本文關(guān)鍵詞： 藍(lán)光照射 線粒體DNA 凋亡 氧化應(yīng)激 人視網(wǎng)膜色素上皮細(xì)胞　出處：《廣州中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:眼底病近年來的研究熱點(diǎn)中,年齡相關(guān)性黃斑病變(Age-related macμlar degeneration,AMD)受到越來越多的關(guān)注,AMD是一種可以造成中心視力下降甚至視力完全喪失的疾病,在疾病早期常無明顯癥狀,但早期往往就會(huì)伴有視網(wǎng)膜色素上皮(retinal pigment epithelium,RPE)的萎縮。國內(nèi)外均有研究表明,光損傷是AMD的重要誘因,其中藍(lán)光是可見光中能量最高的,長時(shí)間暴露于高強(qiáng)度的藍(lán)光下可導(dǎo)致人視網(wǎng)膜的光損傷,現(xiàn)已有研究證實(shí)藍(lán)光可以誘導(dǎo)RPE細(xì)胞發(fā)生氧化損傷和細(xì)胞凋亡,其中線粒體在藍(lán)光損傷RPE細(xì)胞中的具有重要作用,但是線粒體及線粒體DNA在RPE細(xì)胞損傷中的的作用機(jī)制和動(dòng)態(tài)變化仍不清楚。本實(shí)驗(yàn)擬通過體外分離培養(yǎng)人原代RPE細(xì)胞,經(jīng)過藍(lán)光誘導(dǎo)損傷RPE細(xì)胞,觀察在同樣藍(lán)光輻照強(qiáng)度下,處以不同時(shí)長的藍(lán)光照射,觀察RPE細(xì)胞因此產(chǎn)生的損傷變化,同時(shí)深入研究線粒體DNA參與到藍(lán)光誘導(dǎo)RPE細(xì)胞損傷中的作用具體變化。研究方法:取對(duì)數(shù)生長期的原代RPE細(xì)胞用于本次實(shí)驗(yàn)研究,使用一定輻射強(qiáng)度的藍(lán)光進(jìn)行照射。根據(jù)光照時(shí)間將RPE細(xì)胞分為正常對(duì)照組(光照0 h組)和藍(lán)光光照組細(xì)胞,藍(lán)光光照組分別照射0.5 h、1 h、2 h、4 h、6 h、12 h、24h。免疫熒光法檢測(cè)RPE細(xì)胞特有蛋白R(shí)PE65表達(dá)結(jié)果。Western blot蛋白質(zhì)印跡法檢測(cè)不同藍(lán)光照射時(shí)長下RPE 細(xì)胞中的凋亡相關(guān)蛋白 Caspase-3、Cleaved Caspase-3、Caspase9、Cleaved Caspase-9 Bcl-2及Bax的相對(duì)表達(dá)量。RT-PCR技術(shù)檢測(cè)凋亡相關(guān)基因Caspase-3、Caspase-9、Bcl-2及Bax的表達(dá)情況。實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)線粒體DNA拷貝數(shù)的相對(duì)表達(dá)量。普通PCR擴(kuò)增檢測(cè)線粒體DNA4977bp基因缺失的表達(dá)情況。PCR擴(kuò)增產(chǎn)物進(jìn)行基因測(cè)序觀察突變情況。研究結(jié)果:1.不同時(shí)長的藍(lán)光照射人原代RPE細(xì)胞后,Western Blot結(jié)果顯示Bax從lh開始表達(dá)水平均顯著高于對(duì)照組(P0.05);Cleaved Caspase-3從2h開始表達(dá)水平均顯著高于對(duì)照組(P0.05);Caspase-3、Caspase-9 Cleaved Caspase-9 從 4h開始表達(dá)水平均顯著高于對(duì)照組(P0.05);Bcl-2從2h開始表達(dá)水平均較對(duì)照組下調(diào)(P0.05)。2.RT-PCR顯示凋亡相關(guān)基因Bcl-2在光照2h后表達(dá)量上調(diào),光照6h后表達(dá)量低于對(duì)照組(P0.05),Caspase-3、Caspase-9和Bax分別在光照12h、4h和4h后表達(dá)量明顯上調(diào)(P0.05)。3.與對(duì)照組相比,光照2h后,線粒體DNA拷貝數(shù)表達(dá)開始下降,具有顯著差異(*P0.05),呈時(shí)間依賴性降低。4.光照4h后,線粒體DNA4977bp缺失表達(dá)情況與正常組比較呈上升趨勢(shì),并且隨光照時(shí)間延長,缺失情況越加嚴(yán)重,結(jié)果具有顯著差異(*P0.05)。5.基因測(cè)序提示普通PCR擴(kuò)增產(chǎn)物與線粒體DNA發(fā)生4977bp基因缺失后基因型相符。結(jié)論:1.藍(lán)光照射1h后,促凋亡蛋白Bax表達(dá)增加,光照2h后,CleavedCaspase-3開始上升,抑凋亡蛋白Bcl-2開始下降,4h后Caspase-3、Caspase-9、Cleaved Caspase-9開始上升,凋亡相關(guān)基因Bcl-2在光照2h后出現(xiàn)上調(diào),6小時(shí)開始出現(xiàn)下降,Bax、Caspase-9在光照4小時(shí)后開始上調(diào),Caspase-3在12h后出現(xiàn)上升,提示在細(xì)胞經(jīng)過藍(lán)光照射后,凋亡系統(tǒng)被激活,并且是以線粒體介導(dǎo)的凋亡通路為主,從而誘導(dǎo)RPE細(xì)胞的凋亡。2.藍(lán)光照射2h后,線粒體DNA拷貝數(shù)逐步下降,然而在光照4h開始,線粒體DNA4977bp基因缺失明顯增加,證明了藍(lán)光照射可以影響線粒體DNA的復(fù)制并且提高其突變率。這表明藍(lán)光光照2h開始出現(xiàn)線粒體DNA損傷,損傷形式包括線粒體DNA拷貝數(shù)下降以及突變率增加,隨后出現(xiàn)RPE細(xì)胞損傷,繼而出現(xiàn)凋亡以及死亡。
[Abstract]:Background: the research focus in recent years fundus diseases, age-related macular degeneration (Age-related MAC lar degeneration, AMD) has attracted more and more attention, AMD is a can cause the decline of visual acuity and even complete loss of vision disease early in the disease often no obvious symptoms, but often accompanied by early retinal pigment epithelium (retinal pigment epithelium, RPE) atrophy. Some studies at home and abroad show that the optical damage is an important cause of AMD, which is the highest visible light blue energy, long time exposure to high intensity blue light can cause retinal light damage, present studies have confirmed that blue light can induce oxidative damage and apoptosis of RPE cells among them, mitochondrial damage in RPE cells plays an important role in the blue, but the mitochondria and mitochondrial DNA in RPE cell injury mechanism and the dynamic change is still not Clearly. The experiment of in vitro cultured human primary RPE cells after injury of RPE cells induced by blue light, blue light irradiation intensity