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

【摘要】：研究背景 隨著社會經(jīng)濟的發(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,嚴重威脅著人們的身心健康。糖尿病大血管病變,如動脈粥樣硬化、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。長期高血糖是導致血管內(nèi)皮功能障礙的主要危險因素。而內(nèi)皮功能障礙是糖尿病血管病變的始動因素和早期表現(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,防治高糖導致的內(nèi)皮細胞損傷、保護血管內(nèi)皮功能,對防治糖尿病心血管并發(fā)癥有重要意義。 腫瘤壞死因子相關的凋亡誘導配體(TNF-related apoptosis-inducing ligand,TRAIL)是腫瘤壞死因子家族的新成員,在細胞凋亡、炎癥反應和免疫應答過程中有重要的調(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合。TRAIL-R1(DR4)和TRAIL-R2(DR5)作為死亡受體,與TRAIL結合后能夠誘導凋亡信號途徑的激活,導致細胞凋亡；而TPRAIL-R3(DcRl)、TRAIL-R4(DcR2)和OPG (osteoprotegerin)缺少功能性死亡域和誘導細胞凋亡的能力,作為誘導性受體,可與TRAIL-R1(DR4)和TRAIL-R2(DR5)競爭性的與TRAIL結合,具有抑制正常細胞凋亡的作用。 近年來,有實驗數(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可通過激活Akt和ERK通路促進人臍靜脈內(nèi)皮細胞的生存和增殖。同時,有體內(nèi)研究表明,在使用重組TRAIL干預ApoE-／-鼠的糖尿病模型時,可觀察到TRAIL通過增加血管平滑肌細胞的含量以穩(wěn)定動脈粥樣硬化斑塊,并降低斑塊的整體面積；并且有研究表明,TRAIL-/-聯(lián)合Apoe-/-敲除鼠的動脈粥樣硬化發(fā)生率及程度明顯高于Apoe-／-敲除鼠。最近三項體內(nèi)研究已經(jīng)表明,TRAIL的血清水平在糖尿病和動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。然而,迄今為止,TRAIL干預是否對高糖導致血管內(nèi)皮功能障礙有保護作用尚不清楚。因此,我們假設TRAIL可保護高糖導致的血管內(nèi)皮損傷。本研究主要從體內(nèi)和體外兩部分實驗來評估TRAIL對血管內(nèi)皮的作用。 研究目的 本研究旨在明確： 1.研究TRAIL對糖尿病大鼠血管內(nèi)皮功能障礙是否有保護作用； 2.研究TRAIL對體外高糖環(huán)境導致的氧化應激及內(nèi)皮細胞凋亡的作用； 3.探討體外高糖環(huán)境下TRAIL對內(nèi)皮細胞作用的可能信號轉導機制。 方法 一、動物實驗 1.4周齡雄性SD大鼠30只,隨機留取10只作為正常對照(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。診斷標準為空腹血糖(FPG)≥16.7mmol/L。將造模成功大鼠隨機分為對照(DM)組(10只)和TRAIL干預組(10只),同時TRAIL干預組予重組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.實驗結束時,腹腔注射1%戊巴比妥鈉50mg·kg-1麻醉大鼠,迅速開胸,腹主動脈采血后,迅速分離并取出胸主動脈,觀察大鼠離體主動脈對乙酚膽堿(Ahc)依賴性血管舒張反應。ELISA試劑盒檢測血漿TRAIL濃度；Griess重氮化反應法檢測主動脈NO含量；免疫組化SP法檢測主動脈eNOS的活性；Western blot檢測主動脈eNOS和P-eNOS的表達水平。 二、細胞實驗 1.細胞培養(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)皮細胞生長培養(yǎng)基中添加20%FBS,10ug/m1肝素和50ug/mlECGF(血管內(nèi)皮細胞生長因子)。所有實驗,使用第2-4代細胞。 2.細胞分組：實驗分為5組；(1)正糖對照組(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)基預處理30min,余處理同HG+TRAIL組)；(5)HG+TRAIL+L-NAME組(內(nèi)皮細胞先經(jīng)含10Oμmol/L的eNOS的阻斷劑L-NAME的培養(yǎng)基預處理30min,余處理同HG+TRAIL組)。 3.Annexin-V-FITC/PI雙染法和TUNEL法檢測細胞凋亡；以DCFH-DA為探針檢測細胞的ROS；實時定量RT-PCR檢測細胞的SOD,GPx-1的表達；Westernblot分析主動脈Akt,P-Akt,eNOS,P-eNOS的表達。