MicroRNA-218-5p及EGFR在翼狀胬肉發(fā)病過程中的機制研究
發(fā)布時間：2018-12-07 14:27
【摘要】：目的翼狀胬肉作為眼表常見疾病之一,可引起局部刺激癥狀、視力下降等多種不適。手術切除為當前最主要的治療方式,但是術后復發(fā)率高。對于翼狀胬肉發(fā)生及發(fā)展的病理機制雖有多種解釋,但至今尚未明確。MicroRNAs是一類高度保守的單鏈非編碼RNA分子,作為近年來的研究熱點,其在翼狀胬肉中的研究也日益受到重視。本研究的目的是探討microRNA-218-5p(miR-218-5p)在翼狀胬肉中是否表達,以及其與表皮生長因子受體(Epidermal Growth Factor Receptor,EGFR)在翼狀胬肉中的表達關系,驗證miR-218-5p能否成為治療翼狀胬肉新的靶點,進一步明確翼狀胬肉的發(fā)病機制,旨在為翼狀胬肉的治療奠定實驗學基礎。方法1.組織學水平:搜集臨床上人翼狀胬肉組織及正常結膜組織,通過qRT-PCR以及Western Blot分別檢測miR-218-5p及EGFR在翼狀胬肉和正常結膜組織中的表達差異。并通過相關性分析,探討miR-218-5p和EGFR的表達相關性。2.細胞學水平:用組織塊培養(yǎng)法培養(yǎng)原代人翼狀胬肉上皮細胞(human Pterygium Epithelial Cells,hPECs),通過抗角蛋白抗體免疫熒光染色鑒定確認為上皮細胞,并傳代培養(yǎng),第3-5代用于后續(xù)研究。向培養(yǎng)的原代人翼狀胬肉上皮細胞轉染miR-218-5p mimics及inhibitor干預miR-218-5p的表達,通過劃痕實驗探究miR-218-5p的表達水平變化對翼狀胬肉上皮細胞遷移的影響;并通過qRT-PCR以及Western Blot檢測miR-218-5p的表達水平改變對EGFR表達的影響。3.雙熒光素酶基因報告分析:確認EGFR是否為miR-218-5p的下游靶目標。結果1.與人正常結膜組織(n=9)對比,發(fā)現(xiàn)在核酸及蛋白水平,翼狀胬肉組織(n=24)中EGFR的表達均增加(P0.05),而miR-218-5p的表達則降低(P0.05);通過相關性分析發(fā)現(xiàn)miR-218-5p及EGFR在翼狀胬肉中的表達呈明顯負相關(R2=0.9062,P0.01)。2.利用組織塊培養(yǎng)法成功培養(yǎng)出原代人翼狀胬肉上皮細胞,并成功進行了傳代培養(yǎng),經免疫熒光染色鑒定排除結膜下成纖維細胞的影響,確認為純化的翼狀胬肉上皮細胞。3.成功向培養(yǎng)的人翼狀胬肉上皮細胞轉染miR-218-5p mimics,mimics-NC,inhibitor,inhibitor-NC從而干預細胞中miR-218-5p的表達變化。通過qRT-PCR及Western Blotting檢測發(fā)現(xiàn):在人翼狀胬肉上皮細胞中,轉染miR-218-5p mimics上調miR-218-5p的表達,明顯減少EGFR的表達(P0.05);細胞中轉染miR-218-5p inhibitor可以下調miR-218-5p的表達,明顯增加EGFR的表達(P0.05)。4.通過劃痕實驗發(fā)現(xiàn)轉染miR-218-5p mimics后會使細胞的遷移明顯減慢(P0.05);而轉染miR-218-5p inhibitor后細胞的遷移則顯著增快(P0.05)。5.雙熒光素酶基因報告分析:說明EGFR是miR-218-5p的下游靶目標。結論本課題首次闡述了在翼狀胬肉中miR-218-5p與EGFR的表達呈負相關;在細胞學水平發(fā)現(xiàn)miR-218-5p可通過抑制EGFR的表達抑制人翼狀胬肉上皮細胞的遷移;實驗驗證EGFR是miR-218-5p的下游靶目標。實驗揭示miR-218-5p參與翼狀胬肉的發(fā)病過程,miR-218-5p可能成為翼狀胬肉治療的新靶點,為翼狀胬肉臨床研究提供新的實驗依據(jù),也為眼部新生血管疾病的發(fā)病機制研究提供新的線索。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R777.33
本文編號：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R777.33
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