TRAIL对高糖诱导的内皮损伤的保护作用及机制研究

发布时间：2018-04-04 05:54

本文选题：肿瘤坏死因子相关的凋亡诱导配体　切入点：内皮　出处：《南方医科大学》2014年硕士论文

【摘要】：研究背景随着社会经济的发展,人口的老龄化和人们生活水平的提高,糖尿病(Diabetes mellitus, DM)的患病率逐年攀升,严重威胁着人们的身心健康。糖尿病大血管病变,如动脉粥样硬化、血栓性大血管病变所致的冠心病、脑血管及周围血管疾病是糖尿病患者高死亡率和致残的主要原因之一。长期高血糖是导致血管内皮功能障碍的主要危险因素。而内皮功能障碍是糖尿病血管病变的始动因素和早期表现,在糖尿病血管病变的发生发展中起着重要作用。因此,防治高糖导致的内皮细胞损伤、保护血管内皮功能,对防治糖尿病心血管并发症有重要意义。肿瘤坏死因子相关的凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)是肿瘤坏死因子家族的新成员,在细胞凋亡、炎症反应和免疫应答过程中有重要的调控作用。TRAIL可与肿瘤坏死因子家族的五种高亲和力受体结合。TRAIL-R1(DR4)和TRAIL-R2(DR5)作为死亡受体,与TRAIL结合后能够诱导凋亡信号途径的激活,导致细胞凋亡；而TPRAIL-R3(DcRl)、TRAIL-R4(DcR2)和OPG (osteoprotegerin)缺少功能性死亡域和诱导细胞凋亡的能力,作为诱导性受体,可与TRAIL-R1(DR4)和TRAIL-R2(DR5)竞争性的与TRAIL结合,具有抑制正常细胞凋亡的作用。近年来,有实验数据表明,TRAIL在糖尿病及心血管疾病中起着重要的作用。体内和体外研究数据均证明TRAIL有潜在的心血管保护作用。体外研究显示,在缺乏培养基的条件下,重组TRAIL可通过激活Akt和ERK通路促进人脐静脉内皮细胞的生存和增殖。同时,有体内研究表明,在使用重组TRAIL干预ApoE-／-鼠的糖尿病模型时,可观察到TRAIL通过增加血管平滑肌细胞的含量以稳定动脉粥样硬化斑块,并降低斑块的整体面积；并且有研究表明,TRAIL-/-联合Apoe-/-敲除鼠的动脉粥样硬化发生率及程度明显高于Apoe-／-敲除鼠。最近三项体内研究已经表明,TRAIL的血清水平在糖尿病和动脉粥样硬化患者显着降低,并且TRAIL水平降低程度与内皮功能呈正相关。然而,迄今为止,TRAIL干预是否对高糖导致血管内皮功能障碍有保护作用尚不清楚。因此,我们假设TRAIL可保护高糖导致的血管内皮损伤。本研究主要从体内和体外两部分实验来评估TRAIL对血管内皮的作用。研究目的本研究旨在明确： 1.研究TRAIL对糖尿病大鼠血管内皮功能障碍是否有保护作用； 2.研究TRAIL对体外高糖环境导致的氧化应激及内皮细胞凋亡的作用； 3.探讨体外高糖环境下TRAIL对内皮细胞作用的可能信号转导机制。方法一、动物实验 1.4周龄雄性SD大鼠30只,随机留取10只作为正常对照(NC)组,以普通饲料喂养,其余20只用于建立2型糖尿病模型,以高脂饲料喂养。6周后所有大鼠禁食禁水12h,给予高脂组STZ35mg/Kg(用前以0.1mol/L的柠檬酸-柠檬酸钠缓冲液配制成1%STZ溶液)一次性腹腔注射,普通饲料组则给予等容量的柠檬酸盐缓冲液一次性腹腔注射。诊断标准为空腹血糖(FPG)≥16.7mmol/L。将造模成功大鼠随机分为对照(DM)组(10只)和TRAIL干预组(10只),同时TRAIL干预组予重组TRAIL(20ug/只)腹腔注射,每周1次,连续6周。NC与DM组予等量的蒸馏水腹腔注射,连续6周。 2.实验结束时,腹腔注射1%戊巴比妥钠50mg·kg-1麻醉大鼠,迅速开胸,腹主动脉采血后,迅速分离并取出胸主动脉,观察大鼠离体主动脉对乙酚胆碱(Ahc)依赖性血管舒张反应。ELISA试剂盒检测血浆TRAIL浓度；Griess重氮化反应法检测主动脉NO含量；免疫组化SP法检测主动脉eNOS的活性；Western blot检测主动脉eNOS和P-eNOS的表达水平。二、细胞实验 1.细胞培养：使用胶原酶灌注新鲜的脐带提取原代人脐静脉内皮细胞(HUVECs)。HUVECs培养在明胶包被的组织培养板中,并在M199内皮细胞生长培养基中添加20%FBS,10ug/m1肝素和50ug/mlECGF(血管内皮细胞生长因子)。所有实验,使用第2-4代细胞。 2.细胞分组：实验分为5组；(1)正糖对照组(葡萄糖终浓度为5.5mmol/L,NG)；(2)高糖组(葡萄糖终浓度33mmol/L,HG)；(3)HG+TRAIL组(100ng/ml TRAILy预先培养18h,再加入终浓度33mmo1/L的葡萄糖)；(4)HG+TRAIL+LY组(内皮细胞先经含10μmol/L的3一磷脂酰肌醇激酶(PI3K)阻断剂LY294002的培养基预处理30min,余处理同HG+TRAIL组)；(5)HG+TRAIL+L-NAME组(内皮细胞先经含10Oμmol/L的eNOS的阻断剂L-NAME的培养基预处理30min,余处理同HG+TRAIL组)。 3.Annexin-V-FITC/PI双染法和TUNEL法检测细胞凋亡；以DCFH-DA为探针检测细胞的ROS；实时定量RT-PCR检测细胞的SOD,GPx-1的表达；Westernblot分析主动脉Akt,P-Akt,eNOS,P-eNOS的表达。统计分析采用SPSS13.0进行统计学处理。实验数据以x±s表示。两组之间比较使用独立样本t检验。多组间比较使用One-Way ANOVA(单因素方差分析)方法,两组间比较用最小显著差(LSD)t检验,以P0.05为显著性指标。结果一、动物实验结果 1.糖尿病组大鼠血浆TRAIL浓度显著低于正常组(56.6±5.5vs.36.5±3.1,P0.001) 2.TRAIL对糖尿病大鼠主动脉内皮依赖性血管舒张功能的影响与对常对照组比较,糖尿病组大鼠胸主动脉内皮依赖性血管舒张功能明显降低[(63.5±4.6)%vs.(93.5±2.6)%,P0.001]。