本文選題：非小細(xì)胞肺癌 + 微小RNA-183　； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:放療是非小細(xì)胞肺癌治療的主要手段之一,放射抵抗是影響NSCLC放療療效的重要原因,EMT是放射抵抗產(chǎn)生的主要原因。研究表明肺腺癌放射抗拒細(xì)胞發(fā)生了EMT表型的改變,且miR-183在肺腺癌放射抗拒細(xì)胞株中表達(dá)增高,提示miR-183可能介導(dǎo)EMT并在肺腺癌細(xì)胞放射抗拒中發(fā)揮作用。因此對miR-183調(diào)控機(jī)制的深入探討,將有望闡明其與EMT的關(guān)系及二者在肺腺癌細(xì)胞放射抵抗中的作用,為臨床上尋找提高NSCLC放射治療療效的靶點(diǎn)提供新的思路。方法:倒置顯微鏡觀察細(xì)胞形態(tài)學(xué)的改變;CCK-8細(xì)胞增殖實(shí)驗(yàn)比較不同細(xì)胞接受相同劑量照射后細(xì)胞生存率的差異;克隆形成實(shí)驗(yàn)比較細(xì)胞放射抗拒性能之間的差異;劃痕愈合實(shí)驗(yàn)比較細(xì)胞遷移能力的改變;qPCR法檢測細(xì)胞中miR-183的表達(dá),qPCR及Western Blot分別檢測ZEB1及EMT相關(guān)分子標(biāo)志物mRNA及蛋白表達(dá)水平的改變;慢病毒轉(zhuǎn)染實(shí)驗(yàn)實(shí)現(xiàn)對H1299及H1299R細(xì)胞中mi R-183基因的上/下調(diào)。結(jié)果:1.X射線持續(xù)照射后,肺腺癌細(xì)胞的形態(tài)發(fā)生了變化,由原本緊密連接的上皮細(xì)胞形態(tài)變成連接疏松、形態(tài)狹長并伸出偽足的類似成纖維細(xì)胞形態(tài);在課題組前期的研究中:克隆形成實(shí)驗(yàn)表明,相比于H1299細(xì)胞,H1299R細(xì)胞的存活肩區(qū)更寬,放射抗拒性更強(qiáng)(P0.05);CCK-8細(xì)胞增殖實(shí)驗(yàn)表明,相比于H1299細(xì)胞,H1299R細(xì)胞在接受亞致死劑量(6Gy)X射線照射后,表現(xiàn)出更高的生存率(P0.05);qPCR檢測發(fā)現(xiàn),mi R-183、ZEB1、Vimentin在H1299R細(xì)胞中高表達(dá)(P0.05),E-cadherin表達(dá)變化無差異(P0.05);Western Blot檢測發(fā)現(xiàn),ZEB1、Vimentin在H1299R細(xì)胞中高表達(dá),E-cadherin表達(dá)降低(P0.05)。2.成功構(gòu)建干擾miR-183表達(dá)的慢病毒載體,qPCR驗(yàn)證miR-183在H1299R-shRNA-miR183細(xì)胞中表達(dá)降低(P0.05)。進(jìn)一步克隆形成實(shí)驗(yàn)表明,相比于陰性對照H1299R-shRNA-NC細(xì)胞,干擾miR-183表達(dá)后的H1299R-shRNA-miR183細(xì)胞的存活肩區(qū)變窄,放射抗拒性減弱(P0.05);CCK-8細(xì)胞增殖實(shí)驗(yàn)表明,相比于H1299R-shRNA-NC細(xì)胞,H1299R-shRNA-mi R183細(xì)胞在接受4Gy X射線照射后,生存率降低(P0.05);劃痕愈合實(shí)驗(yàn)發(fā)現(xiàn),H1299R-shRNA-miR183細(xì)胞的劃痕愈合時(shí)間相對延長,細(xì)胞遷移能力減弱(P0.05);qPCR及Western Blot檢測發(fā)現(xiàn)在基因及蛋白水平ZEB1、Vimentin在H1299R-shRNA-miR183細(xì)胞中表達(dá)均降低,E-cadherin表達(dá)均相對增高(P0.05)。3.成功構(gòu)建miR-183上調(diào)表達(dá)的慢病毒載體,qPCR驗(yàn)證miR-183在H1299-EGFP-miR183細(xì)胞中表達(dá)增高(P0.05)。進(jìn)一步克隆形成實(shí)驗(yàn)表明,相比于陰性對照H1299-EGFP-NC細(xì)胞,mi R-183上調(diào)表達(dá)后的H1299-EGFP-miR183細(xì)胞的存活肩區(qū)變寬,放射抗拒性增強(qiáng)(P0.05);CCK-8細(xì)胞增殖實(shí)驗(yàn)表明,相比H1299-EGFP-NC細(xì)胞,H1299-EGFP-miR183細(xì)胞在接受4Gy X射線照射后,生存率增高(P0.05);劃痕愈合實(shí)驗(yàn)發(fā)現(xiàn),H1299-EGFP-miR183細(xì)胞的劃痕愈合時(shí)間相對縮短,細(xì)胞遷移能力增強(qiáng)(P0.05);qPCR及Western Blot檢測發(fā)現(xiàn)在基因及蛋白水平ZEB1、Vimentin在H1299-EGFP-miR183細(xì)胞中表達(dá)均增高,而E-cadherin表達(dá)均相對降低(P0.05)。結(jié)論:1.持續(xù)X射線照射過程中,H1299細(xì)胞發(fā)生了EMT,其增殖活性增強(qiáng),放射抗拒能力增加;2.miR-183在H1299細(xì)胞發(fā)生放射抗拒的過程中表達(dá)增高,并在其中發(fā)揮重要作用:過表達(dá)miR-183的表達(dá),可能會(huì)促進(jìn)H1299細(xì)胞發(fā)生EMT,且增強(qiáng)其增殖、遷移及放射抗拒的能力;下調(diào)miR-183的表達(dá),可能會(huì)逆轉(zhuǎn)H1299R細(xì)胞的EMT過程,且減弱其增殖、遷移及放射抗拒的能力。
[Abstract]:Objective: radiotherapy is one of the main means of the treatment of non-small cell lung cancer, radiation resistance is an important factor affecting NSCLC radiotherapy, EMT is the main reason for radiation resistance generated. Studies have shown that the EMT phenotype changes of lung adenocarcinoma radioresistant cell, and miR-183 in lung adenocarcinoma cell lines in radioresistant expression increased, suggesting that miR-183 Can mediate EMT and play a role in lung adenocarcinoma cells. So the radioresistant miR-183 regulation mechanism deeply, is expected to clarify its relationship with EMT and the two in the lung cancer cell radiation resistance in vitro, to improve the targeting of NSCLC treatment to provide new ideas for clinical methods: cells were observed under inverted microscope. The morphological changes of CCK-8 cell proliferation; experimental comparison of different cell difference cell survival rate after irradiation at the same dosage; radiation cell clone formation experiment to resist performance differences between; wound healing