siRNA抑制syk基因对外周T细胞淋巴瘤细胞增殖与凋亡的影响

摘要

目的 探讨siRNA抑制脾酪氨酸激酶(syk)基因后对外周T细胞淋巴瘤细胞株HUT-78细胞增殖与凋亡的影响.方法 针对syk基因特异靶点设计3条siRNA(siRNA-1、siRNA-2和siRNA-3),采用电穿孔法转染外周T细胞淋巴瘤细胞株HUT-78,同时设Mock组和siRNA-NC组,转染48 h后,分别用RT-PCR和Western blot技术检测syk mRNA和蛋白的表达水平,筛选出最有效的syk siRNA;syk siRNA转染HUT-78后分别用软琼脂克隆形成实验检测syk下调后HUT-78细胞的克隆形成能力,MTT法检测干扰24、48、72 h后细胞增殖情况,流式细胞术检测syk下调后HUT-78细胞的凋亡情况.结果 RT-PCR和Western blot结果显示,与Mock组和siRNA-NC组相比,3条siRNA均能有效降低HUT-78细胞中syk mRNA和蛋白的表达,其中syk siRNA-1抑制效果最明显.克隆形成实验显示,下调syk基因表达后HUT-78细胞的克隆形成能力与Mock组相比明显下降(P<0.05);MTT实验结果显示,下调syk基因表达后HUT-78细胞的增殖能力与Mock组相比显著下降(P<0.05);流式细胞术结果显示,下调syk基因表达后HUT-78细胞的凋亡比例明显高于Mock组(P<0.05).结论 siRNA下调syk基因表达后可抑制外周T细胞淋巴瘤细胞的增殖、克隆形成,促进凋亡,推测syk基因在外周T细胞淋巴瘤的发生发展中发挥重要作用,有可能成为外周T细胞淋巴瘤基因治疗的新靶点.%Objective To investigate the influence of down-regulation of syk via siRNA on the proliferation and apoptosis of human peripheral T-cell lymphoma HUT-78 cells.Methods 3 siRNAs targeting syk were designed, synthetized,and transfected into HUT-78 cells via electroporation.Cells transfected with negative control siRNA and untreated cells were used as controls.48 h after transfection, the expression of syk mRNA and protein were examined by RT-PCR and Western blot respectively, and the most effective syk siRNA was screened out.The proliferation of cells after 24 h, 48 h and 72 h interference and colony formation ability were examined by MTT and soft agar assay.Flow cytometry was used to detect the cell apoptosis.Results 3 siRNAs notably down-regulated syk expression at mRNA and protein levels, and the inhibitory effect of siRNA-1 was the most obvious.The colony formation of syk siRNA-1 group was significantly decreased compared with control groups after down-regulationof syk expression(P<0.05).The results of MTT test showed that the proliferation ability of syk siRNA-1 group was significantly decreased compared with control groups(P<0.05).Flow cytometry results showed that the apoptosis ratio of syk siRNA-1 group was about 37%, obviously higher than control groups(P<0.05).Conclusion The down-regulation of syk expression suppressed the cell proliferation and colony formation ability, and promoted the cell apoptosis of peripheral T-cell lymphoma cells.The results showed that syk gene may play a very important role in the occurrence and development of peripheral T-cell lymphoma, and it may be a novel target for gene therapy of peripheral T-cell lymphoma.