本文選題：腫瘤壞死因子相關(guān)的凋亡誘導(dǎo)配體　切入點(diǎn)：內(nèi)皮　出處：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 隨著社會(huì)經(jīng)濟(jì)的發(fā)展,人口的老齡化和人們生活水平的提高,糖尿病(Diabetes mellitus, DM)的患病率逐年攀升,嚴(yán)重威脅著人們的身心健康。糖尿病大血管病變,如動(dòng)脈粥樣硬化、血栓性大血管病變所致的冠心病、腦血管及周圍血管疾病是糖尿病患者高死亡率和致殘的主要原因之一。長(zhǎng)期高血糖是導(dǎo)致血管內(nèi)皮功能障礙的主要危險(xiǎn)因素。而內(nèi)皮功能障礙是糖尿病血管病變的始動(dòng)因素和早期表現(xiàn),在糖尿病血管病變的發(fā)生發(fā)展中起著重要作用。因此,防治高糖導(dǎo)致的內(nèi)皮細(xì)胞損傷、保護(hù)血管內(nèi)皮功能,對(duì)防治糖尿病心血管并發(fā)癥有重要意義。 腫瘤壞死因子相關(guān)的凋亡誘導(dǎo)配體(TNF-related apoptosis-inducing ligand,TRAIL)是腫瘤壞死因子家族的新成員,在細(xì)胞凋亡、炎癥反應(yīng)和免疫應(yīng)答過(guò)程中有重要的調(diào)控作用。TRAIL可與腫瘤壞死因子家族的五種高親和力受體結(jié)合。TRAIL-R1(DR4)和TRAIL-R2(DR5)作為死亡受體,與TRAIL結(jié)合后能夠誘導(dǎo)凋亡信號(hào)途徑的激活,導(dǎo)致細(xì)胞凋亡；而TPRAIL-R3(DcRl)、TRAIL-R4(DcR2)和OPG (osteoprotegerin)缺少功能性死亡域和誘導(dǎo)細(xì)胞凋亡的能力,作為誘導(dǎo)性受體,可與TRAIL-R1(DR4)和TRAIL-R2(DR5)競(jìng)爭(zhēng)性的與TRAIL結(jié)合,具有抑制正常細(xì)胞凋亡的作用。 近年來(lái),有實(shí)驗(yàn)數(shù)據(jù)表明,TRAIL在糖尿病及心血管疾病中起著重要的作用。體內(nèi)和體外研究數(shù)據(jù)均證明TRAIL有潛在的心血管保護(hù)作用。體外研究顯示,在缺乏培養(yǎng)基的條件下,重組TRAIL可通過(guò)激活A(yù)kt和ERK通路促進(jìn)人臍靜脈內(nèi)皮細(xì)胞的生存和增殖。同時(shí),有體內(nèi)研究表明,在使用重組TRAIL干預(yù)ApoE-／-鼠的糖尿病模型時(shí),可觀察到TRAIL通過(guò)增加血管平滑肌細(xì)胞的含量以穩(wěn)定動(dòng)脈粥樣硬化斑塊,并降低斑塊的整體面積；并且有研究表明,TRAIL-/-聯(lián)合Apoe-/-敲除鼠的動(dòng)脈粥樣硬化發(fā)生率及程度明顯高于Apoe-／-敲除鼠。最近三項(xiàng)體內(nèi)研究已經(jīng)表明,TRAIL的血清水平在糖尿病和動(dòng)脈粥樣硬化患者顯著降低,并且TRAIL水平降低程度與內(nèi)皮功能呈正相關(guān)。然而,迄今為止,TRAIL干預(yù)是否對(duì)高糖導(dǎo)致血管內(nèi)皮功能障礙有保護(hù)作用尚不清楚。因此,我們假設(shè)TRAIL可保護(hù)高糖導(dǎo)致的血管內(nèi)皮損傷。本研究主要從體內(nèi)和體外兩部分實(shí)驗(yàn)來(lái)評(píng)估TRAIL對(duì)血管內(nèi)皮的作用。 研究目的 本研究旨在明確： 1.