神经胶质瘤中Notch1基因与化疗药物敏感性关系的研究

发布时间：2018-05-01 09:30

  本文选题：神经胶质瘤 + 化疗药物敏感性　； 参考：《福建医科大学》2014年硕士论文

【摘要】：目的： 1.研究Notch1基因对人胶质瘤U251细胞株化疗药物敏感性的影响。 2.人胶质瘤中Notch1与多药耐药基因MDR、MRP1表达的相关性。 方法： 1.构建NICD高表达质粒载体和Notch1sh-RNA质粒载体，通过慢病毒包装、转染、筛选，分别获得高表达NICD的U251细胞株和Notch1基因干扰的U251细胞株。 2. MTT法检测上述模型细胞及对照U251细胞对化疗药物BCNU、VM-26的敏感性。 3. Q-PCR、Western blot、ICC方法检测U251细胞、NICD高表达的U251细胞及Notch1基因干扰的U251细胞中多药耐药基因MDR1、MRP1的表达，以及BCNU、VM-26处理后，各组细胞Notch1、MDR1、MRP1基因表达的变化，并分析其相关性。 4. IHC检测人胶质瘤组织标本中Notch1、MDR1、MRP1基因表达，分析其相关性。结果： 1.成功建立了高表达NICD和Notch1基因干扰的U251细胞株。 2. MTT实验表明NICD高表达U251细胞（plvx-NICD组）与空载对照细胞（plvx组）相比，其药物的半数抑制浓度IC50值升高，而Notch1基因干扰的U251细胞（sh-NA组）与对照组细胞（sh-NC组）相比，其IC50值降低，表明高表达NICD使U251细胞有更强的药物抗性，低表达Notch1细胞其耐药性减弱，，对两种化疗药物更加敏感。 3. Q-PCR、Western Blot、免疫荧光染色检测均表明NICD高表达U251细胞中耐药基因MDR1、MRP1的表达水平均高于对照组细胞，而Notch1基因干扰的U251细胞，MDR1、MRP1表达均下调。 4.免疫组化实验结果显示人脑胶质瘤组织中存在多药耐药基因MDR1、MRP1的表达，表达水平与胶质瘤病理级别呈正相关。在Ⅰ、Ⅱ、Ⅲ级胶质瘤中与Notch1基因的表达具有相关性。 结论： 1. Notch1基因参与调节神经胶质瘤对化疗药物BCNU、VM-26的敏感性。 2.在人胶质瘤U251细胞中Notch1基因可以影响多药耐药基因MDR1、MRP1的表达，并可能通过调节多药耐药基因MDR1、MRP1的表达导致肿瘤细胞的耐药。 3.人脑胶质瘤组织中存在多药耐药基因MDR1、MRP1的表达，且在Ⅰ、Ⅱ、Ⅲ级胶质瘤中与Notch1基因的表达呈正相关。
[Abstract]:Objective: 1. To study the effect of Notch1 gene on chemosensitivity of human glioma cell line U251. 2. Correlation between Notch1 and multidrug resistance gene MDR- MRP1 expression in human glioma. Methods: 1. NICD high expression plasmid vector and Notch1sh-RNA plasmid vector were constructed. U251 cell lines with high expression of NICD and U251 cell lines interfered with Notch1 gene were obtained by lentivirus packaging, transfection and screening. 2. The sensitivity of U251 cells to BCNUVM-26 was detected by MTT assay. 3. The expression of multidrug resistance gene MDR1 and MRP1 in U251 cells with high expression of NICD and interference with Notch1 gene was detected by Q-PCR- Western blotnICC, and the expression of Notch1, MDR1, MRP1 was analyzed after treated with BCNUVM-26, and the correlation between MDR1 and MDR1MRP1 in U251 cells was analyzed. 4. IHC was used to detect the expression of Notch1, MDR1 and MRP1 gene in human glioma tissues and its correlation was analyzed. Results: 1. U251 cell lines with high expression of NICD and Notch1 genes were successfully established. 2. MTT assay showed that the half inhibitory concentration (IC50) of U251 cells with high expression of NICD (plvx-NICD) was higher than that of unloaded control cells (plvx), but the IC50 value of Notch1 gene interfering U251 cells sh-na) was lower than that of control group (sh-NC). The results showed that high expression of NICD resulted in stronger drug resistance in U251 cells and decreased drug resistance of low expression Notch1 cells, which was more sensitive to two chemotherapeutic drugs. 3. Q-PCR Western blot and immunofluorescence staining showed that the expression level of MDR1- MRP1 in U251 cells was significantly higher than that in control cells, but the expression of MDR1MRP1 was down-regulated in U251 cells with interference of Notch1 gene. 4. The results of immunohistochemistry showed that there was a positive correlation between the expression of MDR1and MRP1 in human glioma tissues and the pathological grade of gliomas. The expression of Notch1 gene in grade 鈪

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