CREB对胶质瘤细胞多药耐药的影响及其机制

发布时间：2018-06-29 03:31

本文选题：胶质瘤 + CREB　；参考：《河北大学》2017年硕士论文

【摘要】：目的:分析CREB(cAMP-response element binding protein,CREB)基因是否参与胶质瘤U251细胞系多药耐药的形成及可能存在的机制,寻求找到一个全新的能够有效逆转胶质瘤细胞多药耐药的分子靶点。方法:免疫组化法检测并比较不同级别脑胶质瘤组织中CREB表达水平差异。培养稳定的胶质瘤U251耐药细胞系U251/TR,CCK-8法检测细胞耐药指数,特异性敲除耐药株中的CREB基因,流式细胞分析术(FCM)检测替莫唑胺(TMZ)作用下U251细胞系及U251/TR的细胞凋亡。Crispr/cas9特异性敲除U251/TR的CREB基因,获得U251/RC细胞株。Western及rt-PCR测定敲除基因前后ABCG2、MGMT、MRP及P-gp基因表达水平。结果:本研究采用分步诱导法成功从U251细胞系中培养出稳定的耐药细胞系U251/TR。在TMZ初始诱导剂量5μmol/L,终末剂量400μmol/L的DMEM培养基中,U251的IC50为(41.36±1.61)μmo L/L,U251/TR的IC50为(293.09±2.46)μmo L/L,U251/TR是U251的7倍。采用Crispr/cas9特异性敲除U251/TR的CREB基因获得U251/RC细胞系,在TMZ干预剂量100μmol/L的培养基中,U251/TRC的细胞凋亡水平远高于U251/TR。Western及rt-PCR测定结果显示,U251/RC的ABCG2、MGMT、MRP及P-gp表达水平均明显降低。结论:CREB基因通过调控下游ABCG2、MGMT、MRP及P-gp的表达水平参与药物转运体、药物靶点、细胞凋亡、细胞修复等过程,影响细胞多药耐药的产生与逆转。这为从根本上解决目前临床胶质瘤治愈及术后复发的双重问题提供了新的方向。
[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 卤1.61) 渭 mol / L and (293.09 卤2.46) 渭 mol / L, respectively. The IC50 of U251 was (293.09 卤2.46) 渭 mol / L, and the IC50 of U251 / TR was (293.09 卤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 卤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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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