摘要
目的探讨抑制细胞周期依赖性激酶CDC2基因对人脑胶质瘤细胞生长的影响．并为其作为胶质瘤发生发展相关基因在胶质瘤治疗方面的应用价值提供实验依据。方法利用本实验室基因重组构建的CDC2shRNA逆转录病毒表达质粒对体外培养的人脑胶质瘤细胞进行转染。倒置显微镜观察细胞形态和数量变化；RT-PCR及荧光实时定量PCR检测CDC2mRNA表达：Westemblot检测CDC2蛋白表达；流式细胞仪检测细胞周期和凋亡的变化；透射电子显微镜观察细胞超微结构。结果人脑胶质瘤细胞系U251在转染针对CDC2的shRNA逆转录病毒表达质粒48h后，贴壁生长的细胞逐渐漂浮，细胞生长受到抑制，CDC2mRNA下调均达98％以上．蛋白表达下调达92％以上，同时出现了明显的细胞G2／M期阻滞．细胞凋亡由0．57％上升到13．01％．并且超微结构受损明显。结论CDC2shRNA逆转录病毒表达质粒介导的RNA干扰能有效的抑制U251细胞的增殖．CDC2可作为胶质瘤基因治疗靶分子作进一步研究。
Objective To investigate the effect of inhibiting CDC2, the cyclin-dependent kinase, on proliferation of human glioma cell line U251, providing experimental evidence for application value of CDC2 in glioma treatment as the gene for the onset and development of gliomas. Methods The recombinant plasmid P-Super-Retro-CDC2 was transfected into human glioma cell line U251. The resulting phenotypic changes of the target gene were investigated. Changes in cell morphology and quantity were observed by inverted microscopy; the expression level of CDC2 mRNA was measured using reverse transcription-polymerase chain reaction （RT-PCR） and real-time fluorescent quantitative PCR; the expression level of CDC2 protein was examined by Western blot; changes in the cell cycle and apoptosis were detected by flow cytometry; ultramicroscopic structure of tumor cells was observed with the transmission electron microscope （TEM）. Results After 48 h transfection of the recombinant retrovirus plasmid expressing shRNA targeting CDC2 into human glioma cell line U251, adherent tumor cells gradually became floating, proliferation and CDC2 mRNA were suppressed by more than 98% and the protein expression was down-regulated by over 92%. Meanwhile, the percentage of cells blocked at G2/M phase was high and that of apoptosis increased from 0.57% to 13.01%. The ultrastructure was obviously injured. Conclusion RNA interference （RNAi） induced by the recombinant retrovirus plasmid expressing shRNA targeting CDC2 may effectively inhibit the proliferation of U251 cells, which can be used for further study on the gene therapy of human gliomas.
出处
《中华神经医学杂志》
CAS
CSCD
2008年第2期109-114,共6页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（30371457）志谢感谢南通大学张天一老师对电镜检测所给予的帮助和指导