与糖尿病组比较,TRAIL干预组血管舒张功能明显改善,[(63.5±4.6)%vs.(78.44±2.7)%(P0.001)]。而NaN02诱导的内皮非依赖性血管舒张反应在三组大鼠中无明显差异。TRAIL干预糖尿病大鼠能明显减轻ACh诱导的内皮依赖性舒张功能损伤而对内皮非依赖性血管舒张功能无明显影响。这些结果显示TRAIL干预明显改善了糖尿病引起的内皮依赖性舒张功能障碍。 3.各组大鼠主动脉NO含量和eNOS免疫组化的结果与NC组比较,TRAIL、DM组大鼠主动脉NO浓度、eNOS免疫组化阳性表达显著降低(P0.001);与糖尿病对照组比较,TRAIL组主动脉NO浓度,主动脉内皮eNOS阳性表达显著升高(p0.001)。 4.TRAIL对大鼠胸主动脉组织中eNOS蛋白表达的影响与正常对照组比较,P-eNOS蛋白水平在糖尿病组明显降低(P＜0.01)。 TRAIL干预6周显著提高糖尿病大鼠胸主动脉组织P-eNOS蛋白表达水平(P0.01)。二、细胞实验结果 1.TUNEL染色：高糖组干预细胞48h后,细胞凋亡率较正糖对照组明显上升(5.9±0.8%vs.37.4±0.9%,p0.001),而高糖+TRAIL组的细胞凋亡率较高糖对照组降低(17.2±0.4%vs.37.4±0.9%,p0.001)。HG+TRAIL+LY组与HG+TRAIL+L-NAME组细胞凋亡率较正糖对照组明显升高,而与高糖对照组无明显差异(p0.001),表明TRAIL抑制细胞的凋亡作用被LY294002和L-NAME阻断。 2. AnnexinVFITC/PI:与正糖对照组相比,高糖对照组内皮细胞调亡率明显增加(p0.01),高糖+TRAIL组的细胞凋亡率较高糖对照组降低。而HG+TRAIL+LY组与HG+TRAIL+L-NAME组细胞凋亡率较正糖对照组明显升高,而与高糖对照组无明显差异(p0.01)。 3.TRAIL对细胞内ROS生成的影响：正常对照组内皮细胞内荧光强度较弱,ROS产生较低；高糖作用于内皮细胞48h后,内皮细胞内荧光强度很强,ROS产生明显多于对照组。TRAIL干预组内皮细胞ROS的生成明显降低；TRAIL+高糖+LY组及TRAIL+高糖+L-NAME组细胞内荧光强度明显高于TRAIL+高糖组,ROS生成增多。 4.实时定量PCR检测细胞内SOD、GPx-1的表达：与正糖对照组相比,高糖对照组SOD、GPx-1表达明显减低(p0.001),高糖+TRAIL组的SOD、GPx-1较高糖对照组升高。而HG+TRAIL+LY组与HG+TRAIL+L-NAME组SOD、GPx-1表达较正糖对照组明显降低,而与高糖对照组无明显差异(p0.001)。 5.TRAIL对细胞内Akt, P-Akt, eNOS, P-eNOS蛋白表达的影响：与正糖对照组相比,高糖对照组P-Akt, P-eNOS表达明显减低(p0.001),高糖+TRAIL组的P-Akt,P-eNOS较高糖对照组升高。而HG+TRAIL+LY组与HG+TRAIL+L-NAME组SOD、GPx-1表达较正糖对照组明显减低,而与高糖对照组无明显差异(p0.001)。TRAIL对细胞内Akt,eNOS的表达无明显影响。结论 1.TRAIL干预可明显改善糖尿病大鼠的主动脉内皮依赖性舒张功能,提示TRAIL对内皮功能有一定的保护作用。由于TRAIL可促P-eNOS的表达和NO的生成,并提示TRAIL保护内皮功能的作用可能是通过促进eNOS的表达和NO的生成而达到的。 2.TRAIL能对抗体外高糖诱导的人脐静脉内皮细胞凋亡,抑制ROS的生成,促进SOD、GPx-1mRNA的表达,并且上调P-Akt,P-eNOS的表达,并且TRAIL的这种保护作用可以被PI3K阻断剂LY294002和eNOS的阻断剂L-NAME消除。从而进一步证明TRAIL对体外高糖诱导内皮细胞损伤的保护足有可能是通过PI3K/Akt/eNOS途径介导的。
[Abstract]:Research background
With the development of social economy, the aging of the population and the improvement of people's living standard, diabetes (Diabetes mellitus, DM) the prevalence rate increased year by year, a serious threat to people's physical and mental health. Diabetic macrovascular disease, such as atherosclerosis, coronary heart disease caused by thrombotic macrovascular disease, cerebrovascular and peripheral vascular disease is one of the most important the reason of high mortality in patients with diabetes and disability. The long-term high blood sugar is the main risk factors. Endothelial dysfunction and endothelial dysfunction is the initiating factor and early manifestation of diabetic angiopathy, in diabetic vasculopathy plays an important role in the development. Therefore, prevention and treatment of injury of endothelial cells induced by high glucose, protection of vascular endothelial function there is an important significance for the prevention of cardiovascular complications of diabetes.
