义。方法:1.采用改良沉淀法提取人血浆低密度脂蛋白(low density lipoprotein,LDL),硫酸铜氧化制备ox-LDL,与U937单核细胞共同孵育,建立泡沫细胞模型。胞内胆固醇定量测定和油红O染色定性评价建模成功。2.采用双重细胞免疫荧光检测TFPI-2蛋白在泡沫细胞中的表达定位。3.在ox-LDL诱导U937源性泡沫细胞形成过程中,分设观察时间点收集细胞,采用逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction,RT-PCR)半定量分析和荧光定量聚合酶链反应(real-time fluorescent quantitativePCR,FQ-PCR)相对定量分析TFPI-2基因的动态变化;采用时间免疫分辨荧光(time resolved fluorometric immunoassay,TRFIA)定量检测TFPI-2蛋白表达水平。4.设rhTFPI-2不同浓度组(1nM,10nM,50nM,100nM,200nM),分别加入含ox-LDL终浓度为80μg/ml,细胞密度为15×10~4个/ml的U937细胞培液中,共同孵育48h后提取细胞脂质,以脂质测定试剂盒检测,计算胞内胆固醇含量。5.将HUVEC和U937细胞共同培养,分别用RT-PCR和TRFIA检测TFPI-2基因和蛋白的表达水平。6.用SPSS 10.0软件进行统计分析,计量资料以均数±标准差((?)±s)表示,组间比较采用成组t检验分析:P＜0.05表明统计学有显著性差异。结果:1.泡沫细胞模型的建立和鉴定:1.1 ox-LDL的制备和鉴定:分离冠心病患者血浆LDL,经5%琼脂糖电泳证实为单一条带。由硫代巴比妥酸反应物质(thiobarbituric acid reactive substance,TBARS)值反映LDL氧化程度。新鲜LDL值为3.81±0.53 nM MDA/mg,而ox-LDL的值为31.95±2.93 nMMDA/mg,ox-LDL的TBARS值大于LDL的5倍以上,两者相比有显著统计学差异(P＜0.05),提示ox-LDL脂质被有效氧化。同时ox-LDL琼脂糖电泳相对迁移率(relmive electrophoresis mobility,REM)升高,亦证实ox-LDL制备成功。1.2 ox-LDL造模浓度确立和泡沫细胞模型鉴定:以细胞内胆固醇定量和油红染色定性,作为评价泡沫细胞的指标。不同浓度组的ox-LDL与U937细胞共同孵育48h后,80mg/L ox-LDL组的胞内胆固醇酯/总胆固醇(CE/TC)值大于50%,光镜下观察油红O染色,胞浆内出现大量红染颗粒和脂质空泡;80mg/L LDL组和60mg/L ox-LDL组的CE/TC值均低于50%;120mg/L ox-LDL组的CE/TC值虽大于50%,但凋亡细胞数量显著增多,不利于后续实验;故选择80mg/Lox-LDL与U937细胞(细胞密度15×10~4个/ml)共孵育48h,确定为最佳建模条件。2.TFPI-2蛋白在泡沫细胞内的表达定位:双重细胞免疫荧光证实,TFPI-2蛋白主要定位于U937源性泡沫细胞胞浆,与TF蛋白有类似分布;与正常U937细胞相比较,泡沫细胞中TFPI-2蛋白和TF蛋白的分布情况更趋一致。HUVEC中也存在类似情况,且在氧化损伤情况下,细胞在形态学上的改变,荧光信号强度较正常情况下稍有减弱。3.泡沫细胞形成过程中TFPI-2蛋白及基因表达的动态变化:TRFIA检测发现,U937与ox-LDL孵育6h后,TFPI-2蛋白表达量明显增高达峰值;而在共同孵育6h至48h期间,TFPI-2蛋白表达量降低;孵育12h后,TFPI-2蛋白表达量基本低于正常水平。以看家基因β-action为内参,FQ-PCR结果显示,U937与ox-LDL孵育6h,TFPI-2 mRNA表达上调达至峰值;而6h至48h期间TFPI-2 mRNA表达下调。FQ-PCR结果与蛋白水平的变化相一致。4.外源性TFPI-2蛋白对泡沫细胞胞内胆固醇的影响:分设rhTFPI-2不同浓度组(1nM,10nM,50nM,100nM,200nM),加入含ox-LDL终浓度为80μg/ml的U937细胞培液中。比较各浓度组与空白组(OnM)之间数据发现:1nM组干预48h后TC和CE/TC水平无明显统计学差异(FC:24.733±1.504 vs 23.130±2.341,P＞0.1:TC::50.775±2.831 vs 52.226±1.662,P＞0.1),CE/TC为51.29%;在10nM,50nM,100nM,200nM组干预48h后,各组FC值分别为21.041±1.702、20.911±2.012、20.790±1.127、20.552±2.733;各组TC值分别为38.707±2.011、34.956±2.562、32.118±1.920、32.168±3.023;各组CE/TC值分别为45.64%、40.18%、35.27%、36.11%,各组TC和CE/TC均明显降低,与空白组(0nM)相比有显著统计学差异(P＜0.05)。外源性TFPI-2蛋白干预可减少泡沫细胞的胞内胆固醇蓄积,该作用呈一定浓度依赖方式,TFPI-2与泡沫细胞形成存在关联。5.内皮细胞对泡沫细胞形成中TFPI-2表达的影响:5.1 ox-LDL对内皮细胞TFPI-2表达的影响:HUVEC与ox-LDL(80mg/L)共同孵育48h,其TFPI-2蛋白表达量呈先升高后降低的动态改变;共同孵育24h后,其TFPI-2蛋白表达量达峰值。RT-PCR结果显示,在ox-LDL作用24h至48h期间,TFPI-2 mRNA的水平持续增高。提示氧化损伤可能导致内皮细胞TFPI-2蛋白表达障碍。5.2内皮细胞对泡沫细胞形成过程中TFPI-2蛋白表达的影响:与单独培养组比较,U937细胞与HUVEC共同培养时TFPI-2的蛋白表达量明显上调;随着ox-LDL氧化损伤时间的延长,尽管共培养组U937细胞的TFPI-2蛋白表达呈下调趋势,但仍高于U937单独培养组(0h:8.106±0.971 vs 6.291±1.272,P＜0.05;24h:6.219±1.704 vs 4.065±0.551,P＜0.05;48h:5.982±1.392vs 4.163±1.110,P＜0.05)。RT-PCR结果显示,U937细胞和HUVEC共同培养48h后,U937细胞TFPI-2 mRNA水平较单独培养组明显上调。结论:1.80 mg/L ox-LDL加入细胞密度为15×10~4个/ml的U937细胞培液中孵育48h,可成功建立U937源性泡沫细胞模型。2.TFPI-2蛋白主要定位于U937源性泡沫细胞胞浆中,与TF蛋白有相似分布。正常U937细胞中有同样的分布规律,但在泡沫细胞中TFPI-2蛋白和TF蛋白的分布更趋一致。3.U937源性泡沫细胞形成过程中,早期TFPI-2蛋白和基因水平迅速上调,而后期TFPI-2蛋白和基因水平明显下调,低于正常值水平。在泡沫细胞形成过程中存在TPFI-2蛋白和基因水平的相对缺乏。4.外源性中高浓度的rhTFPI-2蛋白可改善U937源性泡沫细胞形成过程中胞内胆固醇蓄积,该效应呈一定的浓度依赖方式;低浓度的rhTFPI-2蛋白无此效应,提示TFPI-2与泡沫细胞形成存在联系。5.与HUVEC共同培养,可以改善U937源性泡沫细胞形成过程中存在的TFPI-2蛋白和基因相对缺乏。更多还原

