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背景:外源性Rb基因的导入是抑制视网膜母细胞瘤细胞增殖的有效途径,piggyBac转座子介导的外源基因表达载体高效安全,在基因治疗方面具有广阔的应用前景。目的:探讨piggyBac转座子介导的外源性Rb基因的转染效率及其对视网膜母细胞瘤细胞的抑制作用。方法:采用RT-PCR技术扩增Rb基因编码区Rb cDNA,定向插入到载体pPiggyBac,获得PiggyBac调节的Rb表达质粒pPiggyBac-Rb;通过单独转染或合并pPiggyBac-helper转染人视网膜母细胞瘤细胞SO-RB50。结果与结论:考马斯亮蓝染色证明piggyBac转座子系统的转染效率最高,荧光定量PCR以及免疫荧光实验证明其介导的目的基因Rb与宿主SO-RB50细胞基因组整合效率最高且可在细胞中长期稳定表达,MTT实验证明经其所介导的外源性Rb基因表达对细胞活性影响最为显著。结果提示piggyBac转座子有可能成为视网膜母细胞瘤基因治疗的安全有效载体。
BACKGROUND: The introduction of exogenous Rb gene is an effective way to inhibit the proliferation of retinoblastoma cells. The exogenous gene expression vector mediated by piggyBac transposon is efficient and safe, and has broad application prospect in gene therapy. Objective: To investigate the transfection efficiency of exogenous Rb gene mediated by piggyBac transposon and its inhibitory effect on retinoblastoma cells. Methods: The Rb cDNA of Rb gene was amplified by RT-PCR and inserted into vector pPiggyBac to obtain PiggyBac-regulated Rb expression plasmid pPiggyBac-Rb. Human retinoblastoma cells were transfected with pPiggyBac-helper SO-RB50. RESULTS AND CONCLUSION: Coomassie brilliant blue staining demonstrated that the transfection efficiency of piggyBac transposon system was the highest. Fluorescent quantitative PCR and immunofluorescence assay showed that the gene targeting Rb and host SO-RB50 cells had the highest efficiency of genomic integration and could be expressed in cells Long-term stable expression, MTT experiments show that by its exogenous Rb gene expression on cell activity of the most significant. The results suggest that piggyBac transposon may become a safe and effective vector for gene therapy of retinoblastoma.