逆转录病毒介导的小鼠IL-23基因在鼠高转移性乳腺癌细胞系MA-891中的表达

Retrovirus mediated IL-23 gene expression in mouse high-metastasis mammary cancer cells MA-891

目的:建立表达小鼠IL-23基因的鼠乳腺癌细胞系IL-23/MA-891,探讨外源性IL-23基因对MA-891细胞生长及其生物学性状的影响。方法:将插入IL-23基因的质粒,应用逆转录病毒载体LXSN经感染ψ2(ecotropic)和PA317(amphotropic)两种包装细胞包装,经G418筛选获得表达IL-23分子的PA317阳性细胞克隆,用IL-23/PA317培养上清转染MA-891细胞,获得表达IL-23的IL-23/MA-891细胞。分别用RT-PCR法、ELISA法和免疫组化染色法分析IL-23/MA-891细胞表达IL-23的mRNA和蛋白的水平,筛选出高表达IL-23的IL-23/MA-891细胞克隆用于下层研究中;用流式细胞仪检测MA-891细胞中MHCⅠ、MHCⅡ、CD80、CD86以及FAS蛋白的表达、细胞周期变化及细胞凋亡情况;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MTT比色法检测细胞增殖反应。结果:外源性IL-23基因转染的小鼠乳腺癌细胞系IL-23/MA-891,在mRNA水平和蛋白水平均可获得稳定表达;IL-23/MA-891细胞与LXSN/MA-891和MA-891比较,细胞周期及细胞凋亡率无显著性差异(P>0.05),H-2Kb(MHCⅠ类分子)、I-Ab(MHCⅡ类分子)、CD80、CD86以及FAS蛋白的表达水平无显著性差异(P>0.05);IL-23/MA-891细胞的增殖反应与LXSN/MA-891和MA-891细胞相比虽有所降低,但无显著性差异(P>0.05)。然而,IL-23/MA-891细胞的培养上清可明显促进小鼠脾细胞分泌IFN-γ(P<0.01)。结论:建立的稳定表达IL-23的IL-23/MA-891细胞,具有较强的免疫学调节活性,其生物学形状与MA-891和LXSN/MA-891细胞相比较没有明显差异,但可进一步用于肿瘤相关免疫基因治疗的研究。

Objective： The mouse mammary cancer cell line IL-23/MA-891 expressing IL-23 protein was set up and the effect of IL-23 on growth and other biological character of the cells was studied. Methods： The IL-23 gene was sequencely transfected into two packing cell lines （ecotropic φ2 and amphotropic PA317） by a retroviras vector （LXSN）, and the positive cellular clones were screened by G418. After infection to MA-891 cells with the culture supernatant of IL-23/PA317 cells and selected with G418, the IL-23/MA-891 cells to express IL-23 mRNA and protein was detected with RT-PCR, ELISA and immunocytechemical stairing, respectively. The expression of H-2Kb（MHCⅠ）, Ⅰ-Ab （MHC Ⅱ ）,CD80,CD86 and FAS was examined with flow cytometry. The ability to secrete IFN-γby mouse splenocytes stimulated with the culture supematant of IL-23/MA-891 cells was detected with ELISA. The proliferation of MA-891 ,LXSN/MA-891 and IL-23/MA-891 cells was detected by M1T colorimetry and flow cytometry in vitro. Results： IL-23/MA-891 cells express IL-23 stably in mRNA and protein level. The expressing of H-2Kb（MHC Ⅰ）, Ⅰ-Ab（ MHC Ⅱ ）, CD80, CD86 and FAS protein by flow cytometry were showing no difference in three kinds of cells. The proliferation rate of IL-23/MA-891 cells were showing little decresed,but statistically showing no difference with parental cells. The culture supematant of IL-23/MA-891 cells induced secretion of IFN-γby mouse splenocytes were increased compared to parental cells. Conclusion：The cellular clone IL-23/MA-891 with high level expression of IL-23 was produced.