MicroRNA-218-5p及EGFR在翼狀胬肉發(fā)病過(guò)程中的機(jī)制研究
發(fā)布時(shí)間：2018-12-07 14:27
【摘要】：目的翼狀胬肉作為眼表常見(jiàn)疾病之一,可引起局部刺激癥狀、視力下降等多種不適。手術(shù)切除為當(dāng)前最主要的治療方式,但是術(shù)后復(fù)發(fā)率高。對(duì)于翼狀胬肉發(fā)生及發(fā)展的病理機(jī)制雖有多種解釋,但至今尚未明確。MicroRNAs是一類(lèi)高度保守的單鏈非編碼RNA分子,作為近年來(lái)的研究熱點(diǎn),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(dá),以及其與表皮生長(zhǎng)因子受體(Epidermal Growth Factor Receptor,EGFR)在翼狀胬肉中的表達(dá)關(guān)系,驗(yàn)證miR-218-5p能否成為治療翼狀胬肉新的靶點(diǎn),進(jìn)一步明確翼狀胬肉的發(fā)病機(jī)制,旨在為翼狀胬肉的治療奠定實(shí)驗(yàn)學(xué)基礎(chǔ)。方法1.組織學(xué)水平:搜集臨床上人翼狀胬肉組織及正常結(jié)膜組織,通過(guò)qRT-PCR以及Western Blot分別檢測(cè)miR-218-5p及EGFR在翼狀胬肉和正常結(jié)膜組織中的表達(dá)差異。并通過(guò)相關(guān)性分析,探討miR-218-5p和EGFR的表達(dá)相關(guān)性。2.細(xì)胞學(xué)水平:用組織塊培養(yǎng)法培養(yǎng)原代人翼狀胬肉上皮細(xì)胞(human Pterygium Epithelial Cells,hPECs),通過(guò)抗角蛋白抗體免疫熒光染色鑒定確認(rèn)為上皮細(xì)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(xì)胞轉(zhuǎn)染miR-218-5p mimics及inhibitor干預(yù)miR-218-5p的表達(dá),通過(guò)劃痕實(shí)驗(yàn)探究miR-218-5p的表達(dá)水平變化對(duì)翼狀胬肉上皮細(xì)胞遷移的影響;并通過(guò)qRT-PCR以及Western Blot檢測(cè)miR-218-5p的表達(dá)水平改變對(duì)EGFR表達(dá)的影響。3.雙熒光素酶基因報(bào)告分析:確認(rèn)EGFR是否為miR-218-5p的下游靶目標(biāo)。結(jié)果1.與人正常結(jié)膜組織(n=9)對(duì)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(dá)均增加(P0.05),而miR-218-5p的表達(dá)則降低(P0.05);通過(guò)相關(guān)性分析發(fā)現(xiàn)miR-218-5p及EGFR在翼狀胬肉中的表達(dá)呈明顯負(fù)相關(guān)(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(xì)胞,并成功進(jìn)行了傳代培養(yǎng),經(jīng)免疫熒光染色鑒定排除結(jié)膜下成纖維細(xì)胞的影響,確認(rèn)為純化的翼狀胬肉上皮細(xì)胞。3.成功向培養(yǎng)的人翼狀胬肉上皮細(xì)胞轉(zhuǎn)染miR-218-5p mimics,mimics-NC,inhibitor,inhibitor-NC從而干預(yù)細(xì)胞中miR-218-5p的表達(dá)變化。通過(guò)qRT-PCR及Western Blotting檢測(cè)發(fā)現(xiàn):在人翼狀胬肉上皮細(xì)胞中,轉(zhuǎn)染miR-218-5p mimics上調(diào)miR-218-5p的表達(dá),明顯減少EGFR的表達(dá)(P0.05);細(xì)胞中轉(zhuǎn)染miR-218-5p inhibitor可以下調(diào)miR-218-5p的表達(dá),明顯增加EGFR的表達(dá)(P0.05)。4.通過(guò)劃痕實(shí)驗(yàn)發(fā)現(xiàn)轉(zhuǎn)染miR-218-5p mimics后會(huì)使細(xì)胞的遷移明顯減慢(P0.05);而轉(zhuǎn)染miR-218-5p inhibitor后細(xì)胞的遷移則顯著增快(P0.05)。5.雙熒光素酶基因報(bào)告分析:說(shuō)明EGFR是miR-218-5p的下游靶目標(biāo)。結(jié)論本課題首次闡述了在翼狀胬肉中miR-218-5p與EGFR的表達(dá)呈負(fù)相關(guān);在細(xì)胞學(xué)水平發(fā)現(xiàn)miR-218-5p可通過(guò)抑制EGFR的表達(dá)抑制人翼狀胬肉上皮細(xì)胞的遷移;實(shí)驗(yàn)驗(yàn)證EGFR是miR-218-5p的下游靶目標(biāo)。實(shí)驗(yàn)揭示miR-218-5p參與翼狀胬肉的發(fā)病過(guò)程,miR-218-5p可能成為翼狀胬肉治療的新靶點(diǎn),為翼狀胬肉臨床研究提供新的實(shí)驗(yàn)依據(jù),也為眼部新生血管疾病的發(fā)病機(jī)制研究提供新的線(xiàn)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R777.33
本文編號(hào)：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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R777.33
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