【摘要】：視網(wǎng)膜色素上皮(RPE)細(xì)胞的退化是年齡相關(guān)性黃斑變性(AMD)的臨床標(biāo)志。隨著線粒體氧化磷酸化減少,RPE細(xì)胞代謝障礙及功能異常,從而導(dǎo)致AMD發(fā)生發(fā)展。RPE細(xì)胞的氧化損傷是AMD病理機(jī)制中的主要原因。本論文首先利用H_2O_2建立ARPE-19細(xì)胞氧化損傷模型,通過細(xì)胞活力檢測(cè),細(xì)胞凋亡形態(tài)學(xué)觀察,SOD活性、GSH和MDA含量測(cè)定,ROS及線粒體膜電位檢測(cè),探究了低聚糖復(fù)合物(OSC)在ARPE-19細(xì)胞內(nèi)的抗氧化能力。實(shí)驗(yàn)結(jié)果表明,當(dāng)利用不同濃度的OSC作用后,H_2O_2引起的ARPE-19細(xì)胞的氧化應(yīng)激水平明顯減弱,并呈劑量依賴性。當(dāng)OSC濃度為250μg/mL時(shí),細(xì)胞活力升高了19%,凋亡形態(tài)明顯減弱。SOD活性和GSH含量分別恢復(fù)至對(duì)照組的94.8%和90.1%,MDA含量和ROS含量分別下降至對(duì)照組的1.47倍和1.4倍,線粒體膜電位顯著升高。這一研究表明OSC在ARPE-19細(xì)胞內(nèi)具有一定的抗氧化能力。然后,利用乙醇建立小鼠氧化損傷模型,通過血清中SOD的活性、GSH和MDA的含量測(cè)定,探究了OSC對(duì)乙醇損傷小鼠的抗氧化作用。實(shí)驗(yàn)結(jié)果表明,當(dāng)給予0.8g/kg的OSC時(shí),SOD活性和GSH含量分別恢復(fù)至對(duì)照組的94.1%和100.1%,而MDA含量下降至對(duì)照組的1.49倍。這種作用結(jié)果說明OSC在小鼠體內(nèi)也有很好的抗氧化功能,可以抵御乙醇對(duì)小鼠造成的氧化損傷。最后,為了研究OSC的抗氧化機(jī)制,我們利用Western Blot檢測(cè)ARPE-19細(xì)胞和小鼠的肝臟及眼球中的SIRT1及PGC-1α蛋白表達(dá)水平。實(shí)驗(yàn)發(fā)現(xiàn),當(dāng)OSC濃度為250μg/m L時(shí),ARPE-19細(xì)胞中SIRT1及PGC-1α的表達(dá)量分別比H_2O_2損傷組增加了31.6%和23.7%。當(dāng)給予0.8g/kg的OSC喂食小鼠時(shí),小鼠肝臟和眼球的SIRT1蛋白表達(dá)量分別比對(duì)照組上調(diào)了23.3%和35.2%,PGC-1α蛋白表達(dá)量分別比對(duì)照組上調(diào)了13.6%和11.5%。說明OSC可以顯著活化SIRT1/PGC-1α信號(hào)通路,從而抵御氧化損傷。綜上所述,OSC具有保護(hù)H_2O_2氧化損傷的ARPE-19細(xì)胞和乙醇氧化損傷的小鼠的功能,通過活化SIRT1/PGC-1α信號(hào)通路,抵御氧化損傷。因此,OSC可以成為預(yù)防和治療AMD的一種潛在藥物。
[Abstract]:The degeneration of the retinal pigment epithelium (RPE) cells is a clinical sign of age-related macular degeneration (AMD). With the decrease of mitochondrial oxidative phosphorylation, the metabolic disorder and functional abnormality of RPE cells resulted in the development of AMD. The oxidative damage of RPE cells is the main cause of AMD's pathological mechanism. In this paper, an ARPE-19 cell oxidative damage model was established by H _ 2O _ 2, and the anti-oxidation ability of the oligosaccharide complex (OSC) in the ARPE-19 cells was investigated by the cell viability test, the morphological observation of apoptosis, the activity of SOD, the content of GSH and MDA, the detection of ROS and the mitochondrial membrane potential. The results showed that the oxidative stress level of ARPE-19 cells induced by H _ 2O _ 2 was significantly reduced and dose-dependent after using the OSC of different concentrations. When the OSC concentration was 250. m u.g/ mL, the cell viability was increased by 19% and the apoptosis was significantly reduced. The activity of SOD and the content of GSH were 94.8% and 90.1% in the control group, respectively. The content of MDA and ROS decreased to 1.47 and 1.4 times in the control group, respectively. This study shows that the OSC has a certain antioxidant capacity in the ARPE-19 cells. Then, the oxidative damage model of mice was established by using ethanol, the activity of SOD in serum, the content of GSH and MDA were measured, and the anti-oxidation effect of OSC on the mice with ethanol injury was investigated. The results showed that when the OSC of 0.8 g/ kg was given, the activity of SOD and the content of GSH were 94.1% and 100.1%, respectively, while the content of MDA decreased to 1.49 times in the control group. The results show that the OSC has good anti-oxidation function in the mouse, and can resist the oxidative damage of the ethanol to the mice. Finally, in order to study the anti-oxidation mechanism of OSC, we used the Western Blot to detect the expression levels of SIRT1 and PGC-1 in the liver and the eye of ARPE-19 cells and mice. The results showed that the expression of SIRT1 and PGC-1 in ARPE-19 cells increased by 31.6% and 23.7%, respectively, when the OSC concentration was 250 & mu; g/ m L. When 0.8 g/ kg of OSC was administered to the mice, the expression of the SIRT1 protein in the liver and the eye of the mice increased by 23.3% and 35.2%, respectively, compared with the control group, and the expression of the PGC-1 protein was 13.6% and 11.5% higher than that of the control group, respectively. The OSC can significantly activate the SIRT1/ PGC-1 signal path to resist oxidative damage. In conclusion, the OSC has the function of protecting the mice with the ARPE-19 cell and the ethanol oxidative damage of the H _ 2O _ 2 oxidative damage, and resisting the oxidative damage by activating the SIRT1/ PGC-1 signal path. Thus, the OSC can be a potential drug for the prevention and treatment of AMD.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R774.5

