Hes1和Hes5表达与人神经胶质瘤细胞增殖能力相关性的研究

发布时间：2018-03-11 13:03

  本文选题：神经胶质瘤　切入点：Notch信号通路　出处：《福建医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的: 1、探讨Notch1受体和其下游信号分子Hes1、Hes5在神经胶质瘤中表达特点和相互关系; 2、构建Hes1、Hes5基因RNAi慢病毒载体,筛选获得Hes1、Hes5沉默的神经胶质瘤细胞株; 3、观察Hes1、Hes5基因沉默对神经胶质细胞U251增殖的影响,初步探讨Notch-Hes信号通路调控神经胶质瘤增殖的可能机制。 方法: 1、应用免疫组织化学方法检测人脑星形细胞瘤和正常人脑组织中Notch1和Hes1、Hes5表达情况; 2、设计、合成针对靶基因Hes1、Hes5的shRNA编码序列,将其克隆到pENTR /U6 RNAi入门载体,经瞬时转染筛选获得有效的靶序列。将含有Hes1、Hes5有效干扰序列的入门载体与pLenti6/BLOCK-iT?-DEST目的载体通过Gateway LR重组构建出相应的shRNA慢病毒表达载体,经293FT细胞包装获得慢病毒颗粒。通过慢病毒转导U251细胞后,筛选获得Hes1、Hes5基因沉默的U251细胞株; 3、采用MTT法,平板克隆形成实验,流式细胞术检测Hes1、Hes5基因沉默对U251细胞增殖及细胞周期的影响。 结果: 1、人脑星形细胞瘤中Notch1、Hes1和Hes5的表达均显著强于正常脑组织;在不同病理分级的星形细胞瘤中,Ⅱ～Ⅲ级组Notch1和Hes1的表达水平显著高于Ⅳ级星形细胞瘤组,Hes5的表达在两组间无显著性差异。星形细胞瘤中Notch1与Hes1的表达有较好的一致性,与Hes5的表达一致性较差。 2、成功筛选出Hes1、Hes5基因特异、有效的RNA干扰靶点,构建了针对Hes1、Hes5基因特异性shRNA慢病毒表达载体,并在293FT细胞中包装获得慢病毒颗粒,病毒滴度分别为8.4×104TU/ml、7.2×104TU/ml。 3、建立了Hes1、Hes5基因稳定沉默的神经胶质瘤U251细胞株。 4、沉默Hes1或Hes5均能明显抑制U251细胞增殖及克隆形成能力。 结论: 1、Notch1表达与星形细胞瘤的发生有关,与星形细胞瘤的病理分级无关。Notch1可能主要是通过下游分子Hes1而非Hes5发挥作用。 2、构建的Hes1、Hes5特异性RNAi慢病毒表达载体能有效抑制胶质瘤细胞U251中Hes1、Hes5基因的表达,可作为研究Notch-Hes信号通路及其与胶质瘤关系的重要技术手段。 3、Hes1、Hes5与胶质瘤细胞的增殖密切相关,在体外Hes1、Hes5特异性RNAi能够抑制胶质瘤细胞U251的增殖能力。
[Abstract]:Objective:. 1. To investigate the expression of Notch1 receptor and its downstream signal molecule Hes1, Hes5, in gliomas. 2. The RNAi lentivirus vector of Hes1 hes5 gene was constructed, and the silencing glioma cell line of Hes1 hes5 was obtained. 3. To observe the effect of Hes1 Hes5 gene silencing on the proliferation of glial cell U251, and to explore the possible mechanism of Notch-Hes signaling pathway regulating glioma proliferation. Methods:. 1. The expression of Notch1 and Hes5 in human astrocytoma and normal human brain tissues were detected by immunohistochemical method. 2. Design and synthesize the shRNA coding sequence for Hes1 gene Hes5, and clone it into pENTR / U6 RNAi entry vector, and obtain the effective target sequence by transient transfection screening. The entry vector containing Hes1hes5 effective interference sequence and pLenti6% BLOCK-iTT? ShRNA lentivirus expression vector was constructed by recombinant Gateway LR. Lentivirus particles were obtained by packaging 293FT cells. After lentivirus was transduced into U251 cells, the U251 cell lines with Hes1Hes5 gene silencing were screened. 3. The effect of Hes1 Hes5 gene silencing on the proliferation and cell cycle of U251 cells was detected by MTT assay and plate clone formation assay. Results:. 1. The expression of Notch1Hes1 and Hes5 in human astrocytoma was significantly higher than that in normal brain tissue. The expression of Notch1 and Hes1 in grade 鈪，

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