【Abstract】 BackgroundTissue factor(TF) is a receptor for factor FVⅡ/FVⅡa on a cell surface.It is an important initiator of the exdogenous coagulation cascade,and TF activates exdogenous coagulation cascade and causes thrombosis.TFPI is a member of the Kunitz-type serine protease inhibitor family,which is divided into TFPI-1 and TFPI-2.The key function of TFPI-1 is anti-coagulate,while TFPI-2 as a broad-spectrum serine protease inhibitor can function as an MMP inhibitor.The differences between TFPI-1 and TFPI-2 lead to the discrepancies in the roles they played in a variety of pathological processes in coronary atheroselerotic plaque.Our previous study of animal models vivo suggested that TFPI-2 played a role in the development of atherosclerosis.We intend to properly understand the relationship between foam cell and TFPI-2,through the research on foam cell model.The study will provide a useful reference for the strategy of inhibiting lipid accumulation by upregulating TFPI-2 levels and supply a clue for finding a new therapeutic method.Objective:1.To investigate the location of tissue factor pathway inhibitor-2(TFPI-2) protein in U937 cell-derived foam cells.2.To observe the regularity of expression of tissue factor pathway inhibitor-2 (TFPI-2) in the development of U937 cell-derived foam cells,as well as their mRNA.3.To investigate the effect of different dose groups of rhTFPI-2 protein on the lipid accumulation of U937 cell-derived foam cells.4.To observe the regularity of expression of TFPI-2 in the U937 cell-derived foam cells,when cocultured with HUVEC,as well as their mRNA.Methods:1.Low density lipoprotein was prepared by recipetation,Foam cell model was established by incubating U937 cells with 80mg/L oxidized low density lipoprotein for 48 hours.The lipid within cells was detected with oil red O.Cholestaerol assay kits was used for quantitative analysis of intracellular cholesterol and cholesteryl esters.2.Double immuofluorescence was used to detect the location of TFPI-2 protein. 3.The expression of TFPI-2 protein was examined by TRFIA.The expression of TFPI-2 mRNA was examined by RT-PCR and FQ-PCR.4.Detect the cellular cholesterol contents of U937 cells treated with rhTFPI-2 in different dose groups(1nM,10nM,50nM,100nM,200nM).5.After HUVEC and U937 cells were cultured together for 48 hours,the protein and mRNA level of TFPI-2 were evaluated by TRFIA and RT-PCR,respectively.6.The results were presented as(?)±S,and the differences among the groups were tested by t test.SPSS 10.0 was used in the data processing.A two-tailed P value＜0.05 was considered as significant.Results:1.Preparation and definition of ox-LDL:1.1 LDL was prepared by recipetation,was definited by single band in the 5% gel electrophoresis.The TBARS value of native LDL was 3.81±0.53,while the value of ox-LDL was 31.95±2.93.Two groups showed significant difference(p＜0.05).And the REM of ox-LDL in the 5%gel electrophoresis was increased.1.2 Preparation and definition of foam cell model.After U937 cells were incubated with different doses of ox-LDL respectively for 48 hours,cholestaerol assay confirmed the value of CE/TC in 80mg/L ox-LDL group was over 50%,but the value of CE/TC in 80mg/L LDL group and 60mg/L ox-LDL group