表达白细胞介素-23基因的小鼠乳腺癌细胞系的建立及其对肿瘤的抑制作用

Construction of murine mammary cancer cell line expressing IL-23 gene and its antitumor effects

目的利用逆转录病毒作为载体将小鼠白细胞介素(IL)-23基因转染小鼠乳腺癌细胞(MA-891),建立分泌IL-23的肿瘤细胞株(IL-23/MA-891),观察对其生长特点及体内成瘤情况。方法应用逆转录病毒载体(LXSN)将IL-23基因质粒经ψ2(ecotropic)和PA317(amphotropic)两种包装细胞包装后,转染MA-891细胞,经G418筛选后获得表达IL-23的IL-23/MA-891细胞。采用逆转录-聚合酶链反应(RT-PCR)鉴定转染细胞mRNA的表达;激光共聚焦显微镜(LSCM)观察其蛋白表达;用ELISA法检测IL-23/MA-891细胞培养上清诱导脾细胞分泌IFN-γ的能力;用噻唑蓝(MTT)比色法检测细胞体外增殖能力;用流式细胞仪检测细胞周期与凋亡。小鼠皮下接种转染IL-23的IL-23/MA-891细胞观察其体内致瘤性的变化,小鼠实体瘤模型研究基因转染对肿瘤的生长抑制作用。结果IL-23转染MA-891细胞后,在mRNA水平水平可获得稳定表达;LSCM可观察到其细胞内表达。IL-23基因转染MA-891细胞后,与对照组比较,其体外增殖能力不受影响,致瘤率也无改变,但IL-23基因转染组肿瘤生长明显受到抑制(P<0.05)。结论成功建立表达小鼠IL-23基因的小鼠乳腺癌细胞系(IL-23/MA-891);在体内,IL-23转染组具有明显的抗肿瘤作用。

Objective To construct the murine mammary cancer cell line IL-23/MA-891 expressing IL-23 protein,and investigate the inhibitory effects of IL-23 on the growth of tumor cells in vitro and in vivo. Methods The IL-23 gene was packed with two packing cell lines （ ecotropic ψ2 and amphotropic PA317） by a retrovirus vector （LXSN） , and the positive cell clones were screened by G418. After transfection to MA-891 cells with the culture supernatant of IL-23/PA317 cells and selection with G418, the IL-23/MA-891 cells expressing IL-23 were obtained. The expression of IL-23 mRNA and protein was detected by RT-PCR and laser scanning confocal microscope （LSCM） , respectively. The ability of IL-23/ MA-891 culture supernatant inducing splenocytes to secrete IFN-γ was detected with ELISA. The proliferation of MA-891, LXSN/MA-891 and IL-23/MA-891 cells was detected by MTT colorimetry and flow cytometry in vitro. Mice were subcutaneously inoculated with MA-891 cells which were transfected with IL-23 or empty vector （LXSN） ,and the tumor size in mice was measured. The inhibitory effect of IL-23 gene transfection on mouse solid tumor was observed. Results IL-23/MA-891 cells stable expressed IL-23 mRNA and protein. The culture supernatant of IL-23/MA-891 cells induced secretion of IFN-γ by mouse splenocytes. The proliferation rate of IL-23/MA-891 cells was slightly decresed （ P 〉 0.05 ） , and the percentage of mice with detectable tumor showed no significant difference after subcutaneous inoculation of MA-891 cells transfected with IL-23. Growth rate of tumor in mice model was also inhibited in IL-23 gene therapy group as compared with the control group （ P 〈 0.05 ）. Conclusion IL-23/MA-891 cell line expressing IL-23 was successfully constructed. In in vivo, IL-23 can remarkably inhibit the growth of solid tumor in mice.