本文選題：內(nèi)皮祖細胞　切入點：高糖　出處：《四川醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:糖尿病(Diabetes mellitus,DM)是因遺傳和壞境因素共同作用而引起的高血糖以及碳水化合物、脂肪和蛋白質(zhì)等代謝失衡的慢性疾病。它不僅引起糖代謝紊亂,而且引起血管病變。內(nèi)皮祖細胞(Endothelial progenitor cells,EPCs)在維持血管健康和促進受損血管的重建過程中起著重要作用。當(dāng)前,在糖尿病血管新生受損的機制研究中,EPCs功能失調(diào)相關(guān)因素已經(jīng)受到廣泛重視,其中,氧化應(yīng)激與EPCs功能失調(diào)關(guān)系密切。已有研究表明:糖尿病患者的高血糖環(huán)境可以使EPCs凋亡增多,數(shù)量減少,而且凋亡增加可能是糖尿病血管病變中EPCs減少的主要機制。細胞凋亡是由基因控制的一種自主性、程序性的死亡過程,它可以由多種刺激因素引起并且主要通過死亡受體介導(dǎo)的途徑介導(dǎo)凋亡,在死亡受體介導(dǎo)的凋亡途徑中,腫瘤壞死因子受體(Tumor necrosis factor receptor,TNFR)是當(dāng)前研究發(fā)現(xiàn)的最大的死亡受體家族,其凋亡信號傳遞過程中,主要是由TNFR1介導(dǎo)的。有研究報道高糖能誘導(dǎo)EPCs中TNFR1表達,這種效應(yīng)能被抗氧化劑抑制。因此,本實驗主要通過觀察體外高糖是否通過氧化應(yīng)激反應(yīng)激活TNFR1及其下游的凋亡信號通路,進而對大鼠骨髓源性EPCs凋亡產(chǎn)生影響,來探討高糖對EPCs的作用和影響。方法:用頸部脫臼法處死SD大鼠,將其置于75%酒精中,浸泡并消毒15 min。然后將消毒后的大鼠放置于超凈工作臺上,用動物解剖器械分離大鼠的股骨和脛骨,分離完成后,用含1%肝素的無菌PBS液沖洗骨髓腔,直到?jīng)_洗液變無色透明為止,收集好沖洗液,并用離心機進行高速離心,離心后收集試管底部的細胞,選擇使用Ficoll密度梯度離心法進行大鼠骨髓源性單核細胞的分離;分離后,將其接種于6孔板中,3天后進行首次換液,以后每隔3-4天換液,連續(xù)使用倒置顯微鏡觀察細胞的形態(tài)變化并且使用激光共聚焦顯微鏡觀察鑒定經(jīng)Di I-ac LDL和FITC-UEA-1雙熒光染色的EPCs;用流式細胞儀檢測經(jīng)過不同含糖濃度(5.5、15、30、60 mmol/L)處理后的EPCs凋亡率,挑出最佳濃度;使用高糖(30 mmol/L)和氧化應(yīng)激拮抗劑Tempol、TNFR1受體拮抗MAB430共同作用,流式細胞儀檢測細胞凋亡率;使用分子探針(DCFH-DA)檢測不同糖濃度處理后EPCs的活性氧(Reactive oxygen species,ROS)含量變化;使用Western blotting檢測不同糖濃度和TNFR1受體拮抗MAB430處理EPCs后,其細胞表面TNFR1受體以及由其介導(dǎo)的細胞內(nèi)信號通路相關(guān)蛋白(TRADD、TRAF2、RIP、NF-k Bp65、Caspase3)表達的情況。結(jié)果:在使用M199培養(yǎng)基培養(yǎng)3天后,EPCs大部分貼壁,呈圓形,逐漸增大伸展,培養(yǎng)至7天后,細胞生長迅速,鏡下可見其呈集落樣生長,培養(yǎng)至14天后,觀察可發(fā)現(xiàn)細胞出現(xiàn)“鵝卵石”樣形態(tài)分布。通過激光共聚焦顯微鏡觀察細胞可見經(jīng)過Di I-ac-LDL處理后陽性的EPCs為紅色熒光,結(jié)合了FITC-UEA-1后的EPCs為綠色熒光,雙染法陽性的細胞為正在分化的EPCs,表明所培養(yǎng)的細胞為EPCs。在高糖刺激EPCs的早期(24-48h),高糖組與對照組之間比較,沒有明顯的凋亡增加,而隨著高糖刺激時間的延長,即高糖刺激的晚期(72-96h),可以發(fā)現(xiàn)高糖刺激組與正常對照組比較,凋亡細胞數(shù)目增加,高糖組(15mmol/L,30mmol/L,60mmol/L)與對照組進行組內(nèi)比較,具有統(tǒng)計學(xué)意義(P0.01);在同一時間點,將30mmol/L,60mmol/L的高糖組分別與15mmol/L高糖組相比,早期沒有發(fā)現(xiàn)明顯凋亡,而在刺激的晚期,僅發(fā)現(xiàn)60mmol/L的高糖組與15mmol/L的高糖組比較,細胞凋亡增加,具有統(tǒng)計學(xué)意義(P0.01),在分別使用抗氧化劑Tempol、TNFR1受體拮抗劑MAB430處理EPCs24h、72h后,在與高糖組(30mmol/L)分別在24h,72h比較后,發(fā)現(xiàn)早期凋亡沒有明顯變化,而在晚期(72h)后,發(fā)現(xiàn)凋亡有明顯下降,且數(shù)值具有顯著統(tǒng)計學(xué)意義(P0.01),同時在實驗的早期,高糖組與對照組比較,ROS的值沒有明顯的增加,而隨著刺激時間的延長,刺激的晚期(72h),發(fā)現(xiàn)高糖組ROS增加明顯,與對照組比較,具有顯著統(tǒng)計學(xué)意義(P0.01),在刺激的早期和晚期,分別用Tempol、MAB430(TNFR1受體拮抗劑)處理后,發(fā)現(xiàn)與高糖組比較,早期ROS值沒有明顯的降低,而晚期ROS值有明顯的降低,并且與高糖組比較,差異具有顯著統(tǒng)計學(xué)意義(P0.01),高糖(30mmol/l)刺激早期24h,TNFR1蛋白及其凋亡信號通路相關(guān)蛋白(TRAF2、TRADD、RIP、Caspase3)表達沒有上調(diào),而在刺激的72h后,相關(guān)蛋白表達明顯上調(diào),同時將上述蛋白表達量在72h與24h進行比較,差異具有顯著統(tǒng)計學(xué)意義(P0.01),氧化應(yīng)激拮抗劑Tempol、TNFR1受體拮抗劑MAB430在72h能夠降低上述TNF凋亡信號通路相關(guān)蛋白的表達,蛋白表達量有統(tǒng)計學(xué)意義(P0.01),而隨著高糖(30mmol/l)的刺激,NF-κBp65蛋白相對表達量是逐漸降低的,其蛋白表達量在72h與24h進行比較,差異具有顯著統(tǒng)計學(xué)意義(P0.01),在用Tempol、MAB430處理后,無論是早期還是晚期,與高糖組(30mmol/l)相比,其蛋白表達量是升高的,且差異具有顯著統(tǒng)計學(xué)意義(P0.01)。結(jié)論:1.高糖通過誘發(fā)內(nèi)皮祖細胞氧化應(yīng)激反應(yīng)促進其凋亡,凋亡主要發(fā)生在刺激的后期,且具有濃度和時間的依賴性。2.高糖通過誘發(fā)內(nèi)皮祖細胞氧化應(yīng)激激活其TNFR1受體及其銜接蛋白TRADD,進而可能通過激活NF-κB p65轉(zhuǎn)錄因子,從而使得與凋亡直接相關(guān)的Caspase3蛋白表達增強,導(dǎo)致內(nèi)皮祖細胞發(fā)生凋亡。