were not less than 50%. Although the value of CE/TC in 120mg/L ox-LDL group was over 50%,the number of apoptotic cells was significant raised.Oil red O staining showed a plenty of red particles and lipid droplets accumlated in U937 cells.A novel human monocyte-derived foam cell model was established by incubating U937 cells with 80 mg/L oxidized low density lipoprotein for 48 hours.2.Double immuofluorescenct staining indicated that TFPI-2 protein located mainly in the cytoplasm of U937 cell-derived foam cells,similar as the location of TF protein.The distributions of TFPI-2 protein and TF protein were more coincident in foam cells,compared with normal U937 cells.3.After U937 cells incubated with 80mg/L ox-LDL for 6 hours,TFPI-2 mRNA and protein expression levels reached peak value,then the TFPI-2 levels declined along with time,were lower than the normal value.4.Compared different dose groups of rhTFPI-2 with blank group(0nM),the values of TC and CE/TC in 1nM group were FC:24.733±1.504 vs 23.130±2.341, P＞0.1;TC:50.775±2.831 vs 52.226±1.662,P＞0.1,CE/TC 51.29%,these two groups had no significant difference.The values of FC in 10nM group,50nM group, 100nM group,200nM group were 21.041±1.702,20.911±2.012,20.790±1.127, 20.552±2.733.The value of TC in 10nM group,50nM group,100nM group, 200nM group were 38.707±2.011,34.956±2.562,32.118±1.920,32.168±3.023. The values of CE/TC in 10nM group,50nM group,100nM group,200nM group were 45.64%,40.18%,35.27%,36.11%.The values of TC and CE/TC both decreased, and the data were significantly different compared with the blank group(P＜0.05).5.Influences ofcoculture with HUVEC on foam cells:5.1 After HUVEC incubated with 80mg/L ox-LDL for 24 hours,TFPI-2 protein expression levels reached peak value,then values were decreased in the subsequent 24 hours.However the levels of TFPI-2 mRNA were upregulated continually.5.2 Compared with the expression of TFPI-2 protein of U937 cells,TFPI-2 protein levels were increased in coculture groups,along with the oxidative injury induced by ox-LDL,the protein levels.were decreased gradually,but always higher than those of U937 culture alone(0h∶8.106±0.971 vs 6.291±1.272,P＜0.05;24h∶6.219±1.704 vs 4.065±0.551,P＜0.05;48h∶5.982±1.392 vs 4.163±1.110,P＜0.05).The PT-PCR results showed that TFPI-2 mRNA of U937 cells was upregulated in coculture group.Conclusion:1.A human monocyte-derived foam cell model was established by incubating U937 cells with 80mg/L oxidized low density lipoprotein for 48 hours.2.TFPI-2 protein located mainly in the cytoplasm of U937 cell-derived foam cells,similar as the location of TF protein.The distributions of TFPI-2 protein and TF protein were more coincident in foam cells,compared with normal U937 cells.3.The levels of TFPI-2 expression were enhanced in the early stage of foam cell formation,then reduced in the later stage.There was relative deficiency of TFPI-2 in the development of foam cell.4.The middle and high dose of rhTFPI-2 could reduce the lipid accumulation in the development of foam cell in a dose-dependent manner.5.The coculture of HUVEC with U937 cells could modify the relative deficiency of TFPI-2 mRNA and protein in the development of foam cell.更多还原