胶质瘤中GDNF mRNA的异常选择性剪接及α-pro-GDNF亚型对胶质瘤细胞迁移能力的影响
发布时间：2018-07-17 15:34
【摘要】：目的:探讨胶质瘤组织细胞中GDNF基因在转录过程中发生的选择性剪接异常变化,并揭露这种变化对胶质瘤迁移能力可能存在的影响。方法:针对高度同源的GDNF剪接变异体cDNA的高效引物,应用Real-time PCR检测胶质瘤组织细胞中GDNF选择性剪接体α-pro-GDNF和β-pro-GDNF的mRNA表达水平;利用western blot检测细胞中α-pro-GDNF和β-pro-GDNF蛋白的表达情况,同时选择免疫荧光染色的方法检测胶质瘤细胞中蛋白的表达分布及表达水平。构建过表达和干扰α-pro-GDNF蛋白的慢病毒质粒并转染胶质瘤细胞U251,使α-pro-GDNF蛋白的表达上调或下降,随后利用划痕实验检测转染后的U251胶质瘤细胞迁移能力的变化。结果:Real-time PCR和western blot结果均表明胶质瘤细胞中GDNF基因存在选择性剪接体,且随着胶质瘤WHO级别升高,α-pro-GDNF和β-pro-GDNF的mRNA和蛋白的表达水平均呈逐渐升高趋势,且α-pro-GDNF的mRNA和蛋白的升高趋势较β-pro-GDNF尤为明显,呈优势表达;免疫荧光染色表明α-pro-GDNF和β-pro-GDNF蛋白主要表达于细胞胞质中,荧光强度定量分析进一步提示胶质瘤细胞中蛋白表达量较正常细胞多;慢病毒转染成功后,过表达组α-pro-GDNF蛋白升高,干扰组α-pro-GDNF蛋白降低,划痕实验初步表明α-pro-GDNF蛋白表达的变化会影响胶质瘤细胞的迁移能力。结论:胶质瘤细胞中GDNF基因发生异常选择性剪接,其中α-pro-GDNF优先表达,并与胶质瘤迁移能力密切相关,为胶质瘤的治疗提供新的作用靶点。
[Abstract]:Aim: to investigate the selective splicing abnormal changes of GDNF gene in glioma tissue cells during transcription, and to explore the possible effect of this change on glioma migration ability. Methods: Real-time PCR was used to detect the expression of 伪 -pro-GDNF and 尾 -pro-GDNF in glioma cells, and the expression of 伪 -pro-GDNF and 尾 -pro-GDNF in glioma cells was detected by western blot. At the same time, immunofluorescence staining was used to detect the distribution and level of protein expression in glioma cells. The lentivirus plasmid which overexpressed and interfered with 伪 -pro-GDNF protein was constructed and transfected into glioma cell U251. The expression of 伪 -pro-GDNF protein was up-regulated or down-regulated. The migration ability of transfected U251 glioma cells was detected by scratch test. Results the results of western blot and real-time PCR showed that there was a selective splicing of GDNF gene in glioma cells, and the mRNA and protein levels of 伪 -pro-GDNF and 尾 -pro-GDNF increased gradually with the increase of WHO grade of glioma. The expression of 伪 -pro-GDNF mRNA and protein was more obvious than that of 尾 -pro-GDNF, and immunofluorescence staining showed that 伪 -pro-GDNF and 尾 -pro-GDNF mainly expressed in cytoplasm. The quantitative analysis of fluorescence intensity further indicated that the expression of 伪 -pro-GDNF protein in glioma cells was higher than that in normal cells, and that the 伪 -pro-GDNF protein increased in the overexpression group and decreased in the interference group after lentivirus transfection. Scratch test showed that the expression of 伪 -pro-GDNF protein affected the migration ability of glioma cells. Conclusion: there is abnormal selective splicing of GDNF gene in glioma cells, in which 伪 -pro-GDNF is preferentially expressed and closely related to glioma migration, which provides a new target for the treatment of glioma.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.4
本文编号：2130115
[Abstract]:Aim: to investigate the selective splicing abnormal changes of GDNF gene in glioma tissue cells during transcription, and to explore the possible effect of this change on glioma migration ability. Methods: Real-time PCR was used to detect the expression of 伪 -pro-GDNF and 尾 -pro-GDNF in glioma cells, and the expression of 伪 -pro-GDNF and 尾 -pro-GDNF in glioma cells was detected by western blot. At the same time, immunofluorescence staining was used to detect the distribution and level of protein expression in glioma cells. The lentivirus plasmid which overexpressed and interfered with 伪 -pro-GDNF protein was constructed and transfected into glioma cell U251. The expression of 伪 -pro-GDNF protein was up-regulated or down-regulated. The migration ability of transfected U251 glioma cells was detected by scratch test. Results the results of western blot and real-time PCR showed that there was a selective splicing of GDNF gene in glioma cells, and the mRNA and protein levels of 伪 -pro-GDNF and 尾 -pro-GDNF increased gradually with the increase of WHO grade of glioma. The expression of 伪 -pro-GDNF mRNA and protein was more obvious than that of 尾 -pro-GDNF, and immunofluorescence staining showed that 伪 -pro-GDNF and 尾 -pro-GDNF mainly expressed in cytoplasm. The quantitative analysis of fluorescence intensity further indicated that the expression of 伪 -pro-GDNF protein in glioma cells was higher than that in normal cells, and that the 伪 -pro-GDNF protein increased in the overexpression group and decreased in the interference group after lentivirus transfection. Scratch test showed that the expression of 伪 -pro-GDNF protein affected the migration ability of glioma cells. Conclusion: there is abnormal selective splicing of GDNF gene in glioma cells, in which 伪 -pro-GDNF is preferentially expressed and closely related to glioma migration, which provides a new target for the treatment of glioma.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.4
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