【摘要】：背景及目的：在年齡相關(guān)性黃斑變性(AMD)的發(fā)病過程中,視網(wǎng)膜色素上皮(RPE)細(xì)胞漸進(jìn)性功能障礙或凋亡起著至關(guān)重要的作用。NYD-SP15是胞苷脫氨酶家族成員之一,具有潛在地抑制細(xì)胞增殖的功能。最近發(fā)現(xiàn)該基因在RPE細(xì)胞中呈內(nèi)源性低表達(dá),但是具體功能尚不清楚。本實(shí)驗(yàn)?zāi)康氖浅醪窖芯縉YD-SP15對(duì)ARPE-19細(xì)胞的功能影響及作用機(jī)制,為探求NYD-SP15在AMD發(fā)生發(fā)展的作用提供基礎(chǔ)。方法：構(gòu)建慢病毒穩(wěn)定轉(zhuǎn)染的NYD-SP15過表達(dá)及干擾ARPE-19細(xì)胞系,通過CCK-8法、體外創(chuàng)傷劃痕愈合實(shí)驗(yàn)、流式細(xì)胞術(shù)及caspase-3、caspase-8、caspase-9蛋白活性水平檢測(cè)研究其增殖、遷移能力以及凋亡情況。通過基因芯片初步研究NYD-SP15過表達(dá)ARPE-19細(xì)胞系的全基因組變化及生物學(xué)功能。利用過氧化氫誘導(dǎo)NYD-SP15干擾ARPE-19細(xì)胞系的氧化應(yīng)激損傷模型,通過流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡水平及細(xì)胞內(nèi)ROS含量。蛋白印跡實(shí)驗(yàn)檢測(cè)Akt通路、MAPKs通路和Keap-1/Nrf2/HO-1通路蛋白表達(dá)水平。結(jié)果：成功構(gòu)建并鑒定慢病毒穩(wěn)定轉(zhuǎn)染的NYD-SP15過表達(dá)及干擾的ARPE-19細(xì)胞系。NYD-SP15過表達(dá)ARPE-19細(xì)胞系的細(xì)胞增殖、遷移能力較對(duì)照組明顯減慢,細(xì)胞周期S期到G2/M期阻滯,并出現(xiàn)明顯的細(xì)胞凋亡,伴caspase-3和caspase-9通路活化增加�；蛐酒Y(jié)果發(fā)現(xiàn),NYD-SP15過表達(dá)ARPE-19細(xì)胞與對(duì)照組相比,有254個(gè)基因下調(diào),僅57個(gè)基因上調(diào),呈現(xiàn)普遍全基因組表達(dá)受抑制現(xiàn)象。GO分析發(fā)現(xiàn)NYD-SP15可能影響ARPE-19細(xì)胞的基因轉(zhuǎn)錄調(diào)控功能,而信號(hào)通路分析篩選出Akt以及MAPKs通路可能是NYD-SP15行使其負(fù)調(diào)控功能的主要通路。蛋白免疫印跡結(jié)果表明NYD-SP15過表達(dá)能明顯地抑制ARPE-19細(xì)胞中Akt及MAPKs通路蛋白Erkl/2、p-38和JNK的表達(dá)水平。此外,正常培養(yǎng)的NYD-SP15干擾ARPE-19細(xì)胞增殖能力及細(xì)胞周期沒有明顯變化,但是在饑餓條件下細(xì)胞增殖活性增加。而經(jīng)過氧化氫誘導(dǎo)氧化應(yīng)激損傷后,與對(duì)照組相比,干擾組細(xì)胞生存率提高,且細(xì)胞內(nèi)ROS水平降低,提示細(xì)胞抗氧化應(yīng)激能力增強(qiáng)。蛋白免疫印跡結(jié)果發(fā)現(xiàn)NYD-SP15干擾組細(xì)胞中,核Nrf2及HO-1蛋白表達(dá)明顯增加,Akt、Erk1/2及p38活化水平明顯增加,而JNK活化水平降低。結(jié)論：NYD-SP15能夠明顯地抑制Akt或MAPKs通路蛋白表達(dá)從而影響ARPE-19細(xì)胞功能正常發(fā)揮,或誘導(dǎo)細(xì)胞凋亡。此外,NYD-SP15干擾后可通過激活A(yù)kt途徑依賴的Nrf2/HO-1通路保護(hù)ARPE-19細(xì)胞抵抗氧化應(yīng)激損傷。因此提示NYD-SP15對(duì)ARPE-19細(xì)胞具有負(fù)調(diào)控作用,抑制NYD-SP15病理性高表達(dá)能夠提高ARPE-19細(xì)胞的抗氧化應(yīng)激損傷能力。
[Abstract]:Background & objective: the progressive dysfunction or apoptosis of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of age-related macular degeneration (AMD). NYD-SP15 is a member of cytidine deaminase family. It has the potential to inhibit the proliferation of cells. Recently, it has been found that the gene is endogenous low expression in RPE cells, but the specific function is not clear. The purpose of this study was to study the functional effects of NYD-SP15 on ARPE-19 cells and its mechanism, and to provide a basis for exploring the role of NYD-SP15 in the development of AMD. Methods: lentivirus stable transfection of NYD-SP15 over-expression and interference of ARPE-19 cell line were constructed. CCK-8 assay, wound healing test in vitro, flow cytometry and caspase-3,caspase-8, were performed. The activity level of caspase-9 protein was measured to study the proliferation, migration and apoptosis of the cells. The genomic