本文選題：晚期糖基化終末產(chǎn)物 + 人視網(wǎng)膜色素上皮細(xì)胞��； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：糖尿病視網(wǎng)膜病變是糖尿病最常見的微血管并發(fā)癥之一,己成為致盲的主要眼疾。糖尿病患者體內(nèi)持續(xù)的或不受控制的高血糖會(huì)逐漸損害視網(wǎng)膜血管細(xì)胞、視網(wǎng)膜上皮細(xì)胞、神經(jīng)細(xì)胞和神經(jīng)膠質(zhì),導(dǎo)致糖尿病視網(wǎng)膜病變。隨著糖尿病發(fā)病率逐年增長(zhǎng),越來(lái)越多的糖尿病患者將經(jīng)受糖尿病視網(wǎng)膜病變的折磨。晚期糖基化終末產(chǎn)物(AGEs)是指在非酶促條件下,蛋白質(zhì)、氨基酸、脂類或核酸等的游離氨基與葡萄糖或其他還原糖的醛基通過(guò)Maillard反應(yīng)系列產(chǎn)生一組穩(wěn)定的終末產(chǎn)物；許多研究表明AGEs在糖尿病視網(wǎng)膜病變發(fā)生和發(fā)展的過(guò)程中起著重要作用,但糖尿病視網(wǎng)膜病變的發(fā)病機(jī)制尚未完全闡明,我們的實(shí)驗(yàn)將致力于研究AGEs在誘導(dǎo)人視網(wǎng)膜色素上皮細(xì)胞凋亡時(shí),發(fā)揮了怎樣的作用,為臨床防治糖尿病視網(wǎng)膜病變提供新的思路。第一部分AGEs誘導(dǎo)人ARPE-19細(xì)胞凋亡的有效性研究目的：探討用AGE-BSA處理ARPE-19細(xì)胞,觀察細(xì)胞的生存能力和凋亡情況。方法：1.將培養(yǎng)的ARPE-19細(xì)胞用濃度為50、100、150、200μg/mL的AGE-BSA分別處理24小時(shí)和48小時(shí),陰性對(duì)照組中加入200 μg/mLBSA,采用MTT比色法分析AGE-BSA對(duì)細(xì)胞存活率的影響,采用流式細(xì)胞儀檢測(cè)分析AGE-BSA對(duì)細(xì)胞凋亡率的影響。2.分別在ARPE-19細(xì)胞培養(yǎng)24小時(shí)和48小時(shí)時(shí)加入AGE-BSA處理,對(duì)照組中加入200 μg/mLBSA,采用MTT比色法分析AGE-BSA后處理對(duì)細(xì)胞存活率的影響,采用凋亡檢測(cè)試劑盒分析AGE-BSA對(duì)細(xì)胞凋亡率的影響。結(jié)果：1.與陰性對(duì)照組比較,用150 μg/mL或200 μg/mL AGE-BSA處理的細(xì)胞,細(xì)胞存活比例分別減少了22%(p0.05)和33%(p0.01),差異有統(tǒng)計(jì)學(xué)意義,而且,不同濃度AGE-BSA處理的兩組細(xì)胞之間也有明顯的差異；當(dāng)細(xì)胞中AGE-BSA濃度達(dá)到100μg/mL時(shí),與陰性對(duì)照組相比,細(xì)胞的凋亡比例顯著升高(p0.05),同時(shí)也發(fā)現(xiàn),當(dāng)AGE-BSA濃度超過(guò)100 μg/mL時(shí),細(xì)胞的凋亡比例與其呈正相關(guān)性。2.用AGE-BSA處理的細(xì)胞,和陰性對(duì)照組相比,細(xì)胞的存活比例在24小時(shí)減少了20%,在48小時(shí)減少了40%；而細(xì)胞的凋亡比例在24小時(shí)和48小時(shí)分別上升了60%和110%。結(jié)論：用AGE-BSA處理ARPE-19細(xì)胞可以減少細(xì)胞的存活并有效地誘導(dǎo)細(xì)胞凋亡。第二部分AGEs通過(guò)線粒體功能障礙誘導(dǎo)人視網(wǎng)膜細(xì)胞凋亡的研究目的：研究線粒體功能障礙在AGEs誘導(dǎo)細(xì)胞凋亡過(guò)程中的作用。方法：1.用小鼠線粒體呼吸鏈復(fù)合物Ⅳ試劑盒檢測(cè)經(jīng)AGE-BSA后處理的人ARPE-19細(xì)胞線粒體呼吸鏈復(fù)合物Ⅳ的活性；用線粒體膜電位試劑盒檢測(cè)人ARPE-19細(xì)胞線粒體膜電位水平。2.將ARPE-19細(xì)胞培養(yǎng)48小時(shí)后,用AGE-BSA進(jìn)行處理,加入BSA的細(xì)胞設(shè)為對(duì)照組,采用Western blot技術(shù)檢測(cè)Bcl-2、 Bcl-xl、Bax、Cytc和Caspase3的表達(dá)水平的變化。結(jié)果：1.與陰性對(duì)照組比較,用200μ g/mL AGE-BSA處理的人ARPE-19細(xì)胞的線粒體呼吸鏈復(fù)合體Ⅳ活性降低了30%(p0.01),線粒體膜電位水平降低了25%(p0.05)。2.和陰性對(duì)照組相比,ARPE-19細(xì)胞的Bcl-2和Bcl-xl的表達(dá)水平分別下調(diào)了67%(p0.01)和43%(p0.05),而Bax的表達(dá)水平卻上調(diào)了122%(p0.01)；3.與凋亡相關(guān)的關(guān)鍵分子Cytc的釋放和活化的Caspase 3的表達(dá)與陰性對(duì)照組也有顯著差異,二者的表達(dá)均增加,分別上調(diào)了43%(p0.01)和188%(p0.01)。結(jié)論：AGEs通過(guò)降低線粒體呼吸鏈復(fù)合體Ⅳ活性和線粒體膜電位水平以及下調(diào)Bcl-2、Bcl-xl的表達(dá)水平,而上調(diào)Bax的表達(dá)水平,導(dǎo)致線粒體功能障礙；從而使Cytc釋放增加和Caspase 3活化最終激活人視網(wǎng)膜ARPE-19細(xì)胞的凋亡。第三部分AGEs通過(guò)Fas-FasL信號(hào)誘導(dǎo)人視網(wǎng)膜細(xì)胞凋亡的研究目的：探討Fas-FasL信號(hào)通路在AGEs誘導(dǎo)細(xì)胞凋亡過(guò)程中的作用。方法：1.將ARPE-19細(xì)胞用不同濃度的AGE-BSA處理,將加入不同濃度BSA的細(xì)胞設(shè)為陰性對(duì)照組,采用Western blot技術(shù)檢測(cè)Fas、Fas-L、活化的caspase8和β-actin的表達(dá)水平。2.將ARPE-19細(xì)胞用不同濃度的α-Fas+AGE-BSA共處理,采用Western blot技術(shù)檢測(cè)Cytc釋放、活化caspase8、活化caspase3的表達(dá)水平。3.