U937泡沫细胞中组织因子途径抑制物2的表达及变化规律的实验研究

发布时间：2018-03-07 07:44

本文选题：动脉粥样硬化　切入点：U937源性泡沫细胞　出处：《复旦大学》2008年硕士论文　论文类型：学位论文

【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。
[Abstract]:Background:
Atherosclerotic disease has become one of the main diseases that threaten the health of the Chinese people, and atherosclerosis (atherosclerosis, AS) and the occurrence of thrombosis are closely related to tissue factor (tissue factor, TF) is the initial factor of thrombosis, tissue factor pathway inhibitor complexes (tissue factor pathway inhibitor, TFPI) as a physiological inhibitor of TF. It has two isoforms TFPI-1 and TFPI-2. family, a large number of studies have confirmed that TFPI-1 play an important role in inhibiting thrombosis and vascular remodeling; stability of TFPI-2 and atherosclerotic plaque, and in the formation of atherosclerosis plaque stage has not been studied in our previous animal model study indicates that TFPI-2 has a certain role in the single. Monocyte derived foam cells of atherosclerotic plaque formation is the typical early stage of disease, through the development of AS disease. We hope to build The vertical foam cell model is the breakthrough point of the study, and the relationship between TFPI-2 and AS plaque formation is preliminarily discussed.
Objective:
1. the expression of TFPI-2 protein in U937 mononuclear cells and U937 derived foam cells was studied.
2., we observed the change of TFPI-2 protein expression and gene level during the formation of U937 derived foam cells induced by oxidized low density lipoprotein (oxidized low density lipoprotein (ox-LDL)).
3., we observed the effect of exogenous hrTFPI-2 protein intervention on the intracellular cholesterol accumulation of U937 derived foam cells to determine whether there is a relationship between TFPI-2 and foam cell formation, and to explore the possible mechanism and significance.
4. to observe the formation of foam cells, the influence factors of TFPI-2 expression. With human umbilical vein endothelial cells (human umbilical vein endothelial cell, HUVEC) co culture, the expression of TFPI-2 protein and gene level of U937 derived foam cells, and to explore the possible mechanism of change and significance.
Method:
1., we use improved precipitation method to extract low density lipoprotein (LDL) from human plasma, prepare ox-LDL by copper sulfate oxidation, and incubate with U937 mononuclear cells, and establish foam cell model. Intracellular cholesterol quantitative determination and oil red O staining qualitative evaluation and modeling are successful.
2. the expression of TFPI-2 protein in foamy cells was detected by double cell immunofluorescence (double cell immunofluorescence).
The formation process of 3. in ox-LDL induced U937 derived foam cells, divided into the observation time points were collected by reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction, RT-PCR) semi quantitative analysis and fluorescence quantitative polymerase chain reaction (real-time fluorescent quantitativePCR, FQ-PCR) the relative quantitative analysis of dynamic change of TFPI-2 gene by using time resolved fluorescence (immune; time resolved fluorometric immunoassay TRFIA), the expression level of quantitative detection of TFPI-2 protein.
4., set up different concentrations of rhTFPI-2 (1nM, 10nM, 50nM, 100nM, 200nM), add ox-LDL containing 80 ox-LDL g/ml, and 15 cell 10~4 /ml /ml cells, respectively, and extract the cell lipids after incubation.
5. HUVEC and U937 cells were co cultured, and the expression level of TFPI-2 gene and protein was detected by RT-PCR and TRFIA respectively.
6., statistical analysis was done with SPSS 10 software. The measurement data were expressed by mean + standard deviation ((+) s). The comparison between groups was analyzed by group t test: P < 0.05 showed statistically significant difference.
Result:
1. the establishment and identification of the foam cell model:
1.1 ox-LDL preparation and identification:
Separation of plasma LDL in patients with coronary heart disease, by 5% agarose gel electrophoresis confirmed as single bands. By thiobarbituric acid reactive substances (thiobarbituric acid, reactive substance, TBARS LDL) value to reflect the degree of oxidation. The fresh LDL was 3.81 + 0.53 nM MDA/mg, and the value of ox-LDL is 31.95 + 2.93 nMMDA/mg, ox-LDL value of TBARS more than 5 times LDL, there was a statistically significant difference between the two compared (P < 0.05), suggesting that ox-LDL is effective and lipid oxidation. Ox-LDL agarose gel electrophoresis. The relative migration rate (relmive electrophoresis mobility, REM) increased, also confirmed the successful preparation of ox-LDL.
1.2 ox-LDL model concentration establishment and foam cell model identification:
The cellular cholesterol quantitative and qualitative evaluation of oil red staining, as foam cell index. Different concentrations of ox-LDL and U937 cells were incubated for 48h, total cholesterol / 80mg/L cholesterol ester group ox-LDL intracellular (CE/TC) value is greater than 50%, under the light microscope oil red O staining, the cytoplasm appeared large red dye particles and lipid vacuoles; 80mg/L LDL group and 60mg/L ox-LDL group CE/TC values were less than 50% 120mg/L; group ox-LDL CE/TC value is greater than 50%, but the number of apoptotic cells increased significantly, is not conducive to the subsequent experiment; the 80mg/Lox-LDL and U937 cells (cell density of 15 * 10~4 /ml) Co incubated with 48h. To determine the best condition of modeling.
