嘌呤能P2z受体介导白血病细胞凋亡的研究

Apoptosis of leukemic lymphocytes mediated by purinergic P2z receptors

目的探讨嘌呤能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（－）］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′－末端的ＴｄＴ法检测细胞凋亡；并对ＡＴＰ、ＢｚＡＴＰ、２ＭｅＳＡＴＰ、ＡＴＰ－γＳ与其他核苷的诱导及ＯｘＡＴＰ、ＫＮ－６２的抑制效应做定量研究。结果（１）Ｐ２ｚ（＋）细胞能在ＡＴＰ诱导下呈现典型的细胞凋亡形态和ＤＮＡ梯状条带；（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或其他核苷几乎无诱导能力；（３）Ｐ２ｚ受体的抑制剂Ｏｘ－ＡＴＰ和钙调节蛋白激酶抑制剂ＫＮ－６２可完全阻止诱导剂诱导的细胞凋亡的诱导。结论Ｐ２ｚ受体在介导由ＡＴＰ诱导ＣＬＬ细胞凋亡的过程中起着十分重要的作用。

Objective To investigate the role of purinergic P2z receptors for apoptosis of human leukemic lymphocytes induced by extracellular adenosine triphosphate (ATP). Methods A total of 11 B CLL patients were studied with regard to exposure of leukemic lymphocytes with ( n =8) or without ( n =3) P2z receptors to ATP, benzoylbenzoic ATP (BzATP), 2 methylthio ATP (2MeSATP), adenosine 5′ triphosphate (ATP γS), and other nucleosides for 8 h in vitro. Apoptosis was detected by electron microscopy (EM), agarose gel electrophoresis, and quantitative assay TdT assay. Results Apoptosis was detected only in leukemic lymphocytes with P2z receptors. Using a quantitative assay, we found that ATP induced DNA strand breaks occur specifically with BzATP, ATP and 2MeSATP, but not for analogue ATP γS nor other nucleosides. Meanwhile, ATP induced DNA fragmentation was fully blocked by pretreatment with oxidized ATP (OxATP), a compound recently shown to block P2z receptors. Also, the Ca 2+ /calmodulin complex played a role in the regulation of the apoptosis induced by ATP on CLL cells, 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 induced apoptosis. Conclusion These data indicate that P2z receptors on lymphocytes play an important role in apoptosis induced by ATP in vitro.