影响P2Z受体介导慢性淋巴细胞白血病细胞凋亡的各种因素及其机理研究

Investigation of the Effects of Various Factors Affecting Apoptosis of Chronic Lymphocytic Leukemic Cells Mediated by P2Z Receptors

目的 探讨嘌呤能 P2 Z受体介导慢性淋巴细胞白血病 (CL L )细胞凋亡的影响因素及其机理。方法 在二价阳离子—— 1.0 mm ol/L Mg2 + 、Zn2 + 、Ca2 + 、Sr2 + 、Co2 + 、Ba2 + ,不同浓度的 EDTA或 EGTA,不同温度及在含 15 0 mm ol/L胆碱的介质中 ,将表达 P2 Z受体 [P2 Z(+) ]的 CL L细胞分别同 1.0 mm ol/L三磷酸腺苷 (ATP)或 0 .1m mol/L苯甲酰苯甲酸 ATP(Bz ATP)体外培养 8小时 ,以 DNA凝胶电泳、Td T法和流式细胞分析 (FCA )检测上述条件下细胞凋亡的诱导或抑制效应。结果 Mg2 + 或 Ca2 + 能以剂量依赖性方式促进 ATP诱导 P2 Z(+)细胞凋亡 ,而 EDTA或 EGTA却以相反的方式抑制 P2 Z(+)细胞凋亡的发生 ;1.0 mm ol/L Zn2 + 可完全阻止 ATP诱导 P2 Z(+)细胞凋亡所产生的 DNA片段 ,但其它二价阳离子包括 1.0 mm ol/L Sr2 + 、Co2 + 、Ba2 + 却不影响 ATP的诱导 ;胆碱作为磷脂酶 D(PL D)的抑制剂 ,也可部分抑制 P2 Z(+)细胞凋亡产生的 DNA片段 ;当温度低于 10℃ ,可完全阻止 ATP诱导 P2 Z(+)细胞凋亡产生 DNA片段的发生。结论 P2 Z受体介导 CL L细胞凋亡可能与核酸内切酶 ,PL D的参与密切相关。

Objective To explore the effects of various factors affecting apoptosis of chronic lymphocytic leukemia (CLL) cells mediated by P2Z purinoceptor. Methods In vitro exposure of CLL cells with P2Z receptors to 1 mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP (BzATP) for 8 h in the presence of 1.0 mmol/L Mg 2+ or other bivalent cations, 150 mmol/L choline in media, 1,2,4 mmol/L EDTA or EGTA, or under different temperature. ATP induced apoptosis was detected by agarose gel electrophoresis, TdT assay and flow cytometric analysis (FCA). Results The results showed that ① Extracellular Mg 2+ or Ca 2+ stimulated ATP induced DNA fragmentation in a dose dependent manner, and the compatible evidence was provided by the inhibition of ATP induced DNA fragmentation in the presence of EGTA or EDTA; ② ATP induced DNA fragmentation was completely inhibited by 1.0 mmol/L Zn 2+ ; ③ ATP induced DNA breaks were not affected by Ba 2+ , Sr 2+ , Co 2+ when they were substituted for extracellular Mg 2+ or Ca 2+ ; ④ Choline, an inhibitor of phospholipase D (PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); ⑤ ATP induced DNA fragmentation was completely obliterated when the temperature was lower than 10℃. Conclusion These results suggest that the endonuclease and PLD may be involved in ATP induced apoptosis in human lymphocytes via P2Z receptor.