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目的探讨CD40基因特异性沉默对脑胶质瘤细胞增殖和迁移等恶性生物学行为的影响。方法构建特异性沉默CD40基因的重组表达载体(pSilencer3.1/shRNA-CD40)和随机对照载体(pSilencer3.1/shRNA-control),转染脑胶质瘤细胞株U87。采用RT-PCR和流式细胞术分别在mRNA和蛋白水平的检测CD40基因沉默效率,进而采用细胞计数法和Transwell法观察下调CD40分子表达后的U87细胞在sCD40L作用下增殖能力和迁移能力的改变。结果成功构建了特异性沉默CD40基因的稳定细胞株U87/shRNA-CD40,可显著下调U87细胞CD40 mRNA和蛋白质表达水平(P<0.05)。采用sCD40L激发CD40信号,结果显示,与野生型U87和对照组U87/shRNA-control相比,U87/shRNA-CD40细胞株增殖速率和迁移能力明显下降(均P<0.01)。结论特异性沉默脑胶质瘤细胞CD40分子表达,可以有效削弱肿瘤微环境中CD40信号导致的脑胶质瘤细胞增殖和迁移能力,这为临床治疗脑胶质瘤提供了新的线索。
Objective To investigate the effects of CD40 gene-specific silencing on malignant biological behaviors such as proliferation and migration of glioma cells. Methods The recombinant plasmid pSilencer3.1 / shRNA-CD40 and pSilencer3.1 / shRNA-control were constructed and transfected into glioma cell line U87. RT-PCR and flow cytometry were used to detect the silencing efficiency of CD40 gene at mRNA and protein levels respectively. Then the cell proliferation and migration of U87 cells treated with sCD40L were observed by cell counting and Transwell assay . Results The stable cell line U87 / shRNA-CD40 with specific silence of CD40 gene was successfully constructed and the expression of CD40 mRNA and protein in U87 cells was significantly down-regulated (P <0.05). The results showed that the proliferation rate and migration ability of U87 / shRNA-CD40 cells were significantly decreased compared with U87 / shRNA-control group (all P <0.01). Conclusion The specific silencing of CD40 expression in glioma cells can effectively attenuate the proliferation and migration of glioma cells induced by CD40 in the tumor microenvironment, which provides a new clue for clinical treatment of gliomas.