溶瘤腺病毒介导的IL-18在黑素瘤A375细胞中的表达

目的构建携带人IL-18基因的溶瘤腺病毒,观察在A375细胞中IL-18基因的表达情况。方法将pZD55-IL-18与含有腺病毒右臂的质粒pBHGE3共转染293细胞获得重组腺病毒。PCR鉴定条件增殖腺病毒ZD55- IL-18。以ZD55-IL-18感染人黑素瘤A375细胞系,通过逆转录-聚合酶链反应(RT-PCR)检测IL-18基因mRNA的表达;Western blot法检测腺病毒E1A的表达情况;结晶紫染色法检测细胞杀伤作用;MTY法检测细胞存活情况。结果PCR鉴定表明ZD55-IL-18构建成功;RT-PCR和Western blot结果表明,ZD55-IL-18能高效介导IL-18基因在A375细胞中的表达,并在肿瘤细胞内表达E1A;结晶紫染色和MTT结果表明,ZD55-IL-18对A375细胞有明显的杀伤作用。结论构建成功的溶瘤腺病毒ZD55-IL-18,可在A375细胞中高效表达IL-18基因,并能抑制黑素瘤细胞生长。

Expression of IL-18 Gene in Melanoma A375 Cell Line Mediated by Oncolytic Adenovirus

Objective To construct oncolytic adenovirus expressing human IL-18 gene and research expression of IL-18 gene in A375 cell line.Methods Plasmid pZD55 was constructed through deleting E1B 55KD gene,and plasmid pZD55-IL- 18 was constructed by inserting IL-18 gene into pZD55.Oncolytic adenoviruses ZD55-IL-18 was obtained through homologous recombination using pZD55-IL-18 and the adenovirus packaging plasmid pBHGE3 in 293 cells.The reeombined adenoviruses were verified by PCR.Further ZD55-IL-18 was used to infect A375 cells,the expression of IL-18 gene in mRNA was observed by RT-PCR analysis,respectively.The expression of E1A was detected by Western blot.Finally,cytotoxic effects of ZD55-IL -18 on A375 cell lines were detected by crystal violet staining and MTr assay.Result The analysis of PCR indicated that the re- combinant adenovirus ZD55-IL-18 was successfully constructed.RT-PCR analysis confirmed that ZD55-IL-18 could ex- press IL-18 gene in A375 cell line with high efficiency.Western blot analyses indicated ZD55-IL-18 could express E1A in ad- enovirus-infected A375 cells.Crystal violet staining and MTT assay showed ZD55-IL-18 could induce obviously cytotoxic effects and growth inhibition of A375 cells.Conclusion The recombinant oncolytic adenovirus ZD55-IL-18 has been con- structed successfully and inhibit the proliferation of melanoma A375 cells.