本文選題：PVR + lncRNAs��； 參考：《南京醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的：增殖性玻璃體視網(wǎng)膜病變(Proliferative Vitreoretinopathy, PVR)是視網(wǎng)膜脫離(Retinal detachment, RD)和玻璃體視網(wǎng)膜手術(shù)的嚴(yán)重并發(fā)癥,也是視網(wǎng)膜復(fù)位手術(shù)失敗的主要原因,最終造成視覺(jué)功能的嚴(yán)重?fù)p害。長(zhǎng)鏈非編碼RNA(longnon-coding RNAs, lncRNAs)是一組長(zhǎng)度大于200核苷酸序列(nucleotides, nt)不具有蛋白編碼功能的RNA轉(zhuǎn)錄本,在正常的生理過(guò)程和很多疾病的發(fā)生發(fā)展中具有重要作用。最近研究發(fā)現(xiàn)lncRNAs在很多眼科疾病中表達(dá)異常,然而lncRNAs與PVR之間的相關(guān)性至今沒(méi)有研究報(bào)道,本研究旨在探索lncRNAs在PVR發(fā)生發(fā)展中的作用及其臨床意義。方法：微陣列基因組芯片篩選PVR-ERMs中與PVR發(fā)生相關(guān)的lnc-RNAs(與白內(nèi)障術(shù)后繼發(fā)性ERMs相比),qRT-PCR隨機(jī)驗(yàn)證其中11條表達(dá)量改變超過(guò)2倍的lncRNAs,篩選出與PVR密切相關(guān)的lncRNA,作為PVR的研究靶點(diǎn)。進(jìn)一步收集臨床標(biāo)本,qRT-PCR比較PVR-ERMs、白內(nèi)障術(shù)后繼發(fā)性ERMs和眼外傷患者正常視網(wǎng)膜組織中靶點(diǎn)lncRNA的表達(dá)情況；并將靶點(diǎn)lncRNA的表達(dá)情況和PVR標(biāo)記物血小板源性生長(zhǎng)因子A(Platelet-Derived Growth Factor, PDGFA)、血小板源性生長(zhǎng)因子C (Platelet-Derived Growth Factor, PDGFC)、激肽原1(Kininogen 1, KNG1)和Collagen I的表達(dá)情況做比較；隨后qRT-PCR分別檢測(cè)了三組標(biāo)本中l(wèi)ncRNA-MIAT、RNCR3和TUG1的表達(dá)水平,再次驗(yàn)證靶點(diǎn)lncRNA的選擇。與此同時(shí),qRT-PCR比較不同病理分級(jí)PVR患者血漿和血細(xì)胞中靶點(diǎn)lncRNA的表達(dá)情況。細(xì)胞實(shí)驗(yàn),通過(guò)原味雜交的方法檢測(cè)靶點(diǎn)lncRNA在視網(wǎng)膜色素上皮細(xì)胞(Retinal Pigment Epithelial, RPE)中的表達(dá)情況。通過(guò)siRNA轉(zhuǎn)染的方法下調(diào)RPE細(xì)胞中靶點(diǎn)lncRNA的表達(dá),并分別給予TNF-α/PDGF-A/IGF-1刺激,MTT檢測(cè)各組細(xì)胞的細(xì)胞活力,臺(tái)盼蘭染色檢測(cè)細(xì)胞的增殖情況及死細(xì)胞的數(shù)量,JC-1染色檢測(cè)細(xì)胞的早期凋亡情況,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞的遷移能力。結(jié)果：通過(guò)微陣列基因分析技術(shù)發(fā)現(xiàn),與白內(nèi)障術(shù)后繼發(fā)性ERMs目比,PVR-ERMs中有78條與PVR發(fā)生相關(guān)的lncRNAs,其中表達(dá)上調(diào)的有48條lncRNAs,表達(dá)下調(diào)的有30條lncRNAs。通過(guò)qRT-PCR的方法隨機(jī)驗(yàn)證了其中11條表達(dá)量改變超過(guò)2倍的lncRNAs,并且3種肺腺癌轉(zhuǎn)移相關(guān)轉(zhuǎn)錄本1 (Metastasis-associated lung adenocarcinoma transcript 1, MALAT1)的基因探針檢測(cè)均發(fā)現(xiàn),MALAT1在PVR-ERMs中表達(dá)量明顯上調(diào)。因此我們初步選擇MALAT1作為我們的研究靶點(diǎn)。進(jìn)一步收集臨床標(biāo)本,通過(guò)qRT-PCR的方法比較PVR-ERMs.白內(nèi)障術(shù)后繼發(fā)性ERMs和眼外傷患者正常視網(wǎng)膜組織中MALAT1的表達(dá)情況,結(jié)果表明：與白內(nèi)障術(shù)后繼發(fā)性ERMs和眼部外傷的正常視網(wǎng)膜組織相比,PVR-ERMs中MALAT1的表達(dá)明顯上調(diào)；PDGFA、PDGFC、KNGl和Collagen I等PVR的標(biāo)記物在PVR-ERMs中表達(dá)水平也明顯上調(diào),與MALAT1的上調(diào)趨勢(shì)一致；并且,lncRNA-MIAI、RNCR3和TUG1的表達(dá)水平在三組臨床標(biāo)本中沒(méi)有明顯差異。進(jìn)一步驗(yàn)證MALAT1可以作為PVR的研究靶點(diǎn)。接著,通過(guò)qRT-PCR的方法檢測(cè)PVR患者外周血中MALAT1的表達(dá)情況,結(jié)果表明：與正常健康者相比,PVR患者血漿和血細(xì)胞中MALAT1的表達(dá)量均明顯上調(diào),且上調(diào)程度與PVR的嚴(yán)重程度成正相關(guān)。而完成手術(shù)治療4個(gè)月且未復(fù)發(fā)的PVR患者,其血漿和血細(xì)胞中MALAT1的表達(dá)量明顯減少。細(xì)胞實(shí)驗(yàn)中,通過(guò)原位雜交的方法檢測(cè)發(fā)現(xiàn)MALAT1主要表達(dá)于RPE細(xì)胞的細(xì)胞核。通過(guò)qRT-PCR方法檢測(cè)發(fā)現(xiàn)MALAT1 siRNA能使RPE細(xì)胞中MALAT1的表達(dá)下調(diào)約80%。MTT、臺(tái)盼藍(lán)和JC-1染色的方法表明：給予TNF-a/PDGFA/IGF-1刺激后,RPE的細(xì)胞活力增加、增殖能力增強(qiáng),但下調(diào)MALAT1表達(dá)后RPE的細(xì)胞活力下降、增殖能力減弱,死細(xì)胞數(shù)及早期凋亡增加；過(guò)劃痕實(shí)驗(yàn)表明,給予TNF-α/PDGF-A/IGF-1刺激后,RPE細(xì)胞的遷移能力增強(qiáng),但下調(diào)MALAT1表達(dá)后, RPE細(xì)胞的遷移能力下降。結(jié)論：本研究發(fā)現(xiàn)78條lncRNAs與PVR的發(fā)生密切相關(guān),其中MALAT1在PVR-ERMs和PVR患者血漿和血細(xì)胞中均表達(dá)上調(diào)。細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn)MALAT1參與調(diào)控RPE的細(xì)胞活力、遷移能力,進(jìn)而參與調(diào)節(jié)PVR的病理過(guò)程。研究結(jié)果加深了對(duì)PVR的發(fā)病機(jī)制的認(rèn)識(shí),為PVR的診斷、基因治療和預(yù)后評(píng)估提供新靶點(diǎn)。
