P2z受体在介导慢性淋巴细胞白血病细胞凋亡中的作用

Study on apoptosis of leukemic lymphocytes mediated by purinergic P2z receptors

目的：研究嘌呤能Ｐ２ｚ受体在介导慢性淋巴细胞白血病（ＣＬＬ）细胞凋亡中的作用。方法：将表达Ｐ２ｚ受体［Ｐ２ｚ（＋）］与不含Ｐ２ｚ受体［Ｐ２ｚ（－）］的两组ＣＬＬ细胞分别同１．０ｍｍｏｌ／Ｌ三磷酸腺苷（ＡＴＰ）体外培养８小时，以电镜、ＤＮＡ凝胶电泳和定量ＤＮＡ３′端ＴｄＴ法检测细胞凋亡；并对ＡＴＰ、苯甲酰苯甲酸ＡＴＰ（ＢｚＡＴＰ）、２甲基硫ＡＴＰ（２ＭｅＳＡＴＰ）、γ硫代ＡＴＰ（ＡＴＰγＳ）及其它核苷的诱导效应和氧化型ＡＴＰ（ＯｘＡＴＰ）、１［Ｎ，Ｏ二（５异喹啉碘酰基）Ｎ甲基Ｌ酪氨酰］４苯哌嗪（ＫＮ６２）的抑制效应做定量研究。结果：①Ｐ２ｚ＋细胞能在ＡＴＰ诱导下呈现典型的细胞凋亡形态和ＤＮＡ梯形条带；②不同的Ｐ２ｚ受体激活剂可通过Ｐ２ｚ受体诱导细胞凋亡并产生不同量的ＤＮＡ降解片段，诱导能力的强弱顺序是ＢｚＡＴＰ＞ＡＴＰ＞２ＭｅＳＡＴＰ，而ＡＴＰγＳ或其它核苷几乎无诱导能力；③ＯｘＡＴＰ和ＫＮ６２可完全阻止诱导剂诱导的细胞凋亡。

Objective:To investigate the role of purinergic P2z receptors in human leukemic lymphocyte apoptosis induced by extracellular adenosine triphosphate (ATP). Methods: Eleven BCLL patients were studied. Leukemic lymphocytes with (n=8) or without (n=3) P2z receptors were exposed in vitro to ATP, benzoylbenzoicATP (BzATP), 2methylthioATP(2MeSATP), adenosine5′ triphosphate (ATPγS), and other nucleosides for 8h. Apoptosis was detected by electron microscopy (EM), agarose gel electrophoresis, and quantitative TdT assay. Results:Apoptosis was detected only in leukemic lymphocytes with P2z receptors. By using the quantitative assay, ATPinducing DNA strand breaks were found to occur specifically for BzATP, ATP and 2MeSATP, but not for ATPγS and other nucleosides. Meanwhile, ATPinducing DNA fragmentation was fully blocked by pretreatment with oxidized ATP (OxATP), a compound recently shown to block P2z receptors. Ca^(2+)/Calmodulin complex played a role in the regulation of the CLL cell apoptosis induced by ATP, because an antagonist of this complex, 1-[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62),was found to inhibit the ATPinducing apoptosis. Conclusion: P2z receptors on lymphocytes play an important role in the apoptosis induced by ATP in vitro.