【作者】 周冲；

【导师】 周菊英；

【作者基本信息】 苏州大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:观察miR-21在人脑胶质瘤细胞SHG-44及其辐射抗性模型中的表达差异,探讨敲低miR-21表达联合辐射对SHG-44辐射敏感性的影响,为以miR-21为靶点的分子靶向放疗增敏提供体外研究的理论及实验依据。方法:使用胶质瘤细胞株SHG-44,建立辐射抗性胶质瘤模型SHG-44抗,分别给予6 MV X射线单次照射0、1、2、4、6、8 Gy,多靶单击模型拟合细胞存活曲线,评估所构建模型的辐射抗性效果。使用Taqman探针实时荧光定量PCR法检测SHG-44及SHG-44抗细胞株中miR-21表达,Western Blot法检测信号转导子和转录激活子3蛋白表达(stat3)。使用脂质体介导采用瞬间转染法转染反义miR-21寡核苷酸(AS-miR-21)及阴性对照寡核苷酸于胶质瘤SHG-44细胞中。设空白对照组、阴性对照转染组及AS-miR-21转染组,成克隆实验计算放射增敏比(SER),绘制细胞存活曲线。流式细胞仪检测上述3组及联合单次6 Gy照射后细胞凋亡率、细胞周期变化。吖啶橙/溴乙啶染色法,在激光共聚焦显微镜下观察辐射联合组SHG-44细胞凋亡的形态学特征。结果:成功建立了SHG-44抗辐射抗性细胞株,集落形成实验显示SHG-44细胞D0=2.35、SF2=0.62 ,SHG-44抗细胞D0=3.22,SF2=0.74,两组SF2比较差异有统计学意义(t = 4.13,P < 0.05)。qRT-PCR及Western blot法检测SHG-44抗细胞的miR-21表达及stat3蛋白表达均较SHG-44升高。SHG-44细胞转染AS-miR-21组的D0、Dq及SF2值较空白对照组及阴性转染组降低,放射增敏比SERD0、SERDq分别为1.32、2.10。细胞周期分析示转染组较空白对照及阴性对照组的G0+G1期比例增高,S期比例降低且差异均具有统计学意义(P<0.05)。凋亡分析示照射组、miR-21转染组及联合组的早期凋亡率均高于对照组(P<0.05)。AO/EB染色可见联合组凋亡细胞核固缩、核碎裂,凋亡小体形成。结论:SHG-44为一高表达miR-21细胞株,且SHG-44照射后代细胞miR-21表达增高, stat3蛋白可能参与了miR-21表达的调节。抑制miR-21表达可增加SHG-44细胞放射敏感性,其机制可能与促进细胞凋亡及周期再分布有关。更多还原

【Abstract】 Objective:To observation miR-21 differential expression in the model of radioresistant glioma SHG-44 cells and SHG-44 cells,and investigate the radiosensitizing effect of knock-down the expression of miR-21 by antisense oligonuleotides of miR-21(AS-miR-21) on human SHG-44 glioma cells.Methods:Radioresistant cell model(SHG-44R) were established from SHG-44 glioma cells line Then the radiosensitivity of SHG-44 and SHG-44R were studied by clonogenic assay.Multi-target single-hit model were used to plot the survival curve. Meanwhile,expression levels of miR-21 in SHG-44R and SHG-44 were determined by quantitative real time PCR(qRT-PCR). Stat3 protain were detected by western blot.As-miR-21,mediated by LipofectamineTM2000,were transfected to SHG-44 cells.Three groups were studied:blank control group(mock group),negative control and Antisense transfected group(AS-miR-21 gorup). Cells of each group were irradiated with 6 MV X-ray at the dose of 0,1,2,4,6 and 8 Gy.Dose-Suvivial curve of each group was established by colony-forming assay.The influence of AS-miR-21 on cell cycle and cell apoptosis was analyzed by flow cytometry assay after 6 Gy irradiation.Morphology feature of apoptosis was observed by laser confocal micorscope in SHG-44 cells stained with AO/EB.Results: miR-21 was up-regulation 1.49 fold in SHG-44R cells relative to the SHG-44 cells. The up-regulation of miR-21 expression was shown to be associated with a increase in the level of stat3 protein。The value of D0、Dq and SF2 of AS-miR-21 group declined obviously compared with the mock group and negative control group.Flow cytometric analysis showed that cell cycle distribution changed(G0+G1 phase arrest,S phase decreased) after transfected with AS-miR-21(P<0.05).Apoptosis assay showed the early apoptosis rate was significantely increased in AS-miR-21、irradition alone and combined group than mock or negative control group(P<0.05).Conclusions:miR-21 may represent a new molecular target and may provide a basis for the use of the AS-miR-21 as potential radiosensitizers for treatment of the glioma tumors.更多还原