[Abstract]:Objective: diabetes mellitus (Diabetes, mellitus, DM) is due to genetic and environmental factors caused by high blood sugar and carbohydrate, fat and protein metabolism in chronic disease. It is not only caused by the imbalance of glucose metabolism, but also cause vascular disease. Endothelial progenitor cells (Endothelial progenitor cells, EPCs) plays an important role in maintaining the health of blood vessels and promote injured vascular reconstruction process. At present, the study on Mechanism of diabetes impaired angiogenesis in EPCs dysfunction and related factors have received wide attention, among them, oxidative stress and EPCs dysfunction closely. Previous studies have shown that high levels of blood glucose in patients with diabetes mellitus can make EPCs apoptosis increased, and reduce the number of apoptosis may be the main mechanism of EPCs in diabetic angiopathy. Reduce apoptosis is an independent gene control, programmed death The process, it can be caused by a variety of stimuli and is mainly mediated by death receptor pathway in apoptosis, apoptosis mediated by death receptor, tumor necrosis factor receptor (Tumor necrosis factor receptor, TNFR) is the current findings of the largest death receptor family, the apoptosis signal transduction process, is mainly mediated by TNFR1. Studies have reported that high glucose can induce expression of TNFR1 in EPCs, this effect can be antioxidants. Therefore, this experiment mainly through the observation of the apoptosis signal pathway is mediated by oxidative stress in vitro high glucose activated TNFR1 and its downstream, and thus have an impact on the apoptosis of bone marrow derived EPCs rats, and to explore the influence of the effect of high glucose on EPCs. Methods: SD rats were killed by cervical dislocation, placed in 75% alcohol immersion disinfection, and 15 min. and then sterilized rats placed on the clean bench On animal anatomy instrument isolated from rat femur and tibia, separation after flushing the bone marrow cavity with sterile PBS solution containing 1% heparin, until the rinse solution becomes colorless and transparent, collect the washing liquid, and high-speed centrifugal centrifuge, the centrifugal collection after the bottom of the tube cell, choose to use the Ficoll density gradient centrifugation separation of bone marrow derived mononuclear cells in rats; after separation, it was inoculated in 6 well plates, 3 days after the first medium change after every change of liquid, 3-4 days of continuous use inverted microscope was used to observe the morphological changes of the cells and the use of laser confocal microscope identification by Di I-ac LDL and FITC-UEA-1 double fluorescence EPCs staining; flow cytometry was used to detect the effect of glucose concentration (5.5,15,30,60 mmol/L) EPCs apoptosis rate after treatment, select the best concentration; using high glucose (30 mmol/L) and oxidative stress of TNFR1 receptor antagonist Tempol. The interaction of antagonistic MAB430, cell apoptosis was detected by flow cytometry; using molecular probes (DCFH-DA) EPCs active oxygen in different glucose concentration after treatment (Reactive oxygen species ROS detection) content changes; using Western blotting to detect different concentrations of sugar and TNFR1 receptor antagonism of MAB430 after EPCs treatment, the cell surface receptors and by TNFR1 the mediated intracellular signal pathway related proteins (TRADD, TRAF2, RIP, NF-k, Bp65, Caspase3). Results: the expression of M199 in the culture medium after 3 days of culture, most of the EPCs adherent, rounded, increasing stretch, after 7 days of culture, cells grow rapidly, which is visible under the microscope colony growth, after 14 days of culture, observation can be found in the cells "cobblestone" distribution. By laser confocal microscopy after Di cells treated with I-ac-LDL positive red fluorescence of EPCs, combined with FITC-UE A-1 EPCs after green fluorescence double