溶瘤腺病毒介导的IL-18在黑素瘤A375细胞中的表达

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

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。

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 recombined 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 M＇I＇F 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 MTI＂ assay showed ZD55 - IL - 18 could induce obviously eytotoxic 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.