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,最終造成視覺功能的嚴重損害。長鏈非編碼RNA(longnon-coding RNAs, lncRNAs)是一組長度大于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異常,然而lncRNAs與PVR之間的相關(guān)性至今沒有研究報道,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隨機驗證其中11條表達量改變超過2倍的lncRNAs,篩選出與PVR密切相關(guān)的lncRNA,作為PVR的研究靶點。進一步收集臨床標本,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lncRNA的表達情況；并將靶點lncRNA的表達情況和PVR標記物血小板源性生長因子A(Platelet-Derived Growth Factor, PDGFA)、血小板源性生長因子C (Platelet-Derived Growth Factor, PDGFC)、激肽原1(Kininogen 1, KNG1)和Collagen I的表達情況做比較；隨后qRT-PCR分別檢測了三組標本中l(wèi)ncRNA-MIAT、RNCR3和TUG1的表達水平,再次驗證靶點lncRNA的選擇。與此同時,qRT-PCR比較不同病理分級PVR患者血漿和血細胞中靶點lncRNA的表達情況。細胞實驗,通過原味雜交的方法檢測靶點lncRNA在視網(wǎng)膜色素上皮細胞(Retinal Pigment Epithelial, RPE)中的表達情況。通過siRNA轉(zhuǎn)染的方法下調(diào)RPE細胞中靶點lncRNA的表達,并分別給予TNF-α/PDGF-A/IGF-1刺激,MTT檢測各組細胞的細胞活力,臺盼蘭染色檢測細胞的增殖情況及死細胞的數(shù)量,JC-1染色檢測細胞的早期凋亡情況,劃痕實驗檢測細胞的遷移能力。結(jié)果：通過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,其中表達上調(diào)的有48條lncRNAs,表達下調(diào)的有30條lncRNAs。通過qRT-PCR的方法隨機驗證了其中11條表達量改變超過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)的基因探針檢測均發(fā)現(xiàn),MALAT1在PVR-ERMs中表達量明顯上調(diào)。因此我們初步選擇MALAT1作為我們的研究靶點。進一步收集臨床標本,通過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的表達明顯上調(diào)；PDGFA、PDGFC、KNGl和Collagen I等PVR的標記物在PVR-ERMs中表達水平也明顯上調(diào),與MALAT1的上調(diào)趨勢一致；并且,lncRNA-MIAI、RNCR3和TUG1的表達水平在三組臨床標本中沒有明顯差異。進一步驗證MALAT1可以作為PVR的研究靶點。接著,通過qRT-PCR的方法檢測PVR患者外周血中MALAT1的表達情況,結(jié)果表明：與正常健康者相比,PVR患者血漿和血細胞中MALAT1的表達量均明顯上調(diào),且上調(diào)程度與PVR的嚴重程度成正相關(guān)。而完成手術(shù)治療4個月且未復(fù)發(fā)的PVR患者,其血漿和血細胞中MALAT1的表達量明顯減少。細胞實驗中,通過原位雜交的方法檢測發(fā)現(xiàn)MALAT1主要表達于RPE細胞的細胞核。通過qRT-PCR方法檢測發(fā)現(xiàn)MALAT1 siRNA能使RPE細胞中MALAT1的表達下調(diào)約80%。MTT、臺盼藍和JC-1染色的方法表明：給予TNF-a/PDGFA/IGF-1刺激后,RPE的細胞活力增加、增殖能力增強,但下調(diào)MALAT1表達后RPE的細胞活力下降、增殖能力減弱,死細胞數(shù)及早期凋亡增加；過劃痕實驗表明,給予TNF-α/PDGF-A/IGF-1刺激后,RPE細胞的遷移能力增強,但下調(diào)MALAT1表達后, RPE細胞的遷移能力下降。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患者血漿和血細胞中均表達上調(diào)。細胞實驗發(fā)現(xiàn)MALAT1參與調(diào)控RPE的細胞活力、遷移能力,進而參與調(diào)節(jié)PVR的病理過程。研究結(jié)果加深了對PVR的發(fā)病機制的認識,為PVR的診斷、基因治療和預(yù)后評估提供新靶點。
[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)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R774.1

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