研究TRAIL對(duì)糖尿病大鼠血管內(nèi)皮功能障礙是否有保護(hù)作用； 2.研究TRAIL對(duì)體外高糖環(huán)境導(dǎo)致的氧化應(yīng)激及內(nèi)皮細(xì)胞凋亡的作用； 3.探討體外高糖環(huán)境下TRAIL對(duì)內(nèi)皮細(xì)胞作用的可能信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。 方法 一、動(dòng)物實(shí)驗(yàn) 1.4周齡雄性SD大鼠30只,隨機(jī)留取10只作為正常對(duì)照(NC)組,以普通飼料喂養(yǎng),其余20只用于建立2型糖尿病模型,以高脂飼料喂養(yǎng)。6周后所有大鼠禁食禁水12h,給予高脂組STZ35mg/Kg(用前以0.1mol/L的檸檬酸-檸檬酸鈉緩沖液配制成1%STZ溶液)一次性腹腔注射,普通飼料組則給予等容量的檸檬酸鹽緩沖液一次性腹腔注射。診斷標(biāo)準(zhǔn)為空腹血糖(FPG)≥16.7mmol/L。將造模成功大鼠隨機(jī)分為對(duì)照(DM)組(10只)和TRAIL干預(yù)組(10只),同時(shí)TRAIL干預(yù)組予重組TRAIL(20ug/只)腹腔注射,每周1次,連續(xù)6周。NC與DM組予等量的蒸餾水腹腔注射,連續(xù)6周。 2.實(shí)驗(yàn)結(jié)束時(shí),腹腔注射1%戊巴比妥鈉50mg·kg-1麻醉大鼠,迅速開(kāi)胸,腹主動(dòng)脈采血后,迅速分離并取出胸主動(dòng)脈,觀察大鼠離體主動(dòng)脈對(duì)乙酚膽堿(Ahc)依賴性血管舒張反應(yīng)。ELISA試劑盒檢測(cè)血漿TRAIL濃度；Griess重氮化反應(yīng)法檢測(cè)主動(dòng)脈NO含量；免疫組化SP法檢測(cè)主動(dòng)脈eNOS的活性；Western blot檢測(cè)主動(dòng)脈eNOS和P-eNOS的表達(dá)水平。 二、細(xì)胞實(shí)驗(yàn) 1.細(xì)胞培養(yǎng)：使用膠原酶灌注新鮮的臍帶提取原代人臍靜脈內(nèi)皮細(xì)胞(HUVECs)。HUVECs培養(yǎng)在明膠包被的組織培養(yǎng)板中,并在M199內(nèi)皮細(xì)胞生長(zhǎng)培養(yǎng)基中添加20%FBS,10ug/m1肝素和50ug/mlECGF(血管內(nèi)皮細(xì)胞生長(zhǎng)因子)。所有實(shí)驗(yàn),使用第2-4代細(xì)胞。 2.細(xì)胞分組：實(shí)驗(yàn)分為5組；(1)正糖對(duì)照組(葡萄糖終濃度為5.5mmol/L,NG)；(2)高糖組(葡萄糖終濃度33mmol/L,HG)；(3)HG+TRAIL組(100ng/ml TRAILy預(yù)先培養(yǎng)18h,再加入終濃度33mmo1/L的葡萄糖)；(4)HG+TRAIL+LY組(內(nèi)皮細(xì)胞先經(jīng)含10μmol/L的3一磷脂酰肌醇激酶(PI3K)阻斷劑LY294002的培養(yǎng)基預(yù)處理30min,余處理同HG+TRAIL組)；(5)HG+TRAIL+L-NAME組(內(nèi)皮細(xì)胞先經(jīng)含10Oμmol/L的eNOS的阻斷劑L-NAME的培養(yǎng)基預(yù)處理30min,余處理同HG+TRAIL組)。 3.Annexin-V-FITC/PI雙染法和TUNEL法檢測(cè)細(xì)胞凋亡；以DCFH-DA為探針檢測(cè)細(xì)胞的ROS；實(shí)時(shí)定量RT-PCR檢測(cè)細(xì)胞的SOD,GPx-1的表達(dá)；Westernblot分析主動(dòng)脈Akt,P-Akt,eNOS,P-eNOS的表達(dá)。