Tumor necrosis factor related apoptosis inducing ligand (TNF-related apoptosis-inducing, ligand, TRAIL) is a new member of the tumor necrosis factor family, in the process of cell apoptosis, inflammatory reaction and immune response in five kinds of high affinity receptor important regulatory role in.TRAIL and the tumor necrosis factor family with.TRAIL-R1 (DR4) and TRAIL-R2 (DR5) as the death receptor, when combined with TRAIL can activate apoptosis pathway, leading to cell apoptosis; TPRAIL-R3 (DcRl), TRAIL-R4 (DcR2) and OPG (osteoprotegerin) lacks the ability of functional domain and death inducing apoptosis, as induced receptor, and TRAIL-R1 (DR4) and TRAIL-R2 (DR5) competition the combined with TRAIL can inhibit the apoptosis of normal cells.
In recent years, with the experimental data show that TRAIL plays an important role in diabetes and cardiovascular disease. In vivo and in vitro data have demonstrated that TRAIL has a role in cardiovascular protection potential. In vitro studies have shown that, in the absence of medium conditions, the recombinant TRAIL can activate Akt and ERK pathway to promote the survival and proliferation of human umbilical vein endothelial cells. At the same time, with the in vivo study showed that the use of recombinant TRAIL in diabetic model rats and intervention of ApoE- /, TRAIL could be observed by the content of vascular smooth muscle cells to increase the stability of atherosclerotic plaque, and reduce the overall area of plaque; and studies have shown that TRAIL-/- combined with Apoe-/- knockout mice and the rate of atherosclerosis was obviously higher than that of Apoe- / - knockout mice. Three in vivo studies have recently shown that the serum level of TRAIL in diabetes and atherosclerosis patients were significantly reduced Low, and lower the level of TRAIL was positively related with the degree of endothelial function. However, so far, TRAIL intervention whether endothelial dysfunction has a protective effect on high glucose induced is unclear. Therefore, we hypothesized that TRAIL can protect the vascular endothelial injury induced by high glucose. The study is mainly from two parts in vivo and in vitro experiments to evaluate the effect of TRAIL on blood vessels endothelial.
research objective
The purpose of this study is to clarify:
1. study the protective effect of TRAIL on vascular endothelial dysfunction in diabetic rats.
