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目的探讨RNA聚合酶Ⅱ转录的ALU序列对人胚肾293(HEK293)细胞凋亡的影响以及干扰素(IFN)在此机制中的作用。方法取对数生长期的HEK293细胞,分为6组,ALU-293组(瞬时转染重组质粒pcDNA3.1-ALU)、pcDNA3.1-293组[瞬时转染空质粒pcDNA3.1(-),作为阴性对照]、Poly I∶C-293组[瞬时转染dsRNA的多聚肌苷胞苷酸(Poly I∶C),作为阳性对照]、IFNβ-293组(加入1.65×104 U IFNβ,作为阳性对照)、空白对照组(未经处理的HEK293细胞)和HBs-293组(瞬时转染重组质粒pcDNA3.1-HBs),转染后48 h,采用MTT法检测细胞的增殖活性;Cellular DNA Fragmentation ELISA和DNA Ladder法检测细胞的凋亡情况;Real-time PCR检测细胞中IFNβ基因mRNA的水平。结果瞬时转染重组质粒pcDNA3.1-ALU能够抑制HEK293细胞增殖,并促使其凋亡,且细胞中IFNβmRNA的水平显著上调。结论 RNA聚合酶Ⅱ转录的ALU序列能够通过激活干扰素系统来诱导细胞凋亡。
Objective To investigate the effect of RNA polymerase Ⅱ transcribed ALU sequence on the apoptosis of human embryonic kidney 293 (HEK293) cells and the role of interferon (IFN) in this mechanism. Methods HEK293 cells in logarithmic growth phase were divided into 6 groups: ALU-293 group (pcDNA3.1-ALU), pcDNA3.1-293 group (transient transfection of empty plasmid pcDNA3.1 (-)) , As a negative control], Poly I: C-293 group [poly I: C transiently transfected dsRNA as a positive control], IFNβ-293 group (1.65x104 U IFNβ added, (Untreated HEK293 cells) and HBs-293 group (transiently transfected with recombinant plasmid pcDNA3.1-HBs) 48 h after transfection, MTT assay was used to detect cell proliferation activity; Cellular DNA Fragmentation ELISA and DNA Ladder method were used to detect the cell apoptosis. Real-time PCR was used to detect the mRNA level of IFNβ in the cells. Results Transient transfection of recombinant plasmid pcDNA3.1-ALU could inhibit HEK293 cell proliferation and induce apoptosis, and the level of IFNβ mRNA in the cells was significantly up-regulated. Conclusion RNA polymerase Ⅱ transcribed ALU sequence can induce apoptosis by activating interferon system.