[Abstract]:Objective: proliferative vitreoretinopathy (Proliferative Vitreoretinopathy, PVR) is a serious complication of retinal detachment (Retinal detachment, RD) and vitreoretinal surgery. It is also the main cause of failure of retinal reposition surgery, which ultimately causes serious damage to visual power. Long chain non coded RNA (longnon-coding RNAs, LN). CRNAs) is a set of RNA transcripts with length greater than 200 nucleotide sequences (nucleotides, NT) that do not have protein coding functions. It plays an important role in normal physiological processes and in the development of many diseases. Recent studies have found that lncRNAs is abnormally expressed in many ophthalmological diseases. However, the correlation between lncRNAs and PVR has not been studied. The purpose of this study was to explore the role and clinical significance of lncRNAs in the development of PVR. Methods: a microarray genome chip was used to screen the lnc-RNAs associated with PVR in PVR-ERMs (compared with secondary ERMs after cataract surgery). QRT-PCR was used to verify that 11 of them changed more than 2 times of lncRNAs, and were closely related to PVR. LncRNA, as a target for the study of PVR. Further collection of clinical specimens, qRT-PCR comparison of PVR-ERMs, the expression of target lncRNA in normal retina of patients with secondary ERMs and ocular trauma after cataract surgery, and the expression of the target lncRNA and the A (Platelet-Derived Growth Factor) of the PVR marker, the platelet derived growth factor (Platelet-Derived Growth Factor), The expression of platelet-derived growth factor C (Platelet-Derived Growth Factor, PDGFC), the expression of kinin 1 (Kininogen 1, KNG1) and Collagen I were compared. Then qRT-PCR respectively detected the lncRNA-MIAT, RNCR3, and the expression levels in the three groups. The expression of target lncRNA in plasma and blood cells of PVR patients. Cell test was used to detect the expression of target lncRNA in retinal pigment epithelial cells (Retinal Pigment Epithelial, RPE) by the method of original flavor hybridization. The expression of lncRNA in RPE cells was downregulated by siRNA transfection, and TNF- alpha /PDGF-A/IGF-1 was given respectively. Stimulation, MTT detection of cell viability, trypan blue staining detection of cell proliferation and the number of dead cells, JC-1 staining detection of early apoptosis of cells, scratch test to detect cell migration ability. Results: microarray gene analysis technique was found to be compared with secondary ERMs mesh after cataract surgery, and there were 78 in PVR-ERMs LncRNAs associated with PVR, in which 48 lncRNAs were up-regulated, and 30 lncRNAs. expressed by qRT-PCR, 11 of them