2. study the effect of TRAIL on oxidative stress and endothelial cell apoptosis induced by high glucose in vitro.
3. to investigate the possible signal transduction mechanism of TRAIL on endothelial cells in high glucose environment in vitro.
Method
First, animal experiment
1.4 week old male SD 30 rats were randomly selected from 10 rats as normal control group (NC), fed with normal diet, the other 20 were used to establish the model of type 2 diabetes mellitus, with high fat diet for.6 weeks all rats after fasting 12h, given the high fat group STZ35mg/Kg (using 0.1mol/L the citric acid sodium citrate buffer solution into 1%STZ solution) by intraperitoneal injection, normal diet group were treated citrate buffer by intraperitoneal injection. The capacity of diagnostic criteria for fasting blood glucose (FPG) aged 16.7mmol/L. rats were randomly divided into control group (DM) (10) and the intervention group (TRAIL = 10), and TRAIL treated group were treated by recombinant TRAIL (20ug/) by intraperitoneal injection, 1 times a week, intraperitoneal injection of distilled water for 6 weeks.NC and DM group were given, for 6 consecutive weeks.
2. at the end of the experiment, intraperitoneal injection of 1% pentobarbital sodium 50mg kg-1 rats were anesthetized, quickly open the chest, abdominal aorta, rapid separation and remove the thoracic aorta of rats were observed in isolated aorta of acetylcholine (Ahc) - dependent vasorelaxation.ELISA kit to detect the concentration of plasma TRAIL; detection of aortic NO content Griess diazotization reaction method; SP immunohistochemical method to detect aortic eNOS activity; the expression level of Western blot and P-eNOS eNOS was detected.
Two, cell experiment
1. cell culture: use fresh umbilical cord collagenase extracted from primary human umbilical vein endothelial cells (HUVECs) of.HUVECs cultured on gelatin coated tissue culture plates, and add 20%FBS M199 in endothelial cell growth medium, 10ug/m1 heparin and 50ug/mlECGF (vascular endothelial growth factor). All experiments, the use of the 2-4 generation of cells.
The 2. group: cells were divided into 5 groups; (1) normal glucose control group (glucose final concentration of 5.5mmol/L, NG); (2) high glucose group (glucose final concentration of 33mmol/L, HG); (3) group HG+TRAIL (100ng/ml TRAILy 18h pre culture, adding a final concentration of 33mmo1/L glucose (4); (group HG+TRAIL+LY) by endothelial cells 3 phosphatidylinositol kinase containing 10 mol/L (PI3K) blocker LY294002 medium pretreatment 30min, I deal with the HG+TRAIL group); (5) group HG+TRAIL+L-NAME (endothelial cells by blocking agent L-NAME medium containing 10O 30min pretreatment, mol/L I deal with the eNOS HG+TRAIL group).
3.Annexin-V-FITC/PI double staining and TUNEL method were used to detect apoptosis. DCFH-DA was used to detect ROS in cells. Real-time quantitative RT-PCR was used to detect SOD and GPx-1 expression. Westernblot analysis was used to analyze the expression of Akt, P-Akt, eNOS and Akt in the aorta.
Statistical analysis were carried out by SPSS13.0. The experimental data expressed by X + s. The comparison between the two groups using independent samples t test. Comparison among multiple groups using One-Way ANOVA (ANOVA) method, two groups using least significant difference (LSD) t test, with P0.05 as the significant indexes. Results a animal. The experimental results
1. the plasma TRAIL concentration in the diabetic rats was significantly lower than that in the normal group (56.6 + 5.5vs.36.5 + 3.1, P0.001)
Effect of 2.TRAIL on endothelium-dependent vasodilatation of aorta in diabetic rats
With the constant control group, diabetic rats aorta endothelium dependent vasodilation significantly reduced [(63.5 + 4.6)%vs. (93.5 + 2.6)%, compared with diabetic group P0.001]., TRAIL group vascular diastolic function improved obviously, [(63.5 + 4.6)%vs. (78.44 + 2.7)% (P0.001). And NaN02 induced endothelium dependent vasodilation in rats of the three groups had no significant difference in.TRAIL intervention diabetic rats can significantly reduce ACh induced endothelium dependent diastolic function and damage of endothelium dependent vasodilation without significant impact. These results suggest that endothelial TRAIL intervention significantly improved diabetes due to the dependence of diastolic dysfunction.