staining method was positive for differentiation of EPCs, showed that the cultured cells of early EPCs. in high glucose stimulated EPCs (24-48h), a comparison between the high glucose group and the control group, no significant increase of apoptosis, with high glucose stimulation time extended, which is stimulated by high glucose late (72-96h), can be found in high glucose group compared with normal control group, the number of apoptotic cells increased in high glucose group (15mmol/L, 30mmol/L, 60mmol/L) and control group for comparison group, with statistical significance (P0.01); at the same time point, 30mmol/L, 60mmol/L respectively compared with high glucose group 15mmol/L high glucose group early, there is no obvious apoptosis in stimulation of late, only found relatively high glucose group high glucose group and 15mmol/L 60mmol/L, increased apoptosis, with statistical significance (P0.01), respectively in the use of antioxidants Tempol, TNFR1 receptor antagonist MAB430 EPCs24h, 72h, and in the high glucose group (30mmol/L) respectively in 24h, 72h, found that early apoptosis did not change significantly, whereas in the late phase (72h), found that apoptosis has decreased significantly, with statistical significance (P0.01), and the value at the same time early in the experiment, compared with the high glucose group control, ROS was not significantly increased, with the prolongation of stimulation time, stimulation of the late (72h), high glucose group ROS increased significantly, compared with the control group, there was a statistically significant (P0.01), in the early and late stimulation, respectively Tempol, MAB430 (TNFR1 receptor antagonist) treatment compared with high glucose group, found that early ROS value was not obviously decreased, and the late ROS value decreased significantly, and compared with high glucose group, the difference was statistically significant (P0.01), high glucose (30mmol/l) stimulation of the early 24h, TNFR1 protein and apoptosis signal pathway related protein (TRAF2, TRADD, RIP, Caspase3) expression did not increase, and in the stimulation of 72h, protein expression was up-regulated, and compare the amount of 72h and 24h in the protein expression, the difference was statistically significant (P0.01), oxidative stress and expression of TNFR1 receptor antagonist Tempol and antagonist MAB430 can reduce the TNF the apoptosis signal pathway related proteins in 72h, protein expression was statistically significant (P0.01), with high glucose (30mmol/l) stimulation, NF- kappa expression of Bp65 protein was decreased, compared with 24h in 72h protein expression, the difference was statistically significant (P0.01), in Tempol after MAB430 treatment, whether early or late, compared with the high glucose group (30mmol/l), the protein content is increased, and the difference was statistically significant (P0.01). Conclusion: 1. high glucose oxidation by endothelial progenitor cells induced by stress induced In the late apoptosis, the apoptosis in stimulation, and has a time and concentration dependent.2. high glucose activation of the TNFR1 receptor and its adaptor protein TRADD induced by oxidative stress and endothelial progenitor cells, possibly through activation of NF- kappa B transcription factor p65, which is directly related to apoptosis and Caspase3 protein expression, leading to endothelial progenitor cell apoptosis.

【學(xué)位授予單位】：四川醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

【共引文獻】

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