統(tǒng)計(jì)分析 采用SPSS13.0進(jìn)行統(tǒng)計(jì)學(xué)處理。實(shí)驗(yàn)數(shù)據(jù)以x±s表示。兩組之間比較使用獨(dú)立樣本t檢驗(yàn)。多組間比較使用One-Way ANOVA(單因素方差分析)方法,兩組間比較用最小顯著差(LSD)t檢驗(yàn),以P0.05為顯著性指標(biāo)。結(jié)果一、動(dòng)物實(shí)驗(yàn)結(jié)果 1.糖尿病組大鼠血漿TRAIL濃度顯著低于正常組(56.6±5.5vs.36.5±3.1,P0.001) 2.TRAIL對(duì)糖尿病大鼠主動(dòng)脈內(nèi)皮依賴性血管舒張功能的影響 與對(duì)常對(duì)照組比較,糖尿病組大鼠胸主動(dòng)脈內(nèi)皮依賴性血管舒張功能明顯降低[(63.5±4.6)%vs.(93.5±2.6)%,P0.001]。與糖尿病組比較,TRAIL干預(yù)組血管舒張功能明顯改善,[(63.5±4.6)%vs.(78.44±2.7)%(P0.001)]。而NaN02誘導(dǎo)的內(nèi)皮非依賴性血管舒張反應(yīng)在三組大鼠中無(wú)明顯差異。TRAIL干預(yù)糖尿病大鼠能明顯減輕ACh誘導(dǎo)的內(nèi)皮依賴性舒張功能損傷而對(duì)內(nèi)皮非依賴性血管舒張功能無(wú)明顯影響。這些結(jié)果顯示TRAIL干預(yù)明顯改善了糖尿病引起的內(nèi)皮依賴性舒張功能障礙。 3.各組大鼠主動(dòng)脈NO含量和eNOS免疫組化的結(jié)果 與NC組比較,TRAIL、DM組大鼠主動(dòng)脈NO濃度、eNOS免疫組化陽(yáng)性表達(dá)顯著降低(P0.001);與糖尿病對(duì)照組比較,TRAIL組主動(dòng)脈NO濃度,主動(dòng)脈內(nèi)皮eNOS陽(yáng)性表達(dá)顯著升高(p0.001)。 4.TRAIL對(duì)大鼠胸主動(dòng)脈組織中eNOS蛋白表達(dá)的影響 與正常對(duì)照組比較,P-eNOS蛋白水平在糖尿病組明顯降低(P＜0.01)。 TRAIL干預(yù)6周顯著提高糖尿病大鼠胸主動(dòng)脈組織P-eNOS蛋白表達(dá)水平(P0.01)。二、細(xì)胞實(shí)驗(yàn)結(jié)果 1.TUNEL染色：高糖組干預(yù)細(xì)胞48h后,細(xì)胞凋亡率較正糖對(duì)照組明顯上升(5.9±0.8%vs.37.4±0.9%,p0.001),而高糖+TRAIL組的細(xì)胞凋亡率較高糖對(duì)照組降低(17.2±0.4%vs.37.4±0.9%,p0.001)。HG+TRAIL+LY組與HG+TRAIL+L-NAME組細(xì)胞凋亡率較正糖對(duì)照組明顯升高,而與高糖對(duì)照組無(wú)明顯差異(p0.001),表明TRAIL抑制細(xì)胞的凋亡作用被LY294002和L-NAME阻斷。 2. AnnexinVFITC/PI:與正糖對(duì)照組相比,高糖對(duì)照組內(nèi)皮細(xì)胞調(diào)亡率明顯增加(p0.01),高糖+TRAIL組的細(xì)胞凋亡率較高糖對(duì)照組降低。而HG+TRAIL+LY組與HG+TRAIL+L-NAME組細(xì)胞凋亡率較正糖對(duì)照組明顯升高,而與高糖對(duì)照組無(wú)明顯差異(p0.01)。 