分化抑制蛋白1在小鼠树突状细胞肉瘤细胞分化中的作用

Effect of inhibitor of differentiation-1 on murine dendritic cell sarcoma cells

目的 研究降低分化抑制蛋白1（Id-1）的表达在树突状细胞肉瘤（DCS）细胞分化中的作用。方法 通过RNA干扰技术来降低DCS细胞株中Id-1蛋白的表达，Western blot和real-time PCR分别检测该蛋白在蛋白水平和mRNA水平上的变化；倒置显微镜下观察降低Id-1蛋白表达后DCS细胞形态的变化；Casy细胞计数分析仪检测细胞大小的分布情况；流式细胞仪检测细胞周期的变化。通过Westernblot和免疫组织化学检测树突状细胞的分化标志如Id-2、CD86、CD11c、MHC Ⅱ的变化。生长曲线及四甲基偶氮唑盐（MTT）法检测DCS细胞的增殖情况的变化；通过Transwell和克隆形成率测定分别观察降低Id-1蛋白前后细胞侵袭、成瘤能力的变化。以不加siRNA的空白组和无关对照siRNA作阴性对照，以诱导分化剂丁酸钠作为阳性对照。结果 降低Id-1蛋白表达后DCS细胞分枝增多，细胞体积明显增大，核质比下降，G0／G1期细胞增多，S期细胞减少（P〈0．01），树突状细胞分化标志Id-2、CD86等表达上调，细胞增殖受到抑制，侵袭、成瘤能力减弱（P〈0．01）。结论 通过RNA干扰技术降低Id-1基因的表达，可以促进恶性实体肿瘤细胞的分化，降低恶性程度。

Objective To investigate the effect of down-expression of inhibitor of differentiation-1 （Id-1） on the differentiation of dendritic cell sarcoma （DCS） cells in vitro. Methods Down-regulation of the expression of Id-1 in DCS cells was performed by RNAi, and confirmed by protein and mRNA quantitative analyses. Cellular differentiation and biological behavior including malignant phenotypes of the cells were evaluated. All experiments included negative （ no treatment group and no-target siRNA） and positive （induction-differentiation drug sodium butyrate） controls. Results When the expression of Id-1 was down regulated, the DCS cells showed more mature morphology including cell enlargement, longer cellular extensions, more branches, and decreased nuclear/plasma ratio. Differentiation marker expression （ Id-2 and CD86） was also increased. RNAi treated cells at 24 and 48 hours, showed increase percentage of cells at G0/G1 phase and less cells at S phase （ P 〈 0. 01 ）. Importantly, the abilities of cell proliferation, colony formation and invasiveness were significantly decreased （ P 〈 0. 01 ）, as evidenced by MTT, colony formation and transwell assays respectively. Conclusion RNAi inhibition of Id-1 protein can induce differentiation of malignant solid tumor cells along with reversion of their malignant phenotype.