統(tǒng)計分析 采用SPSS13.0進行統(tǒng)計學處理。實驗數(shù)據(jù)以x±s表示。兩組之間比較使用獨立樣本t檢驗。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,以P0.05為顯著性指標。結果一、動物實驗結果 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對糖尿病大鼠主動脈內(nèi)皮依賴性血管舒張功能的影響 與對常對照組比較,糖尿病組大鼠胸主動脈內(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誘導的內(nèi)皮非依賴性血管舒張反應在三組大鼠中無明顯差異。TRAIL干預糖尿病大鼠能明顯減輕ACh誘導的內(nèi)皮依賴性舒張功能損傷而對內(nèi)皮非依賴性血管舒張功能無明顯影響。這些結果顯示TRAIL干預明顯改善了糖尿病引起的內(nèi)皮依賴性舒張功能障礙。 3.各組大鼠主動脈NO含量和eNOS免疫組化的結果 與NC組比較,TRAIL、DM組大鼠主動脈NO濃度、eNOS免疫組化陽性表達顯著降低(P0.001);與糖尿病對照組比較,TRAIL組主動脈NO濃度,主動脈內(nèi)皮eNOS陽性表達顯著升高(p0.001)。 4.TRAIL對大鼠胸主動脈組織中eNOS蛋白表達的影響 與正常對照組比較,P-eNOS蛋白水平在糖尿病組明顯降低(P＜0.01)。 TRAIL干預6周顯著提高糖尿病大鼠胸主動脈組織P-eNOS蛋白表達水平(P0.01)。二、細胞實驗結果 1.TUNEL染色：高糖組干預細胞48h后,細胞凋亡率較正糖對照組明顯上升(5.9±0.8%vs.37.4±0.9%,p0.001),而高糖+TRAIL組的細胞凋亡率較高糖對照組降低(17.2±0.4%vs.37.4±0.9%,p0.001)。HG+TRAIL+LY組與HG+TRAIL+L-NAME組細胞凋亡率較正糖對照組明顯升高,而與高糖對照組無明顯差異(p0.001),表明TRAIL抑制細胞的凋亡作用被LY294002和L-NAME阻斷。 2. AnnexinVFITC/PI:與正糖對照組相比,高糖對照組內(nèi)皮細胞調(diào)亡率明顯增加(p0.01),高糖+TRAIL組的細胞凋亡率較高糖對照組降低。而HG+TRAIL+LY組與HG+TRAIL+L-NAME組細胞凋亡率較正糖對照組明顯升高,而與高糖對照組無明顯差異(p0.01)。 3.TRAIL對細胞內(nèi)ROS生成的影響：正常對照組內(nèi)皮細胞內(nèi)熒光強度較弱,ROS產(chǎn)生較低；高糖作用于內(nèi)皮細胞48h后,內(nèi)皮細胞內(nèi)熒光強度很強,ROS產(chǎn)生明顯多于對照組。TRAIL干預組內(nèi)皮細胞ROS的生成明顯降低；TRAIL+高糖+LY組及TRAIL+高糖+L-NAME組細胞內(nèi)熒光強度明顯高于TRAIL+高糖組,ROS生成增多。 4.實時定量PCR檢測細胞內(nèi)SOD、GPx-1的表達：與正糖對照組相比,高糖對照組SOD、GPx-1表達明顯減低(p0.001),高糖+TRAIL組的SOD、GPx-1較高糖對照組升高。而HG+TRAIL+LY組與HG+TRAIL+L-NAME組SOD、GPx-1表達較正糖對照組明顯降低,而與高糖對照組無明顯差異(p0.001)。 5.TRAIL對細胞內(nèi)Akt, P-Akt, eNOS, P-eNOS蛋白表達的影響：與正糖對照組相比,高糖對照組P-Akt, P-eNOS表達明顯減低(p0.001),高糖+TRAIL組的P-Akt,P-eNOS較高糖對照組升高。而HG+TRAIL+LY組與HG+TRAIL+L-NAME組SOD、GPx-1表達較正糖對照組明顯減低,而與高糖對照組無明顯差異(p0.001)。TRAIL對細胞內(nèi)Akt,eNOS的表達無明顯影響。結論 1.TRAIL干預可明顯改善糖尿病大鼠的主動脈內(nèi)皮依賴性舒張功能,提示TRAIL對內(nèi)皮功能有一定的保護作用。由于TRAIL可促P-eNOS的表達和NO的生成,并提示TRAIL保護內(nèi)皮功能的作用可能是通過促進eNOS的表達和NO的生成而達到的。 2.TRAIL能對抗體外高糖誘導的人臍靜脈內(nèi)皮細胞凋亡,抑制ROS的生成,促進SOD、GPx-1mRNA的表達,并且上調(diào)P-Akt,P-eNOS的表達,并且TRAIL的這種保護作用可以被PI3K阻斷劑LY294002和eNOS的阻斷劑L-NAME消除。從而進一步證明TRAIL對體外高糖誘導內(nèi)皮細胞損傷的保護足有可能是通過PI3K/Akt/eNOS途徑介導的。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R587.2

【參考文獻】

相關期刊論文 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];中國實驗動物學報;2003年03期

，

本文編號：1708650