MicroRNA-218-5p及EGFR在翼状胬肉发病过程中的机制研究
发布时间：2018-12-07 14:27
【摘要】：目的翼状胬肉作为眼表常见疾病之一,可引起局部刺激症状、视力下降等多种不适。手术切除为当前最主要的治疗方式,但是术后复发率高。对于翼状胬肉发生及发展的病理机制虽有多种解释,但至今尚未明确。MicroRNAs是一类高度保守的单链非编码RNA分子,作为近年来的研究热点,其在翼状胬肉中的研究也日益受到重视。本研究的目的是探讨microRNA-218-5p(miR-218-5p)在翼状胬肉中是否表达,以及其与表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)在翼状胬肉中的表达关系,验证miR-218-5p能否成为治疗翼状胬肉新的靶点,进一步明确翼状胬肉的发病机制,旨在为翼状胬肉的治疗奠定实验学基础。方法1.组织学水平:搜集临床上人翼状胬肉组织及正常结膜组织,通过qRT-PCR以及Western Blot分别检测miR-218-5p及EGFR在翼状胬肉和正常结膜组织中的表达差异。并通过相关性分析,探讨miR-218-5p和EGFR的表达相关性。2.细胞学水平:用组织块培养法培养原代人翼状胬肉上皮细胞(human Pterygium Epithelial Cells,hPECs),通过抗角蛋白抗体免疫荧光染色鉴定确认为上皮细胞,并传代培养,第3-5代用于后续研究。向培养的原代人翼状胬肉上皮细胞转染miR-218-5p mimics及inhibitor干预miR-218-5p的表达,通过划痕实验探究miR-218-5p的表达水平变化对翼状胬肉上皮细胞迁移的影响;并通过qRT-PCR以及Western Blot检测miR-218-5p的表达水平改变对EGFR表达的影响。3.双荧光素酶基因报告分析:确认EGFR是否为miR-218-5p的下游靶目标。结果1.与人正常结膜组织(n=9)对比,发现在核酸及蛋白水平,翼状胬肉组织(n=24)中EGFR的表达均增加(P0.05),而miR-218-5p的表达则降低(P0.05);通过相关性分析发现miR-218-5p及EGFR在翼状胬肉中的表达呈明显负相关(R2=0.9062,P0.01)。2.利用组织块培养法成功培养出原代人翼状胬肉上皮细胞,并成功进行了传代培养,经免疫荧光染色鉴定排除结膜下成纤维细胞的影响,确认为纯化的翼状胬肉上皮细胞。3.成功向培养的人翼状胬肉上皮细胞转染miR-218-5p mimics,mimics-NC,inhibitor,inhibitor-NC从而干预细胞中miR-218-5p的表达变化。通过qRT-PCR及Western Blotting检测发现:在人翼状胬肉上皮细胞中,转染miR-218-5p mimics上调miR-218-5p的表达,明显减少EGFR的表达(P0.05);细胞中转染miR-218-5p inhibitor可以下调miR-218-5p的表达,明显增加EGFR的表达(P0.05)。4.通过划痕实验发现转染miR-218-5p mimics后会使细胞的迁移明显减慢(P0.05);而转染miR-218-5p inhibitor后细胞的迁移则显著增快(P0.05)。5.双荧光素酶基因报告分析:说明EGFR是miR-218-5p的下游靶目标。结论本课题首次阐述了在翼状胬肉中miR-218-5p与EGFR的表达呈负相关;在细胞学水平发现miR-218-5p可通过抑制EGFR的表达抑制人翼状胬肉上皮细胞的迁移;实验验证EGFR是miR-218-5p的下游靶目标。实验揭示miR-218-5p参与翼状胬肉的发病过程,miR-218-5p可能成为翼状胬肉治疗的新靶点,为翼状胬肉临床研究提供新的实验依据,也为眼部新生血管疾病的发病机制研究提供新的线索。
[Abstract]:Objective To study the effect of pterygoid meat as one of the diseases of ocular surface disease, which can cause local irritation and decrease of vision. Surgical resection is the most important mode of treatment, but the recurrence rate is high. Although there are many explanations for the pathogenesis and development of pterygoid meat, it has not yet been clear. MicroRNAs are a highly conserved single-chain non-coding RNA molecule as a hot spot in recent years. The purpose of this study is to investigate the expression of microRNA-218-5p (miR-218-5p) in the pterygoid meat and its relationship with the expression of the epidermal growth factor receptor (EGFR) in the pterygoid meat, to verify whether the miR-218-5p can be a new target for the treatment of pterygoid meat, and to further define the pathogenesis of the pterygoid meat. and aims to lay an experimental basis for the treatment of the pterygoid meat. Method 1. Histologic level: The expression of miR-218-5p and EGFR in pterygoid and normal conjunctival tissues was detected by qRT-PCR and Western Blot respectively. The expression of miR-218-5p and EGFR was discussed by correlation analysis. Cytological level: The primary human pterygidium-specific cells (hPECs) were cultured by tissue culture method, and the epithelial cells were identified by the immunofluorescence staining of the anti-keratin antibody, and the culture was carried out. The third-fifth generation was used for the follow-up study. The expression of miR-218-5p and the expression of miR-218-5p were examined by the expression of miR-218-5p, and the effect of the expression level of miR-218-5p on the expression of EGFR was investigated by means of qRT-PCR and Western Blot. The double luciferase gene report analysis: confirm that the EGFR is the downstream target of miR-218-5p. Results 1. In contrast to normal conjunctival tissue (n = 9), the expression of EGFR in the level of nucleic acid and protein (n = 24) was increased (P0.05), while the expression of miR-218-5p was decreased (P0.05); and the expression of miR-218-5p and EGFR in the wing-like meat was found to be negatively correlated with the correlation analysis (R2 = 0.9062, P0.01).2. The primary human pterygoid meat epithelial cells were successfully cultured by the tissue block culture method, and the culture was successfully carried out. The effects of the subconjunctival fibroblasts were identified by immunofluorescence staining and confirmed as the purified pterygoid-like meat epithelial cells. The expression of miR-218-5p in the cells was analyzed by the successful transfection of miR-218-5p mimetics, mimics-NC, inhibisitor, inhibitor-NC into the cultured human-winged human-like meat epithelial cells. The expression of miR-218-5p was up-regulated by transfection of miR-218-5p mimics in human-winged human-like meat epithelial cells by qRT-PCR and Western Blotting. The expression of miR-218-5p could be reduced by the transfection of miR-218-5p in the cells, and the expression of EGFR was significantly increased (P0.05). After the transfection of miR-218-5p imics, the migration of the cells was significantly slowed by the scratch test (P0.05); and the migration of the cells transfected with miR-218-5p imics increased significantly (P0.05). The double luciferase gene report analysis: indicates that the EGFR is the downstream target of miR-218-5p. Conclusion The first time in this study, the expression of miR-218-5p in the wing-like meat is negatively correlated with the expression of EGFR, and the expression of miR-218-5p can be inhibited by inhibiting the expression of EGFR in the cytologic level, and the target of the downstream target of miR-218-5p can be verified by experiments. The experiment revealed that miR-218-5p was involved in the pathogenesis of pterygoid meat, and miR-218-5p could be a new target for the treatment of pterygoid meat.