【相似文獻(xiàn)】

相關(guān)期刊論文 前3條

1 龐利民,孫二丹,張小猛,龐慧民,張?zhí)熨Y,李曼麗;曲安奈德對(duì)人視網(wǎng)膜色素上皮細(xì)胞(ARPE-19)增生抑制作用的實(shí)驗(yàn)研究[J];眼科;2005年04期

2 周青;陳志鈞;趙麗;戈應(yīng)濱;王斌;袁志蘭;;血管內(nèi)皮生長因子mRNA表達(dá)及ARPE-19細(xì)胞增生與缺氧的誘導(dǎo)效應(yīng)[J];中國組織工程研究與臨床康復(fù);2007年19期

3 ;[J];;年期

相關(guān)會(huì)議論文 前1條

1 劉慶淮;高娟玉;王珍;蔡建園;;新的TPR蛋白TPR99和熱休克蛋白70在ARPE-19細(xì)胞中的相互作用[A];中國眼底病論壇·全國眼底病專題學(xué)術(shù)研討會(huì)論文匯編[C];2008年

相關(guān)博士學(xué)位論文 前1條

1 岳巖;缺氧人視網(wǎng)膜色素上皮細(xì)胞株ARPE-19差異MicroRNA表達(dá)及鑒定[D];中國人民解放軍軍醫(yī)進(jìn)修學(xué)院;2009年

相關(guān)碩士學(xué)位論文 前5條

1 褚文麗;加減駐景方對(duì)Akt基因轉(zhuǎn)染后ARPE-19細(xì)胞表達(dá)VEGF的影響及機(jī)制研究[D];中國中醫(yī)科學(xué)院;2016年

2 郝曉寧;白藜蘆醇對(duì)人視網(wǎng)膜色素上皮細(xì)胞遷移的影響[D];暨南大學(xué);2016年

3 盛雪;低聚糖復(fù)合物對(duì)ARPE-19細(xì)胞及小鼠的抗氧化活性研究[D];吉林大學(xué);2017年

4 孫二丹;曲安奈德對(duì)人視網(wǎng)膜色素上皮細(xì)胞（ARPE-19）生長的影響[D];吉林大學(xué);2005年

5 劉海燕;人巨細(xì)胞病毒及其IE86蛋白對(duì)ARPE-19細(xì)胞周期影響的研究[D];青島大學(xué);2010年

，

本文編號(hào)：2452665