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目的检测独立生长因子1(GFI-1)在Sézary综合征患者和正常人外周血中的表达,为开发针对GFI-1基因靶点的治疗提供实验依据。方法利用流式细胞术分离纯化7例Sézary综合征患者外周血中CD4~+CD7-的Sézary细胞(SS细胞)作为实验组,以10例正常人外周血CD4~+T细胞、Sézary综合征来源细胞系Hut78细胞和人急性T细胞白血病细胞系Jurkat细胞为对照组。应用qPCR检测各组细胞中GFI-1 mRNA的表达,蛋白质印迹法检测GFI-1蛋白表达。用干扰素α2b(IFN-α2b)诱导Hut78细胞凋亡后,采用MTS法测定细胞增殖情况,用qPCR检测GFI-1、细胞周期依赖性蛋白激酶抑制因子P21、肿瘤坏死因子相关的凋亡诱导配体(TRAIL)和Caspase-3mRNA的表达情况,用流式细胞术检测细胞凋亡情况。结果 Sézary综合征患者外周血SS细胞GFI-1mRNA表达水平高于Jurkat细胞和正常人外周血CD4~+T细胞(P均<0.05)。SS细胞和Hut78细胞的GFI-1蛋白表达水平高于Jurkat细胞和正常人外周血CD4~+T细胞(P均<0.05)。IFN-α2b能够抑制Hut78细胞增殖,且其抑制作用呈时间和浓度依赖性。IFN-α2b处理Hut78细胞12h和24h后GFI-1mRNA的表达水平呈时间依赖性降低,P21、TRAIL和Caspase-3mRNA的表达水平呈时间依赖性增加(P<0.05)。IFN-α2b处理Hut78细胞12h和24h后细胞的凋亡水平增加(P<0.05)。结论 GFI-1基因在Sézary综合征患者外周血SS细胞中表达增加,IFN-α2b能抑制Hut78细胞GFI-1基因的表达,表明GFI-1基因可能在Sézary综合征患者SS细胞的肿瘤性增殖中发挥重要调控作用。
Objective To detect the expression of GFI-1 in peripheral blood of patients with Sézary syndrome and normal controls, and to provide experimental evidence for the development of GFI-1 gene therapy. Methods Sézary cells (SS cells) from peripheral blood of 7 patients with Sézary syndrome were isolated and purified by flow cytometry as experimental group. Ten normal human peripheral blood CD4 ~ + T cells, Sézary syndrome origin The cell line Hut78 cells and the human acute T cell leukemia cell line Jurkat cells were control groups. The expression of GFI-1 mRNA in each group was detected by qPCR, and the protein expression of GFI-1 was detected by Western blotting. After induced apoptosis of Hut78 cells with interferon α2b (IFN-α2b), the cell proliferation was determined by MTS assay. The apoptosis induced by GFI-1, P21 and TNF-related apoptosis were detected by qPCR (TRAIL) and Caspase-3 mRNA expression, using flow cytometry to detect apoptosis. Results The expression of GFI-1mRNA in peripheral blood of Sézary syndrome patients was higher than that of Jurkat cells and normal individuals (all P <0.05). The GFI-1 protein expression of SS cells and Hut78 cells was higher than that of Jurkat cells and normal human CD4 ~ + T cells (all P <0.05). IFN-α2b can inhibit Hut78 cell proliferation, and its inhibitory effect in a time-and concentration-dependent manner. The expression of GFI-1 mRNA in Hut78 cells treated with IFN-α2b decreased in a time-dependent manner at 12h and 24h, and the expression of P21, TRAIL and Caspase-3 mRNA increased in a time-dependent manner (P <0.05). The apoptosis of Hut78 cells treated with IFN-α2b for 12h and 24h increased (P <0.05). Conclusions GFI-1 gene expression is increased in peripheral blood of Sézary syndrome patients, IFN-α2b can inhibit the expression of GFI-1 gene in Hut78 cells, indicating that GFI-1 gene may be involved in the neoplastic development of SS cells in patients with Sézary syndrome Play an important regulatory role.