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R777.33
本文编号：2367342
[Abstract]:Objective To study the effect of pterygoid meat as one of the diseases of ocular surface disease, which can cause local irritation and decrease of vision. Surgical resection is the most important mode of treatment, but the recurrence rate is high. Although there are many explanations for the pathogenesis and development of pterygoid meat, it has not yet been clear. MicroRNAs are a highly conserved single-chain non-coding RNA molecule as a hot spot in recent years. The purpose of this study is to investigate the expression of microRNA-218-5p (miR-218-5p) in the pterygoid meat and its relationship with the expression of the epidermal growth factor receptor (EGFR) in the pterygoid meat, to verify whether the miR-218-5p can be a new target for the treatment of pterygoid meat, and to further define the pathogenesis of the pterygoid meat. and aims to lay an experimental basis for the treatment of the pterygoid meat. Method 1. Histologic level: The expression of miR-218-5p and EGFR in pterygoid and normal conjunctival tissues was detected by qRT-PCR and Western Blot respectively. The expression of miR-218-5p and EGFR was discussed by correlation analysis. Cytological level: The primary human pterygidium-specific cells (hPECs) were cultured by tissue culture method, and the epithelial cells were identified by the immunofluorescence staining of the anti-keratin antibody, and the culture was carried out. The third-fifth generation was used for the follow-up study. The expression of miR-218-5p and the expression of miR-218-5p were examined by the expression of miR-218-5p, and the effect of the expression level of miR-218-5p on the expression of EGFR was investigated by means of qRT-PCR and Western Blot. The double luciferase gene report analysis: confirm that the EGFR is the downstream target of miR-218-5p. Results 1. In contrast to normal conjunctival tissue (n = 9), the expression of EGFR in the level of nucleic acid and protein (n = 24) was increased (P0.05), while the expression of miR-218-5p was decreased (P0.05); and the expression of miR-218-5p and EGFR in the wing-like meat was found to be negatively correlated with the correlation analysis (R2 = 0.9062, P0.01).2. The primary human pterygoid meat epithelial cells were successfully cultured by the tissue block culture method, and the culture was successfully carried out. The effects of the subconjunctival fibroblasts were identified by immunofluorescence staining and confirmed as the purified pterygoid-like meat epithelial cells. The expression of miR-218-5p in the cells was analyzed by the successful transfection of miR-218-5p mimetics, mimics-NC, inhibisitor, inhibitor-NC into the cultured human-winged human-like meat epithelial cells. The expression of miR-218-5p was up-regulated by transfection of miR-218-5p mimics in human-winged human-like meat epithelial cells by qRT-PCR and Western Blotting. The expression of miR-218-5p could be reduced by the transfection of miR-218-5p in the cells, and the expression of EGFR was significantly increased (P0.05). After the transfection of miR-218-5p imics, the migration of the cells was significantly slowed by the scratch test (P0.05); and the migration of the cells transfected with miR-218-5p imics increased significantly (P0.05). The double luciferase gene report analysis: indicates that the EGFR is the downstream target of miR-218-5p. Conclusion The first time in this study, the expression of miR-218-5p in the wing-like meat is negatively correlated with the expression of EGFR, and the expression of miR-218-5p can be inhibited by inhibiting the expression of EGFR in the cytologic level, and the target of the downstream target of miR-218-5p can be verified by experiments. The experiment revealed that miR-218-5p was involved in the pathogenesis of pterygoid meat, and miR-218-5p could be a new target for the treatment of pterygoid meat.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R777.33
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