本文選題：膠質(zhì)瘤 + CREB　； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：目的:分析CREB(cAMP-response element binding protein,CREB)基因是否參與膠質(zhì)瘤U251細(xì)胞系多藥耐藥的形成及可能存在的機(jī)制,尋求找到一個(gè)全新的能夠有效逆轉(zhuǎn)膠質(zhì)瘤細(xì)胞多藥耐藥的分子靶點(diǎn)。方法:免疫組化法檢測(cè)并比較不同級(jí)別腦膠質(zhì)瘤組織中CREB表達(dá)水平差異。培養(yǎng)穩(wěn)定的膠質(zhì)瘤U251耐藥細(xì)胞系U251/TR,CCK-8法檢測(cè)細(xì)胞耐藥指數(shù),特異性敲除耐藥株中的CREB基因,流式細(xì)胞分析術(shù)(FCM)檢測(cè)替莫唑胺(TMZ)作用下U251細(xì)胞系及U251/TR的細(xì)胞凋亡。Crispr/cas9特異性敲除U251/TR的CREB基因,獲得U251/RC細(xì)胞株。Western及rt-PCR測(cè)定敲除基因前后ABCG2、MGMT、MRP及P-gp基因表達(dá)水平。結(jié)果:本研究采用分步誘導(dǎo)法成功從U251細(xì)胞系中培養(yǎng)出穩(wěn)定的耐藥細(xì)胞系U251/TR。在TMZ初始誘導(dǎo)劑量5μmol/L,終末劑量400μmol/L的DMEM培養(yǎng)基中,U251的IC50為(41.36±1.61)μmo L/L,U251/TR的IC50為(293.09±2.46)μmo L/L,U251/TR是U251的7倍。采用Crispr/cas9特異性敲除U251/TR的CREB基因獲得U251/RC細(xì)胞系,在TMZ干預(yù)劑量100μmol/L的培養(yǎng)基中,U251/TRC的細(xì)胞凋亡水平遠(yuǎn)高于U251/TR。Western及rt-PCR測(cè)定結(jié)果顯示,U251/RC的ABCG2、MGMT、MRP及P-gp表達(dá)水平均明顯降低。結(jié)論:CREB基因通過(guò)調(diào)控下游ABCG2、MGMT、MRP及P-gp的表達(dá)水平參與藥物轉(zhuǎn)運(yùn)體、藥物靶點(diǎn)、細(xì)胞凋亡、細(xì)胞修復(fù)等過(guò)程,影響細(xì)胞多藥耐藥的產(chǎn)生與逆轉(zhuǎn)。這為從根本上解決目前臨床膠質(zhì)瘤治愈及術(shù)后復(fù)發(fā)的雙重問(wèn)題提供了新的方向。
[Abstract]:Aim: to investigate whether the cAMP-response element binding protein (CREB) gene is involved in the formation of multidrug resistance in U251 glioma cell line and its possible mechanism, and to find a novel molecular target that can effectively reverse multidrug resistance in human glioma cell line U251. Methods: immunohistochemical method was used to detect and compare the expression of CREB in different grade gliomas. Stable glioma cell line U251 resistant cell line U251% TRCCK-8 was used to detect the drug resistance index, and the CREB gene was specifically knocked out in the resistant cell line. Flow cytometry (FCM) was used to detect the CREB gene of U251 cell line and U251% tr cell line induced by temozolidomide (TMZ). The CREB gene of U251% TR was specifically knocked out by Crispr-rc9. The expression levels of ABCG2MGMTMRP and P-gp genes were detected by Western and rt-PCR. Results: a stable drug resistant cell line U251 / TR was successfully cultured from U251 cell line by stepwise induction. The IC50 of U251 was (41.36 鹵1.61) 渭 mol / L and (293.09 鹵2.46) 渭 mol / L, respectively. The IC50 of U251 was (293.09 鹵2.46) 渭 mol / L, and the IC50 of U251 / TR was (293.09 鹵2.46) 渭 mol / L / L ~ (251) / L ~ (251TR) in DMEM medium with a final dose of 400 渭 mol 路L ~ (-1) 路L ~ (-1). The IC50 of U251 was (41.36 鹵1.61) 渭 mo / L ~ (-1). U251% RC cell line was obtained by using CREB gene of Crispr-cas9 specific knockout of U251% tr. The apoptosis level of U251% TRC in TMZ medium was much higher than that of U251% TR.Western and rt-PCR. The results showed that the expression of ABCG2MGMTMRP and P-gp in U251% RC was significantly decreased. Conclusion the expression of MGMTP and P-gp in the downstream ABCG2MGMTP is involved in the process of drug transporter, drug target, cell apoptosis, cell repair and so on, which may influence the production and reversal of multidrug resistance. This provides a new direction for solving the dual problems of clinical glioma cure and postoperative recurrence.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 黃從剛;段發(fā)亮;陳謙學(xué);吳京雷;黃喬春;陳曉斌;閔強(qiáng);羅明;李乾鋒;宋平;;X線照射后耐替莫唑胺U251細(xì)胞株中多藥耐藥基因MDR1及P-gp蛋白的表達(dá)[J];中華神經(jīng)醫(yī)學(xué)雜志;2016年01期

相關(guān)博士學(xué)位論文 前1條

1 胡汪來(lái);黑色素瘤治療的抗藥性機(jī)制研究[D];中國(guó)科學(xué)技術(shù)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 解靖;CREB在腦膠質(zhì)瘤中的表達(dá)及敲除后對(duì)腫瘤細(xì)胞生物學(xué)特性的影響[D];河北大學(xué);2016年

2 王培軍;CREB在急性髓細(xì)胞白血病和骨髓增生異常綜合征中的表達(dá)及其臨床意義[D];華中科技大學(xué);2011年

，

本文編號(hào)：2080657