發(fā)布時(shí)間：2018-07-05 11:39

  本文選題：膀胱癌 + Notch信號(hào)通路　； 參考：《南昌大學(xué)》2014年碩士論文

【摘要】：目的： 探討microRNA-200b調(diào)控膀胱癌發(fā)生EMT與Notch信號(hào)通路關(guān)系的內(nèi)在分子機(jī)制。 方法： 將has-miR-200b mimics及陰性對(duì)照組(scramble)用脂質(zhì)體Lip2000轉(zhuǎn)染膀胱癌T24、5637細(xì)胞，通過(guò)倒置顯微鏡觀察膀胱癌T24、5637細(xì)胞生長(zhǎng)及形態(tài)的變化；采用qRT-PCR檢測(cè)轉(zhuǎn)染效率；運(yùn)用RT-PCR及western blot技術(shù)檢測(cè)E-cadherin、N-cadherin、vimentin、alpha-smooth muscle actin、Notch-1、Jagged-1的mRNA及蛋白表達(dá)變化；MTT檢測(cè)轉(zhuǎn)染后膀胱癌細(xì)胞株耐藥性的改變；采用Hoechst染色法檢測(cè)抗凋亡能力的改變；細(xì)胞凋亡率的改變通過(guò)流式細(xì)胞學(xué)檢測(cè)；轉(zhuǎn)染后的膀胱癌細(xì)胞株侵襲能力的變化運(yùn)用transwell來(lái)評(píng)估。 結(jié)果： 實(shí)驗(yàn)組的膀胱癌細(xì)胞數(shù)量變少，分布更分散，細(xì)胞的體積明顯增大，形態(tài)更加不規(guī)則；qRT-PCR結(jié)果示：轉(zhuǎn)染48h后，miR-200b顯著性上調(diào)（P＜0.05）；RT-PCR及western blot結(jié)果表明：E-cadherin mRNA和蛋白表達(dá)顯著上調(diào)（P＜0.05），N-cadherin、vimentin、alpha-smooth muscle actin、Notch-1、Jagged-1mRNA和蛋白顯著下調(diào)（P＜0.05）；MTT結(jié)果提示:實(shí)驗(yàn)組細(xì)胞對(duì)順鉑的抵抗力降低（P＜0.05）；Hoechst33342表明:實(shí)驗(yàn)組的膀胱癌細(xì)胞株抗凋亡能力減弱（P＜0.05）；流式細(xì)胞學(xué)結(jié)果示：實(shí)驗(yàn)組的早期晚期凋亡率顯著增加，差異均具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；Transwell表明實(shí)驗(yàn)組膀胱癌細(xì)胞的侵襲能力明顯下調(diào)（P＜0.05）。 結(jié)論： 上調(diào)microRNA-200b能通過(guò)抑制Notch-1、Jagged-1的mRNA及蛋白表達(dá)，，從而阻斷Notch信號(hào)通路，進(jìn)而抑制膀胱癌發(fā)生EMT；同時(shí)對(duì)膀胱癌細(xì)胞的耐藥性、抗凋亡能力、侵襲性產(chǎn)生影響。
[Abstract]:Aim: to investigate the molecular mechanism of microRNA-200b regulating the relationship between EMT and Notch signaling pathway in bladder cancer. Methods: has-miR-200b mimics and negative control group (scramble) were transfected with liposome Lip2000 into bladder cancer cell line T2465637. The growth and morphology of T24567 cells were observed by inverted microscope, and the transfection efficiency was detected by qRT-PCR. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect the changes of mRNA and protein expression of E-cadherin N-cadherin alpha-smooth muscle actinus Notch-1 and Jagged-1. The changes of drug resistance of bladder cancer cell lines after transfection were detected by MTT assay, the changes of anti-apoptosis ability were detected by Hoechst staining, and the changes of apoptosis rate were detected by flow cytometry. Changes in invasive ability of bladder cancer cell lines after transfection were evaluated by transwell. Results: in the experimental group, the number of bladder cancer cells decreased, the distribution became more dispersed, the volume of the cells increased significantly, and the morphology became more irregular. The results of qRT-PCR showed that miR-200b was significantly up-regulated 48 hours after transfection (P < 0.05). The results of RT-PCR and western blot showed that the expression of E-cadherin mRNA and protein was significantly up-regulated (P < 0. 05). (P < 0. 05). The results of western blot showed that the resistance to cisplatin was decreased in the experimental group (P < 0. 05), and the expression of Jagged-1 mRNA and protein was significantly down-regulated (P < 0. 05). Hoechst33342 showed that the anti-apoptotic ability of bladder cancer cell line in the experimental group was decreased (P < 0.05), and the apoptosis rate in the early and late stage of the experimental group was significantly increased (P < 0.05), and the apoptosis rate in the early and late stage of the experimental group was significantly higher than that in the control group (P < 0.05). Transwell showed that the invasion ability of bladder cancer cells in the experimental group was significantly down-regulated (P < 0.05). Conclusion: upregulation of microRNA-200b can block Notch signaling pathway by inhibiting the expression of Notch-1 and Jagged-1 mRNA and protein, and then inhibit the development of EMTs in bladder cancer, and affect the resistance, anti-apoptosis and invasiveness of bladder cancer cells.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

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本文編號(hào)：2100084