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目的构建能高效敲减分化抑制因子2(Id2)基因的短发夹状小干扰RNA(shRNA)慢病毒载体,并观察敲减Id2对胶质瘤细胞株U251增殖和凋亡的影响。方法设计并合成特异性针对Id2基因的shRNA序列,将其构建到慢病毒载体pGCSIL-GFP中。以含有Id2基因的质粒为模板,扩增Id2基因cDNA序列的特异性片段,插入真核表达载体pEGFP-N1-3FLAG。构建含Id2基因质粒和不同靶点的RNAi慢病毒载体,共转染入工具细胞293T,筛选出适合浓度慢病毒感染U251细胞株。MTT法检测敲减Id2后胶质瘤细胞增殖的改变,RT-PCR检测Id2敲减后U251细胞caspase 3的表达。结果构建后载体的PCR鉴定及DNA测序结果与预期一致。与对照组相比,转染Id2 shRNA的U251细胞增殖降低,凋亡率升高,caspase3表达升高,差异有统计学意义(P<0.05)。结论成功构建的针对Id2基因的RNA干扰慢病毒载体体外转染可抑制胶质瘤细胞增殖并促进其凋亡。
Objective To construct short hairpin RNA interference lentiviral vectors capable of efficiently knockdown of Id2 gene and to observe the effects of knockdown of Id2 on the proliferation and apoptosis of glioma cell line U251. Methods The shRNA sequence specific to Id2 gene was designed and synthesized, which was constructed into lentiviral vector pGCSIL-GFP. The specific fragment of Id2 gene cDNA sequence was amplified by using the plasmid containing Id2 gene as a template and inserted into the eukaryotic expression vector pEGFP-N1-3FLAG. The RNAi lentiviral vector containing Id2 gene and different targets was constructed and transfected into 293T cells. The lentivirus-infected U251 cell line was screened out. The proliferation of glioma cells after Id2 knockdown was detected by MTT assay. The expression of caspase 3 in U251 cells after Id2 knockdown was detected by RT-PCR. Results After construction, the PCR identification and DNA sequencing of the vector were as expected. Compared with the control group, U251 cells transfected with Id2 shRNA had lower proliferation, higher apoptosis rate and higher caspase3 expression (P <0.05). Conclusion The successful construction of Id2 gene RNA interference lentiviral vector transfected in vitro can inhibit glioma cell proliferation and promote apoptosis.