氨茶碱对淋巴瘤细胞系增殖与凋亡的影响及机制探讨

Effects of Aminophylline on Proliferation and Apoptosis in Raji Lympho-blastoid Cell Line

为探讨磷酸二酯酶 (phosphodiesterase ,PDE)抑制剂在淋巴瘤细胞增殖与凋亡中的作用及机制 ,研究了非选择性PDE抑制剂氨茶碱 (aminophylline,AM)对Burkitt淋巴瘤细胞系Raji的影响。采用MTT检测 ,光学显微术 ,电镜术及膜联蛋白V染色观察细胞增殖、细胞凋亡的形态学变化及凋亡率 ;用流式细胞术和RT PCR技术检测细胞周期 ,周期蛋白B1和Bcl 2的表达及线粒体跨膜电位的变化。结果显示 ,氨茶碱对Raji细胞增殖呈浓度依赖性抑制作用 ;形态学观察发现随氨茶碱浓度增加 ,细胞体积明显缩小 ,核固缩 ,核染色质核膜下聚集 ,出现凋亡小体 ;细胞涂片显示 ,核染色质凝聚、核碎裂 ;电镜观察有典型凋亡细胞 ;膜联蛋白V染色显示氨茶碱诱导Raji细胞凋亡率增加 ,线粒体跨膜电位 (△ψm)呈浓度依赖性下降 ;流式细胞术及RT PCR显示氨茶碱下调Bcl 2蛋白及mRNA的表达 ;细胞周期分析显示经氨茶碱处理的细胞出现S期阻滞、细胞周期蛋白B1表达下调。结论 :氨茶碱可通过S期阻滞抑制细胞增殖、下调Bcl 2的表达及降低线粒体膜电位而诱导Raji细胞凋亡。

The aim of this study was to investigate whether and how phosphodiesterase (PDE) inhibitors modulate the proliferation, cell cycle and apoptosis in lymphoma cells. The effects of aminophylline (AM), a non specific PDE inhibitor, on Raji cells were explored in vitro. MTT assay, light and transmission electron microscopy and annexin V staining were used to observe cell proliferation, morphologic changes and apoptosis rate in AM treated cells, and FCM and RT PCR techniques were adopted to detect the effect on cell cycle, the expression of cyclin B1 and Bcl 2 and mitochondrial transmembrane potential in AM treated cells. The results showed that AM inhibited the growth of Raji cells in a concentration dependent manner. Morphologic observations showed apoptosis changes in AM treated cells, including cytoplamic shrinkage, cytoplasmic bubbling, karyopyknosis and nuclear fragmentation. FCM and RT PCR detection showed that AM intervention increased the fraction of annexin V + cells, reduced the value of mitochondrial transmembrane potential, induced S phase arrest, and down regulated the expression of Bcl 2 at both mRNA and protein level and cyclin B1 protein in a concentration dependent manner. It is concluded that PDE inhibitor aminophylline may induce Raji cell growth inhibition, S phase arrest, apoptosis via down regulation of Bcl 2 and reduction of mitochondrial transmembrane potential.