增殖性玻璃体视网膜病变相关长链非编码RNA的研究

发布时间：2018-06-18 23:53

本文选题：PVR + lncRNAs　；参考：《南京医科大学》2016年博士论文

【摘要】：目的：增殖性玻璃体视网膜病变(Proliferative Vitreoretinopathy, PVR)是视网膜脱离(Retinal detachment, RD)和玻璃体视网膜手术的严重并发症,也是视网膜复位手术失败的主要原因,最终造成视觉功能的严重损害。长链非编码RNA(longnon-coding RNAs, lncRNAs)是一组长度大于200核苷酸序列(nucleotides, nt)不具有蛋白编码功能的RNA转录本,在正常的生理过程和很多疾病的发生发展中具有重要作用。最近研究发现lncRNAs在很多眼科疾病中表达异常,然而lncRNAs与PVR之间的相关性至今没有研究报道,本研究旨在探索lncRNAs在PVR发生发展中的作用及其临床意义。方法：微阵列基因组芯片筛选PVR-ERMs中与PVR发生相关的lnc-RNAs(与白内障术后继发性ERMs相比),qRT-PCR随机验证其中11条表达量改变超过2倍的lncRNAs,筛选出与PVR密切相关的lncRNA,作为PVR的研究靶点。进一步收集临床标本,qRT-PCR比较PVR-ERMs、白内障术后继发性ERMs和眼外伤患者正常视网膜组织中靶点lncRNA的表达情况；并将靶点lncRNA的表达情况和PVR标记物血小板源性生长因子A(Platelet-Derived Growth Factor, PDGFA)、血小板源性生长因子C (Platelet-Derived Growth Factor, PDGFC)、激肽原1(Kininogen 1, KNG1)和Collagen I的表达情况做比较；随后qRT-PCR分别检测了三组标本中lncRNA-MIAT、RNCR3和TUG1的表达水平,再次验证靶点lncRNA的选择。与此同时,qRT-PCR比较不同病理分级PVR患者血浆和血细胞中靶点lncRNA的表达情况。细胞实验,通过原味杂交的方法检测靶点lncRNA在视网膜色素上皮细胞(Retinal Pigment Epithelial, RPE)中的表达情况。通过siRNA转染的方法下调RPE细胞中靶点lncRNA的表达,并分别给予TNF-α/PDGF-A/IGF-1刺激,MTT检测各组细胞的细胞活力,台盼兰染色检测细胞的增殖情况及死细胞的数量,JC-1染色检测细胞的早期凋亡情况,划痕实验检测细胞的迁移能力。结果：通过微阵列基因分析技术发现,与白内障术后继发性ERMs目比,PVR-ERMs中有78条与PVR发生相关的lncRNAs,其中表达上调的有48条lncRNAs,表达下调的有30条lncRNAs。通过qRT-PCR的方法随机验证了其中11条表达量改变超过2倍的lncRNAs,并且3种肺腺癌转移相关转录本1 (Metastasis-associated lung adenocarcinoma transcript 1, MALAT1)的基因探针检测均发现,MALAT1在PVR-ERMs中表达量明显上调。因此我们初步选择MALAT1作为我们的研究靶点。进一步收集临床标本,通过qRT-PCR的方法比较PVR-ERMs.白内障术后继发性ERMs和眼外伤患者正常视网膜组织中MALAT1的表达情况,结果表明：与白内障术后继发性ERMs和眼部外伤的正常视网膜组织相比,PVR-ERMs中MALAT1的表达明显上调；PDGFA、PDGFC、KNGl和Collagen I等PVR的标记物在PVR-ERMs中表达水平也明显上调,与MALAT1的上调趋势一致；并且,lncRNA-MIAI、RNCR3和TUG1的表达水平在三组临床标本中没有明显差异。进一步验证MALAT1可以作为PVR的研究靶点。接着,通过qRT-PCR的方法检测PVR患者外周血中MALAT1的表达情况,结果表明：与正常健康者相比,PVR患者血浆和血细胞中MALAT1的表达量均明显上调,且上调程度与PVR的严重程度成正相关。而完成手术治疗4个月且未复发的PVR患者,其血浆和血细胞中MALAT1的表达量明显减少。细胞实验中,通过原位杂交的方法检测发现MALAT1主要表达于RPE细胞的细胞核。通过qRT-PCR方法检测发现MALAT1 siRNA能使RPE细胞中MALAT1的表达下调约80%。MTT、台盼蓝和JC-1染色的方法表明：给予TNF-a/PDGFA/IGF-1刺激后,RPE的细胞活力增加、增殖能力增强,但下调MALAT1表达后RPE的细胞活力下降、增殖能力减弱,死细胞数及早期凋亡增加；过划痕实验表明,给予TNF-α/PDGF-A/IGF-1刺激后,RPE细胞的迁移能力增强,但下调MALAT1表达后, RPE细胞的迁移能力下降。结论：本研究发现78条lncRNAs与PVR的发生密切相关,其中MALAT1在PVR-ERMs和PVR患者血浆和血细胞中均表达上调。细胞实验发现MALAT1参与调控RPE的细胞活力、迁移能力,进而参与调节PVR的病理过程。研究结果加深了对PVR的发病机制的认识,为PVR的诊断、基因治疗和预后评估提供新靶点。
[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.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1

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