changes and biological functions of NYD-SP15 overexpressing ARPE-19 cell lines were studied by microarray. The oxidative stress injury model of ARPE-19 cell line induced by hydrogen peroxide was induced by NYD-SP15. The level of apoptosis and the content of ROS in the cell line were detected by flow cytometry. The protein expression levels of Akt pathway, MAPKs pathway and Keap-1/Nrf2/HO-1 pathway were detected by Western blot assay. Results: the ARPE-19 cell line stably transfected with lentivirus was successfully constructed and identified. The cell proliferation and migration ability of NYD-SP15 overexpressing ARPE-19 cell line were significantly slower than those of the control group. Cell cycle arrest from S phase to G2 / M phase showed obvious apoptosis, accompanied by increased activation of caspase-3 and caspase-9 pathways. The results of gene chip showed that there were 254 genes down-regulated and only 57 genes up-regulated in NYD-SP15-overexpressing ARPE-19 cells compared with the control group. Go analysis showed that NYD-SP15 might affect the transcriptional regulation of ARPE-19 cells. Signal pathway analysis showed that Akt and MAPKs pathway may be the main pathways for NYD-SP15 to exercise its negative regulatory function. Western blot showed that the overexpression of NYD-SP15 could significantly inhibit the expression of Akt and MAPKs pathway proteins Erkl/2,p-38 and JNK in ARPE-19 cells. In addition, NYD-SP15 interfered with the proliferation and cell cycle of ARPE-19 cells in normal culture, but the cell proliferation activity increased under the condition of starvation. After oxidative stress injury induced by hydrogen oxide, the survival rate of the cells in the interference group was higher than that in the control group, and the level of ROS in the cells decreased, suggesting that the ability of anti-oxidative stress of the cells was enhanced. Western blot showed that the expression of nuclear Nrf2 and HO-1 protein increased significantly, while the activation level of Akt,Erk1/2 and p38 increased significantly, while the level of JNK activation decreased in NYD-SP15 interference group. Conclusion: NYD-SP15 can significantly inhibit the expression of Akt or MAPKs pathway proteins and thus affect the normal function of ARPE-19 cells or induce apoptosis. In addition, NYD-SP15 interference can protect ARPE-19 cells from oxidative stress damage by activating Akt pathway-dependent Nrf2/HO-1 pathway. It is suggested that NYD-SP15 has a negative regulatory effect on ARPE-19 cells, and inhibiting the pathological high expression of NYD-SP15 can improve the ability of anti-oxidative stress damage of ARPE-19 cells.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R774.5

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