將合成的scramble SiRNA、SiRNA-Fas和SiRNA-FasL轉(zhuǎn)染到ARPE-19細(xì)胞,通過(guò)Western blot技術(shù)檢測(cè)Fas和Fas-L的mRNA表達(dá)水平的變化。4.分別將上述3種SiRNA轉(zhuǎn)染到用200 μ g/mL AGE-BSA處理的人ARPE-19細(xì)胞,采用Western blot技術(shù)檢測(cè)Fas、Fas-L、活化Caspase8和活化Caspase 3的表達(dá)情況。結(jié)果：1.當(dāng)細(xì)胞中AGE-BSA的濃度超過(guò)100 μ g/mL時(shí),相較于空白組、BSA組和50 μ g/mLAGE-BSA組,Fas和FasL的表達(dá)均有顯著的統(tǒng)計(jì)學(xué)差異；200μg/mL AGE-BSA處理組的FasL的表達(dá)比100 μ g/mLAGE-BSA處理組升高了50%(p0.01),200 μ g/mL AGE-BSA處理組的活化caspase8的表達(dá)比其他組提升了42.8%(p0.01)。2. Cytc釋放、活化caspase8和活化caspase3隨α-Fas劑量的增加而增加；50ng/ml α-Fas共處理組相比于20ng/ml α-Fas共處理組,Cytc釋放升高了60%(p0.01)；活化caspase8的表達(dá)在50ng/ml α-Fas共處理組比10ng/ml α-Fas共處理組增加了40%(p0.01)；而活化caspase3的表達(dá)在20ng/ml α-Fas共處理組和50ng/ml α-Fas共處理組分別上調(diào)了45%(p0.01)和45%(p0.01)相比于10ng/ml α-Fas共處理組。3. SiRNA-Fas和SiRNA-FasL有效地抑制了Fas和Fas-L的mRNA表達(dá)水平。4. SiRNA-Fas+AGE-BSA處理組Fas的表達(dá)下降了20%(p0.01), SiRNA-FasL+AGE-BSA處理組FasL的表達(dá)降低了13%(p0.01)；因此,SiRNA-Fas+AGE-BSA處理組和SiRNA-FasL+AGE-BSA處理組均有效減少了活化Caspase8和活化Caspase 3的表達(dá)。結(jié)論：AGE-BSA處理細(xì)胞,能夠促進(jìn)Fas、Fas-L的表達(dá)及Fas-FasL信號(hào)誘導(dǎo)活化Caspase8;外源性的α-Fas能加強(qiáng)AGE-BSA誘導(dǎo)Cytc釋放、活化caspase8和活化caspase3的作用；SiRNA介導(dǎo)的Fas或FasL基因沉默能夠有效地抑制AGE-BSA誘導(dǎo)的Caspase 8和Caspase 3的激活。
[Abstract]:Diabetic retinopathy is one of the most common microvascular complications of diabetes. It has become a major blindness disease. Continuous or uncontrolled hyperglycemia in diabetic patients will gradually damage retinal vascular cells, retinal epithelial cells, nerve cells, and neuroglia, leading to diabetic retinopathy. The rate of disease is increasing year by year. More and more diabetic patients will suffer from diabetic retinopathy. Late glycosylation end product (AGEs) refers to a group of stable terminals through the Maillard reaction series of free amino groups of protein, amino acids, lipids, or nucleic acids, such as protein, amino acids, lipids, or nucleic acids, or other reductive glycosylated aldehyde groups. Many studies have shown that AGEs plays an important role in the development and development of diabetic retinopathy, but the pathogenesis of diabetic retinopathy has not been fully elucidated. Our experiments will be devoted to the role of AGEs in inducing apoptosis of human retinal pigment epithelial cells and to prevent and cure diabetes in clinical practice. Disease retinopathy provides new ideas. Part one AGEs induced the effectiveness of apoptosis in human ARPE-19 cells. Objective: To explore the use of AGE-BSA to treat ARPE-19 cells and to observe the viability and apoptosis of the cells. Methods: 1. the cultured ARPE-19 cells were treated with AGE-BSA with a concentration of 50100150200 mu g/mL for 24 hours and 48 hours, respectively. The negative control group was added to 200 g/mLBSA, and the effect of AGE-BSA on the cell survival rate was analyzed by MTT colorimetry. The effect of AGE-BSA on the apoptosis rate was analyzed