3. NO content of aorta and immunohistochemical results of eNOS in rats of each group
Compared with group NC, the NO concentration in aorta and eNOS immunohistochemical expression in TRAIL and DM groups were significantly decreased (P0.001). Compared with diabetic control group, the NO concentration of aorta and the positive expression of eNOS in aorta were significantly increased (p0.001).
The effect of 4.TRAIL on the expression of eNOS protein in the thoracic aorta of rats
Compared with the normal control group, the level of P-eNOS protein in the diabetic group was significantly decreased (P < 0.01). TRAIL intervention significantly increased the expression level of P-eNOS protein in the thoracic aorta tissue of diabetic rats for 6 weeks (P0.01). Two, the results of cell experiment.
1.TUNEL staining: the high glucose group intervention after 48h cells, the apoptosis rate was significantly increased than glucose control group (5.9 + 0.8%vs.37.4 + 0.9%, p0.001), and the apoptosis rate of +TRAIL group compared with high glucose high glucose control group decreased (17.2 + 0.4%vs.37.4 + 0.9%, p0.001) of.HG+TRAIL+LY group and HG+TRAIL group +L-NAME cells apoptosis rate than normal glucose the control group was significantly increased, while no significant difference between the control group and high glucose (p0.001), showed that the apoptosis of TRAIL cells by LY294002 and L-NAME block.
2. AnnexinVFITC/PI: normal glucose group, high glucose control group endothelial cell apoptosis rate increased significantly (P0.01), the apoptosis rate of +TRAIL group compared with high glucose and high glucose control group HG+TRAIL+LY decreased. The apoptosis rate of group HG+TRAIL+L-NAME was normal glucose control group increased significantly, but no significant difference between the control group and high glucose (P0.01).
Effect of 3.TRAIL on intracellular ROS generation: normal control group of weak fluorescence intensity in endothelial cells, ROS production is low; the effect of high glucose on endothelial cells after 48h, endothelial cells with strong fluorescence intensity, ROS produced significantly more than the control group.TRAIL group generated endothelial cells ROS significantly decreased; TRAIL+ high glucose group +LY and TRAIL+ high glucose group +L-NAME fluorescence intensity in cells of high glucose group was significantly higher than that of TRAIL+, ROS production increased.
4. real time quantitative PCR detection of SOD cells, the expression of GPx-1: normal glucose group, high glucose control group SOD, GPx-1 expression was significantly reduced (p0.001), high glucose group +TRAIL SOD GPx-1, compared with the high glucose control group increased. While the HG+TRAIL+LY group and HG+TRAIL+L-NAME group SOD, the expression of GPx-1 was positive in sugar control group was significantly reduced. But with the high glucose control group had no significant difference (p0.001).
5.TRAIL on intracellular Akt, P-Akt, eNOS, P-eNOS protein expression: compared with normal glucose control group, high glucose control group P-Akt, P-eNOS expression was significantly reduced (p0.001), high glucose group +TRAIL P-Akt P-eNOS, compared with the high glucose control group increased. While the HG+TRAIL+LY group and HG+TRAIL+L-NAME group SOD, the expression of GPx-1 was positive glucose control group decreased significantly, and high glucose control group had no significant difference (p0.001) of.TRAIL Akt in cells, no significant effect on the expression of eNOS. Conclusion
1.TRAIL intervention can significantly improve diabetic rat aortic endothelium dependent diastolic function, suggesting that TRAIL has a protective effect on endothelial function. The expression of NO and TRAIL can promote the formation of P-eNOS, and suggests that the endothelial function of TRAIL protection effect might be achieved by generating NO and promote the expression of eNOS.
2.TRAIL can antagonize the in vitro high glucose induced apoptosis of human umbilical vein endothelial cells, inhibit ROS generation, promote the expression of GPx-1mRNA, SOD, and up regulation of P-Akt P-eNOS expression, and the protective effect of TRAIL could be blocked by PI3K blocking agent L-NAME LY294002 elimination and eNOS. In order to further prove the protection of TRAIL on high glucose induced endothelial in vitro cell injury foot may be mediated by PI3K/Akt/eNOS pathway.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2

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