migration ability of experimental cells; the expression of miR-183 in qPCR cell was detected by qPCR, Western and Blot were detected in Z EB1 and EMT molecular marker mRNA expression and protein level changes; lentiviral transfection experiments achieve the down-regulation of the H1299 and H1299R cells in MI / R-183 gene. Results: 1.X ray continued after irradiation, changes in lung adenocarcinoma cell morphology, from the original tight junction on the skin cells into loose connections the elongated shape and extending. A fibroblast morphology similar to actin; research group in the colony forming experiments show that, compared with H1299 cells, H1299R cells survival shoulder region wider, stronger radiation resistance (P0.05); CCK-8 showed that cell proliferation experiment, compared with H1299 cells, H1299R cells at sublethal dose (6Gy X) after irradiation, showing Higher survival rates (P0.05); qPCR mi R-183, detected ZEB1, Vimentin expression in H1299R cells (P0.05), the expression of E-cadherin had no difference (P0.05); Western Blot ZEB1, detected the high expression of Vimentin in H1299R cells, the expression of E-cadherin decreased (P0.05) lentiviral vector.2. the successful construction of miR-183 interference expression, qPCR verification mi The decreased expression of R-183 protein in H1299R-shRNA-miR183 cells (P0.05). Further cloning experiments show that, compared to the negative control H1299R-shRNA-NC cells, narrow interference miR-183 expression of H1299R-shRNA-miR183 cells after the survival of shoulder region, radiation resistance decreased; (P0.05) showed that CCK-8 cell proliferation experiment, compared with H1299R-shRNA-NC cells, H129 9R-shRNA-mi R183 4Gy X in cells after irradiation, the survival rate decreased (P0.05); wound healing experiment found that relatively prolonged H1299R-shRNA-miR183 cell wound healing time, cell migration ability decreased (P0.05); qPCR and Western Blot detected ZEB1 in gene and protein level, Vimentin expression in H1299R-shRNA-miR183 cells Reduced expression of E-cadherin was relatively higher (P0.05).3. successfully constructed lentiviral vector miR-183 expression, qPCR expression in H1299-EGFP-miR183 cells increased in the verification of miR-183 (P0.05). Further cloning experiments show that, compared to the negative control of H1299-EGFP-NC cells, the expression of MI R-183 increased after the survival of H1299-EGFP-miR183 cells Shoulder width, radiation resistance enhancement; (P0.05) showed that CCK-8 cell proliferation experiment, compared with H1299-EGFP-NC cells, H1299-EGFP-miR183 cells in 4Gy X after irradiation, the survival rate increased (P0.05); wound healing experiment found that H1299-EGFP-miR183 cells relative to shorten the wound healing time, enhance the ability of cell migration (P0.05) and qPCR; West Ern Blot detected ZEB1 in gene and protein level, the expression of Vimentin in H1299-EGFP-miR183 cells was increased, and the expression of E-cadherin was decreased (P0.05). Conclusion: 1. continuous X irradiation process, the EMT H1299 cells, enhance the proliferation activity, anti radiation ability to increase; 2.miR-183 radiation resistance in H1299 cell The expression process, and plays an important role in the overexpression of miR-183 may promote H1299 expression, EMT cells, and enhance the ability of proliferation, migration and radioresistant; down regulating the expression of miR-183 and EMT may reverse H1299R cell, and decrease the proliferation, migration and the ability of radiation resistance.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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