by flow cytometry..2. was added to AGE-BSA treatment at 24 hours and 48 hours in ARPE-19 cells, and the control group was added to 200 Mu g/mLBSA, and AGE-BS was analyzed by the MTT colorimetry. The effect of A postprocessing on cell survival rate was analyzed by the apoptosis detection kit. Results: 1. compared with the negative control group, the cell survival ratio decreased by 22% (P0.05) and 33% (P0.01) in the cells treated with 150 g/mL or 200 mu g/mL AGE-BSA, and the difference was statistically significant, and the different concentrations of AGE-BS were statistically significant. There was also significant difference between the two groups of cells treated with A. When the concentration of AGE-BSA in the cell reached 100 g/mL, the cell apoptosis ratio was significantly higher than that in the negative control group (P0.05). At the same time, it was found that when the concentration of AGE-BSA exceeded 100 u g/mL, the cell apoptosis ratio was positively correlated with the.2. treated cells with AGE-BSA, and negative pairs. The survival ratio of cells decreased by 20% in 24 hours and 40% in 48 hours, while the percentage of cell apoptosis increased by 60% and 110%. in 24 hours and 48 hours, respectively. The use of AGE-BSA to treat ARPE-19 cells could reduce cell survival and induce apoptosis effectively. Second part of AGEs was induced by mitochondrial dysfunction. Study on the apoptosis of human retinal cells: study the role of mitochondrial dysfunction in the process of AGEs induced apoptosis. Methods: 1. the viability of mitochondrial respiratory chain complex IV in human ARPE-19 cells treated by AGE-BSA was detected by the mitochondrial respiratory chain complex IV kit in mice; the mitochondrial membrane potential kits were used to detect the viability of human ARPE-19 cells. The mitochondrial membrane potential level of human ARPE-19 cells.2. cells were cultured for 48 hours and treated with AGE-BSA. The cells added to BSA were set as the control group. The changes in the expression of Bcl-2, Bcl-xl, Bax, Cytc and Caspase3 were detected by Western blot technique. Results: 1. compared with the negative control group, the persons treated with 200 mu blot were treated. The cell mitochondrial respiratory chain complex IV activity decreased by 30% (P0.01), the mitochondrial membrane potential level decreased by 25% (P0.05).2. and the negative control group, the expression level of Bcl-2 and Bcl-xl in ARPE-19 cells decreased by 67% (P0.01) and 43% (P0.05), but the level of Bax (P0.01) was up 122% (P0.01); 3. key molecules associated with apoptosis The expression of Cytc release and activation of Caspase 3 was also significantly different from that in the negative control group. The expression of the two was increased by 43% (P0.01) and 188% (P0.01). Conclusion: AGEs increased the expression of Bax by lowering the level of