changed more than 2 times of lncRNAs, and 3 kinds of adenocarcinoma associated transcriptional transcript 1 (Metastasis-associated lung adenocarcinoma transcript 1, MALAT1) It was found that the expression of MALAT1 in PVR-ERMs was obviously up-regulated. Therefore, we initially chose MALAT1 as our target. Further collect clinical specimens and compare the expression of MALAT1 in the normal retina of patients with secondary ERMs and ocular trauma after PVR-ERMs. cataract surgery by qRT-PCR. The expression of MALAT1 in PVR-ERMs was significantly up-regulated in secondary ERMs after cataract surgery and in normal retina of ocular trauma, and the expression level of PVR in PVR-ERMs, such as PDGFA, PDGFC, KNGl and Collagen I, was also up obviously up to the upward trend of MALAT1. There was no significant difference in this study. Further validation of MALAT1 could be used as a target for PVR research. Then, the expression of MALAT1 in peripheral blood of PVR patients was detected by qRT-PCR. The results showed that the expression of MALAT1 in plasma and blood cells of PVR patients was up to rise compared with those of normal healthy people, and the up-regulation degree was more serious than that of PVR. Positive correlation. And the expression of MALAT1 in plasma and blood cells decreased significantly in PVR patients who had been treated for 4 months without recurrence. In cell experiments, in situ hybridization method detected that MALAT1 was mainly expressed in the nucleus of RPE cells. The qRT-PCR method detected that MALAT1 siRNA could reduce the expression of MALAT1 in RPE cells. About 80%.MTT, trypan blue and JC-1 staining showed that after the stimulation of TNF-a/PDGFA/IGF-1, the cell vitality of RPE increased and the proliferation ability increased, but the cell vitality of RPE decreased, the proliferation ability weakened, the number of dead cells and the early apoptosis increased after the down regulation of MALAT1 expression. The cross scratch test showed that TNF- alpha /PDGF-A/IGF-1 stimulated, RPE cells were given, and RPE cells were given TNF- alpha /PDGF-A/IGF-1. The migration ability of the RPE cells decreased after the downregulation of MALAT1. Conclusion: This study found that 78 lncRNAs were closely related to the occurrence of PVR, in which MALAT1 was up-regulated in both plasma and blood cells of PVR-ERMs and PVR patients. The pathological process of PVR has deepened the understanding of the pathogenesis of PVR and provided a new target for PVR diagnosis, gene therapy and prognosis evaluation.
【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)】：R774.1

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6 陶方方;白內(nèi)障術(shù)后盲及低視力的原因和防治方法[D];鄭州大學(xué);2014年

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8 廖振奇;白內(nèi)障術(shù)后影響視力的因素[D];福建醫(yī)科大學(xué);2010年

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10 陳永鈴;光學(xué)相干斷層掃描對(duì)青光眼聯(lián)合白內(nèi)障術(shù)后黃斑厚度變化的初步觀察[D];中南大學(xué);2010年

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