重组p16腺病毒的构建及其对人白血病细胞的抑制作用

为了探讨腺病毒载体用于基因治疗的可行性及野生型 p1 6基因的抗肿瘤特性 ,构建了复制缺陷型重组 p1 6腺病毒 .首先将 p1 6全长 c DNA插入穿梭质粒 p Ad CMV产生重组质粒 p Ad-CMV- p1 6,然后通过脂质体介导与 p JM1 7共转染 2 93细胞 ,经同源重组产生 E1区缺失的重组腺病毒空斑 .用纯化后的腺病毒感染人白血病细胞株 HL- 60后 ,PCR及 Western blot分析显示在感染细胞中有外源性 p1 6 c DNA存在和 p1 6蛋白表达 ;被感染的 HL- 60细胞的生长受到明显抑制 ,而未感染细胞及对照腺病毒感染的细胞没有受到抑制 .结果表明 ,腺病毒作为一种新型基因转移载体 ,可有效地介导肿瘤抑制基因 p1 6的表达 ,在肿瘤基因治疗方面具有很大的应用前景 .

Construction of Recombinant p16 Adenovirus and its Suppressive Role in the Growth of Human Leukemia Cells

To explore the suppressive function of wild type p16 gene in the growth of tumor cells and the potential of adenovirus vector in gene therapy,an E1 deficient and replication defective recombinant p16 adenovirus was generated.The virus,Ad p16 ,carried human CMV promotor, p16 cDNA and SV40 polyadenylation signal.First, p16 cDNA was cloned into shuttle plasmid pAdCMV to construct recombinant plasmid pAdCMV p16 ,which was cotransfected with pJM17 into the 293 packaging cells by lipofectine mediated transfection.The prepared recombinant adenovirus was identified by dot blot hybridization and purified by CsCl gradient ultracentrifuge.The leukemia cell line HL 60,which had a hemizygous deletion and point mutation of p16 gene,was infected with recombinant adenovirus at a multiplicity of infection(M.O.I.)of 50 100 plaque forming unit(pfu)/cell.PCR and Western blot indicated that the presence of p16 cDNA and p16 protein was expressed in the infected HL 60;Growth of the Ad p16 virus infected HL 60 was inhibited markedly,whereas that of noninfected cells and the cells infected with the control virus Ad LacZ was not inhibited.These results suggest that adenovirus is an efficient vector for mediating transfer and expression of tumour suppressor gene in human tumor cells and the prepared recombinant p16 adenovirus could be used for cancer gene therapy.