3.TRAIL對(duì)細(xì)胞內(nèi)ROS生成的影響：正常對(duì)照組內(nèi)皮細(xì)胞內(nèi)熒光強(qiáng)度較弱,ROS產(chǎn)生較低；高糖作用于內(nèi)皮細(xì)胞48h后,內(nèi)皮細(xì)胞內(nèi)熒光強(qiáng)度很強(qiáng),ROS產(chǎn)生明顯多于對(duì)照組。TRAIL干預(yù)組內(nèi)皮細(xì)胞ROS的生成明顯降低；TRAIL+高糖+LY組及TRAIL+高糖+L-NAME組細(xì)胞內(nèi)熒光強(qiáng)度明顯高于TRAIL+高糖組,ROS生成增多。 4.實(shí)時(shí)定量PCR檢測(cè)細(xì)胞內(nèi)SOD、GPx-1的表達(dá)：與正糖對(duì)照組相比,高糖對(duì)照組SOD、GPx-1表達(dá)明顯減低(p0.001),高糖+TRAIL組的SOD、GPx-1較高糖對(duì)照組升高。而HG+TRAIL+LY組與HG+TRAIL+L-NAME組SOD、GPx-1表達(dá)較正糖對(duì)照組明顯降低,而與高糖對(duì)照組無(wú)明顯差異(p0.001)。 5.TRAIL對(duì)細(xì)胞內(nèi)Akt, P-Akt, eNOS, P-eNOS蛋白表達(dá)的影響：與正糖對(duì)照組相比,高糖對(duì)照組P-Akt, P-eNOS表達(dá)明顯減低(p0.001),高糖+TRAIL組的P-Akt,P-eNOS較高糖對(duì)照組升高。而HG+TRAIL+LY組與HG+TRAIL+L-NAME組SOD、GPx-1表達(dá)較正糖對(duì)照組明顯減低,而與高糖對(duì)照組無(wú)明顯差異(p0.001)。TRAIL對(duì)細(xì)胞內(nèi)Akt,eNOS的表達(dá)無(wú)明顯影響。結(jié)論 1.TRAIL干預(yù)可明顯改善糖尿病大鼠的主動(dòng)脈內(nèi)皮依賴性舒張功能,提示TRAIL對(duì)內(nèi)皮功能有一定的保護(hù)作用。由于TRAIL可促P-eNOS的表達(dá)和NO的生成,并提示TRAIL保護(hù)內(nèi)皮功能的作用可能是通過(guò)促進(jìn)eNOS的表達(dá)和NO的生成而達(dá)到的。 2.TRAIL能對(duì)抗體外高糖誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞凋亡,抑制ROS的生成,促進(jìn)SOD、GPx-1mRNA的表達(dá),并且上調(diào)P-Akt,P-eNOS的表達(dá),并且TRAIL的這種保護(hù)作用可以被PI3K阻斷劑LY294002和eNOS的阻斷劑L-NAME消除。從而進(jìn)一步證明TRAIL對(duì)體外高糖誘導(dǎo)內(nèi)皮細(xì)胞損傷的保護(hù)足有可能是通過(guò)PI3K/Akt/eNOS途徑介導(dǎo)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.2

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相關(guān)期刊論文 前1條

1 楊架林,李果,劉優(yōu)萍,張芳林,崔九蘭,張迪,周文中,羅敏;長(zhǎng)期高脂飲食加小劑量鏈脲佐霉素建立人類普通2型糖尿病大鼠模型的研究[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)報(bào);2003年03期

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本文編號(hào)：1708650