mitochondrial respiratory chain complex IV and mitochondrial membrane potential and the expression level of Bcl-2, Bcl-xl. Leveling, resulting in mitochondrial dysfunction, resulting in increased Cytc release and activation of Caspase 3 activation eventually activating the apoptosis of human retinal ARPE-19 cells. Third the purpose of part AGEs to induce apoptosis in human retinal cells through Fas-FasL signal: explore the role of Fas-FasL signaling pathway in the process of apoptosis induced by AGEs. Method: 1. ARPE-19 The cells were treated with different concentrations of AGE-BSA, and the cells with different concentrations of BSA were set into negative control groups. The Western blot technique was used to detect Fas, Fas-L, activated caspase8 and the expression level of beta -actin. ARPE-19 cells were treated with alpha -Fas+AGE-BSA in different concentrations. The expression level of Caspase3.3. transfected the synthesized scramble SiRNA, SiRNA-Fas and SiRNA-FasL into ARPE-19 cells. The Western blot technique was used to detect the changes of Fas and Fas-L mRNA expression levels. The expression of spase8 and activated Caspase 3. Results: 1. when the concentration of AGE-BSA in the cells exceeded 100 g/mL, the expressions of Fas and FasL were significantly different compared to those in the blank group, BSA and 50 mu g/mLAGE-BSA, and the FasL expression in the 200 micron AGE-BSA treatment group was 50% higher than that of the 100 micron g/. 200 mu. The expression of activated caspase8 in the g/mL AGE-BSA treatment group increased by 42.8% (P0.01).2. Cytc than in the other groups, and activated caspase8 and activated Caspase3 increased with the increase of the alpha -Fas dose; 50ng/ml alpha -Fas co processing group was 60% higher than that of 20ng/ml alpha co processing group. The co processing group increased by 40% (P0.01), while the expression of activated Caspase3 increased by 45% (P0.01) and 45% (P0.01) in the 20ng/ml alpha -Fas co processing group and the 50ng/ml alpha -Fas co processing group, respectively. The expression of Fas in the iRNA-Fas+AGE-BSA treatment group decreased by 20% (P0.01), and the expression of FasL in the SiRNA-FasL+AGE-BSA treatment group decreased by 13% (P0.01); therefore, both the SiRNA-Fas+AGE-BSA treatment group and the SiRNA-FasL+AGE-BSA treatment group effectively reduced the expression of activated Caspase8 and activated Caspase 3. Conclusion: AGE-BSA processing cells can promote the tables of Fas. Fas-FasL signals induce activation of Caspase8, and exogenous alpha -Fas can enhance AGE-BSA induced Cytc release, activation of caspase8 and activation of Caspase3; SiRNA mediated Fas or FasL gene silencing can effectively inhibit AGE-BSA induced Caspase 8 and activating 3.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R774.1;R587.2

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