鞘鞍醇激酶-1对胰腺癌SW1990细胞增殖和凋亡的影响

摘要

目的 探讨鞘鞍醇激酶-1(SPK1)对胰腺癌SWl990细胞增殖和凋亡的影响.方法 培养的胰腺癌SW1990细胞株,实验分为DMS组、PMA组和对照组,各组细胞接种于孔板中,每组设3个复孔,每个实验至少重复3次.DMS组加入N,N-二甲基鞘氨醇(DMS,50μmol/L),PMA组加入佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA,100 nmol/L),对照组按常规培养,采用MTT法和克隆形成实验检测细胞生长增殖的变化,流式细胞术检测细胞凋亡,RT-PCR检测SPK1 mRNA的表达,Western blot检测SPK1蛋白的表达.结果 PMA组细胞的增殖活力[OD值:(1.37±0.03),细胞克隆数目:(292.45±10.68)]较对照组[OD:(1.00±0.01),细胞克隆数目:(215.34±12.23)]显著增加,DMS组细胞的增殖活力[OD值:(0.65±0.02),细胞克隆数目:(130.56±15.6)]较对照组显著降低,差异均有统计学意义(P<0.05).PMA组细胞凋亡率[(9.7±0.62)%]较对照组[(16.71±1.53)%]明显下降,DMS组细胞凋亡率[(31.7±1.32)%]较对照组明显升高,差异均有统计学意义(P<0.01),PMA组SPK1 mRNA表达水平(0.202±0.013)及蛋白表达水平(0.258±0.015)较对照组[SPK1 mRNA:(0.148±0.006),蛋白:(0.182±0.044)]显著增加,而DMS组SPK1 mRNA表达水平(0.112±0.022)及SPK1蛋白表达水平(0.101±0.034)较对照组显著降低,差异均有统计学意义(P<0.05).结论 SPK1与胰腺癌细胞的增殖和凋亡密切相关,SPK1的激活可促进胰腺癌细胞的增殖并抑制细胞的凋亡.%Objective To investigate the effect of sphingosine kinase 1 (SPK1) on the proliferation and apopto-sis of pancreatic cancer line SW1990. Methods Cultured SW1990 cell were divided into three groups: PMA group, DMS group and the control group. The cells of each group were seeded in the orifice plate, with 3 wells in each group, at least 3 replicates for each experiment. The cells of the PMA group were treated with 100 nmol/L of phorbol 12-my-ristate13-acetate (PMA);the DMS group was treated with 50 μmol/L N, N-dimethylsphingosine (DMS);while the con-trol group was cultured routinely. After the treatment, cell proliferation was determined by MTT (3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide) assay and colony formation assay, cell apoptosis was detected by flow cy-tometry, and mRNA and protein expression of SPK1 were detected by RT-PCR and Western blot. Results The cell via-bility in PMA group was the OD value of (1.37±0.03) and the number of cell clones of (292.45±10.68), which was signif-icantly higher than the OD value of (1.00±0.01) and the number of cell clones of (215.34±12.23) in the control group (P<0.05). The cell viability in DMS group was the OD value of (0.65±0.02) and the number of cell clones of (130.56±15.6), which was significantly higher than that in the control group (P<0.05). The apoptosis rate in PMA group was (9.7 ± 0.62)%, which was significantly lower than (16.71 ± 1.53)%in the control group, and the apoptosis rate in DMS group was (31.7 ± 1.32)%, which was significantly higher than that in the control group (P<0.01). The mRNA and protein ex-pression of SPK1 in PMA group were respectively (0.202 ± 0.013) and (0.258 ± 0.015), which were significantly higher than (0.148 ± 0.006) and (0.182 ± 0.044) in the control group, and the mRNA and protein expression of SPK1 in DMS group were (0.112 ± 0.022) and (0.101 ± 0.034), which were significantly lower than those in the control group (P<0.05). Conclusion SPK1 is closely related to the proliferation and apoptosis of pancreatic cancer cells. The activation of SPK1 can promote the proliferation of pancreatic cancer cells and inhibit the apoptosis.