目的:将小鼠IL-23基因转染小鼠乳腺癌细胞MA-891,检测该细胞在体内外的凋亡变化,探讨IL-23抗肿瘤作用机制.方法:应用逆转录病毒载体(LXSN)将含 IL-23 基因质粒经ψ2(亲嗜性)和PA317(双嗜性) 2种细胞包装,G418筛选获得携带 IL-23 基因的病毒,后者转染MA-891细胞,经G418筛选后获得IL-23/MA-891阳性克隆.用ELISA法检测IL 23/MA-891细胞分泌IL-23的能力,用MTT比色法检测细胞体外增殖能力.小鼠皮下接种转染IL-23/MA-891细胞,观察其体内致瘤性变化;用流式细胞术及TUNEL法检测肿瘤细胞凋亡情况;用RT-PCR和Western 印迹法检测肿瘤组织Fas和survivin的表达.结果:IL-23 基因成功转染MA-891细胞,获得了IL-23高表达细胞IL-23/MA-891.转染 IL-23 基因不影响MA-891细胞的体外生长和细胞凋亡,但体内 IL-23 基因转染组肿瘤生长明显受到抑制,肿瘤组织细胞凋亡率增高(P < 0.01),细胞表面Fas的表达水平明显增加(P < 0.01),survivin表达水平明显降低(P < 0.01).结论:转染小鼠 IL-23 基因后对小鼠乳腺癌细胞MA-891的体外凋亡无影响,但在体内 IL-23 可能通过降低survivin表达和上调Fas表达,诱导细胞凋亡而产生抗肿瘤作用.

Objective: To investigate the changes in apoptosis of mouse mammary cancer cells (MA-891) after transfection with IL-23 gene in vitro and in vivo and explore the anti-tumor mechanism of IL-23. Methods: IL-23 gene was transferred into two packing cell lines (ectotrophic ψ2 and amphotrophic PA317) in sequence by a retrovirus vector (LXSN), and the positive cellular clones that can produce retroviruses carrying IL-23 gene were screened by G418. The retroviruses were used to transduce them IL-23 gene into mouse mammary cancer cells (MA-891). After being screened by G418, IL-23/MA-891 cells expressing IL-23 protein were obtained. The expression of IL-23 protein in IL-23/MA-891 cells was detected by ELISA method. The proliferations of MA-891, LXSN/MA-891 and IL-23/MA-891 cells were detected by MTT assay in vitro. Mice were subcutaneously inoculated with IL-23/MA-891, LXSN/MA-891 and the parental MA-891 cells and the solid tumor formation was observed in vivo. The apoptosis of tumor cells from mice injected with different MA-891 cells was detected by TUNEL and flow cytometry analysis. The expressions of Fas and survivin were determined by RT-PCR and Western blotting in vivo. Results: IL-23 gene was successfully transfected into MA-891 cells. The stable IL-23/MA-891 cells with over-expression of IL-23 was obtained. Transfection with IL-23 gene had significant effects on the proliferation and apoptosis of MA-891 cells in vitro (P > 0.05). But the tumor growth in mice was greatly inhibited and apoptosis ratio of tumor tissues was significantly increased in the IL-23 gene-transfected group compared with the control group (P < 0.01). The expression of Fas on the surface of cells was up-regulated and the expression of survivin was significantly decreased (P < 0.01). Conclusion: Transfection with IL-23 gene has no significant effects on the apoptosis of MA-891 cells in vitro. But IL-23 exerts its anti-tumor effect by up-regulating the expression of Fas and down-regulating the expression of survivin in vivo.