Expression of 2.TFPI-2 protein in foamy cells:
Double immunofluorescence confirmed that TFPI-2 protein was mainly localized in U937 derived foam cells have a similar distribution with TF protein; compared with normal U937 cells, the situation is similar to the distribution of more consistent.HUVEC TFPI-2 protein and TF protein in the foam cells, and oxidative damage in the case of cells in morphology the change of fluorescence signal intensity is normally weakened slightly.
3. the dynamic changes of TFPI-2 protein and gene expression during the formation of foamy cells:
TRFIA detection, U937 and ox-LDL after 6h incubation, the expression of TFPI-2 protein was obviously increased up to the peak; and during the incubation of 6h to 48h, the expression of TFPI-2 protein decreased; after 12h incubation, TFPI-2 protein expression was lower than the normal level. The basic housekeeping gene beta -action as a reference, the results of FQ-PCR showed that U937 and ox-LDL were incubated with 6h, TFPI-2 expression of mRNA was up-regulated to peak; consistent during 6h to 48h TFPI-2 mRNA expression and down-regulation of.FQ-PCR protein level results.
4. the effect of exogenous TFPI-2 protein on intracellular cholesterol in foam cells:
RhTFPI-2 is divided into different concentration groups (1nM, 10nM, 50nM, 100nM, 200nM, ox-LDL) with a final concentration of U937 cell culture medium 80 g/ml. Compared with each concentration group and blank control group (OnM) data found no statistically significant between group 1nM and CE/TC TC after the intervention of 48h (24.733 + FC: level difference 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%; in 10nM, 50nM, 100nM, 200nM group 48h after the intervention, the FC values of each group were 21.041 + 1.702,20.911 + 2.012,20.790 + 1.127,20.552 + 2.733; group TC = 38.707 + 2.011,34.956. 2.562,32.118 + 1.920,32.168 + 3.023; the CE/TC values of each group were 45.64%, 40.18%, 35.27%, 36.11%, TC and CE/TC groups were significantly decreased, and the blank group (0nM) compared with significant difference (P < 0.05). The intervention of exogenous TFPI-2 protein can reduce the intracellular cholesterol less foam cell accumulation, and the effect was In a certain concentration dependent manner, TFPI-2 is associated with the formation of foam cells.
5. the effect of endothelial cells on the expression of TFPI-2 in the formation of foam cells:
The effect of 5.1 ox-LDL on the expression of TFPI-2 in endothelial cells:
HUVEC and ox-LDL (80mg/L) were incubated with 48h, the expression of TFPI-2 protein showed the dynamic change increased first and then decreased; after 24h incubation, the expression of TFPI-2 protein reached the peak.RT-PCR results show that during the ox-LDL 24h to 48h, TFPI-2 continued to increase the level of mRNA. Suggested that oxidative damage may lead to endothelial expression the expression of TFPI-2.
5.2 the effect of endothelial cells on the expression of TFPI-2 protein during the formation of foam cells:
With the single culture group, U937 and HUVEC cells co cultured with the TFPI-2 protein expression was significantly up-regulated; with the prolonged oxidative ox-LDL damage, although the co culture group U937 cells TFPI-2 protein expression decreased, but still higher than that of U937 cultured alone. (0h:8.106 + 0.971 vs 6.291 + 1.272, P < 0.05; 24h:6.219 4.065 + 1.704 vs + 0.551, P < 0.05; 48h:5.982 + 1.392vs 4.163 + 1.110, P < 0.05) the results of.RT-PCR showed that U937 and HUVEC cells after cultured 48h U937 cells, TFPI-2 mRNA levels were significantly increased compared with single culture.
Conclusion:
1.80 mg/L ox-LDL incubated 48h in the U937 cell culture of 15 x 10~4 /ml cells, and the U937 derived foam cell model could be successfully established.
2.TFPI-2 protein is mainly located in cytoplasm of U937 derived foam cells, and has similar distribution with TF protein. Normal U937 cells have the same distribution pattern, but the distribution of TFPI-2 protein and TF protein is more consistent in foam cells.
In the process of 3.U937 derived foam cell formation, the TFPI-2 protein and gene level increased rapidly at the early stage, while the TFPI-2 protein and gene level in the later stage were significantly down regulated, which was lower than the normal level. There was a relative lack of TPFI-2 protein and gene level in the foam cell formation process.
4., exogenous high and high concentration of rhTFPI-2 protein can improve intracellular cholesterol accumulation during the formation of U937 derived foam cells. The effect is in a concentration dependent manner. Low concentration of rhTFPI-2 protein has no such effect, suggesting that TFPI-2 is related to foam cell formation.
The co culture of 5. with HUVEC can improve the relative lack of TFPI-2 protein and gene in the formation of U937 derived foam cells.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【相似文献】