observed at the same time, a blue light, so the observation of damage caused by the changes of RPE cells, and studies on mitochondrial DNA involved in blue light induced RPE cell damage in concrete change. Methods: primary RPE cells in logarithmic growth phase were used in this experiment, using the radiation intensity of blue light irradiation. According to the illumination time of RPE cells were divided into normal control group (H group, 0 light) and blue light group cells, blue light group were irradiated 0.5 h. 1 h, 2 h, 4 h, 6 h, 12 h, detection of RPE cell specific protein RPE65 24h. immunofluorescence detection of.Western protein expression results of blot blotting in different time under blue light irradiation in RPE cell apoptosis related protein Caspase-3, Cleaved Casp Ase-3, Caspase9, Cleaved Caspase-9 and Bax Bcl-2 relative expression of apoptosis related gene Caspase-3.RT-PCR technology, Caspase-9, expression of Bcl-2 and Bax. The relative expression of real-time fluorescence quantitative PCR detection of mitochondrial DNA copy number amplification. Mitochondrial DNA4977bp gene deletion PCR expression of.PCR amplified products were sequenced to observe the mutation. Results: 1. blue light irradiation time of primary RPE cells, Western Blot showed that Bax began expression level were significantly higher than the control group from LH (P0.05); Cleaved Caspase-3 expression levels were significantly higher than those in the control group from 2H (P0.05); Caspase-3 Caspase-9 Cleaved Caspase-9, the expression level began were higher than the control group from 4h (P0.05); Bcl-2 expression levels than those in the control group decreased from 2H (P0.05).2.RT-PCR showed apoptosis related gene Bcl-2 in After 2H light upregulated after 6h light expression was lower than that of the control group (P0.05), Caspase-3, Caspase-9 and Bax respectively according to 12h in light, up-regulated 4H and 4H (P0.05).3. compared with the control group, after 2H light mitochondrial DNA copy number expression began to decrease significantly the difference (*P0.05), a time dependent decrease in.4. after 4H light expression of mitochondrial DNA4977bp deletion compared with the normal group showed an upward trend, and with the increase of irradiation time, the lack of more results showed significant difference (*P0.05).5. gene sequencing suggested that genotype PCR amplified products and consistent common mitochondrial DNA 4977bp after gene deletion. Conclusion: 1. blue light 1H, the pro apoptotic protein Bax expression increased after 2H light, CleavedCaspase-3 began to rise, the antiapoptotic protein Bcl-2 began to decline after 4h Caspase-3, Caspase-9, Cleaved and Caspase-9 began to rise, apoptosis related genes Because Bcl-2 was up-regulated after 2H light 6 hours began to decline, Bax, Caspase-9 began to increase in light after 4 hours, Caspase-3 increased after 12h, suggesting that the cell apoptosis after blue light irradiation, the system is activated, and the mitochondria mediated apoptosis pathway in the apoptosis of.2.. The blue light irradiation 2H can induce RPE cells, mitochondrial DNA copy number decreased gradually, but in the light of 4h, mitochondrial DNA4977bp deletion increased significantly, that blue light can affect mitochondrial DNA replication and increase the mutation rate. This indicates that blue light began to appear 2H mitochondrial DNA damage, including damage of mitochondrial DNA copy number the mutation rate decreased and increased, followed by RPE cell injury, and apoptosis and death.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R774.5

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