目的：探讨程序性细胞死亡因子4（PDCD4）的抑癌机制。方法：以含有全长 PDCD4的 cDNA 为模板，扩增 PDCD4全长基因，融合 PDCD4－GFP 融合基因真核表达载体，将其转染 Hela 细胞，采用激光共聚焦显微镜观察融合蛋白在细胞中的表达，以荧光定量 PCR 和 Western －blot 检测 PDCD4在细胞内的表达以及抗凋亡基因 Bcl －2的表达情况。结果：经酶切图谱分析、PCR 扩增及 DNA 测序证实融合基因真核表达载体构建正确；荧光显微镜可观察到细胞内 PDCD4－GFP 融合蛋白的表达；RT －PCR 证实经 PDCD4基因转染的细胞内 PDCD4 mRNA 表达增高。PDCD4的过表达能够导致 Bcl －2基因表达下调。结论：PDCD4基因表达于宫颈癌细胞核和胞浆内，并且 PDCD4能够抑制 Bcl －2基因的表达水平。%Objective:To understand the function of anti -tumor gene -programmed cell death 4(PDCD4),and provide a useful tool for the prevention of human cervical tumor.Methods:The full length of PDCD4 was amplified by polymerase chain reaction(PCR),using cDNA as a template.The recombinant plasmid PDCD4 -pEGFP -N1 was confirmed by restricted endonuclease mapping,PCR amplification and DNA sequencing.Hela cells were transfected with PDCD4 -pEGFP -N1 to investigate the expressions of PDCD4.Fluorescence microscopy was employed to ob-serve the expression of PDCD4 -GFP fusion protein,Real -time PCR was used to analyze the expression of PDCD4 gene in Hela cells,and protein levels of Bcl -2.Results:The expression vector PDCD4 -pEGFP -N1 was success-fully constructed and resulted in simultaneously cellular expression of both PDCD4 and GFP when transfected into He-la cells.Fluorescence microscopy confirmed the GFP expression in the cells and RT -PCR revealed that the expres-sion of PDCD4 mRNA was increased in PDCD4 gene transfected cells,PDCD4 overexpression lead to reduced level of Bcl -2 mRNA.Conclusion:It is demonstrated the location of PDCD4 and simultaneously express PDCD4 in Hela cells which lead to decrease of Bcl -2 expression,which provide the evidence that how PDCD4 function as a tumor suppress gene.
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