蟾蜍灵在诱导HL60细胞凋亡过程中对Bcl2和PKC的影响_医学论文.docVIP

蟾蜍灵在诱导HL细胞凋亡过程中对Bcl和PKC的影响_医学论文 蟾蜍灵在诱导HL细胞凋亡过程中对Bcl和PKC的影响_医学论文 【摘要】 为了研究蟾蜍灵在诱导HL细胞凋亡过程中对Bcl表达与裂解和磷酸化的作用，及对蛋白激酶C（PKC）活性与PKCs亚细胞定位的影响，分别采用台盼蓝拒染法检测细胞生存率，细胞染色法观察凋亡形态，流式细胞术分析细胞周期，琼脂糖凝胶电泳检测凋亡DNA片段化，［γ］同位素掺入法测定PKC活性，Western blot分析Bcl及PKC蛋白表达。结果表明：①蟾蜍灵抑制HL细胞增殖，24、48及72小时的IC50分别为(25.8±2.1)、(8.0±1.2)及 (2.3±0.3) nmol/L ②50 nmol/L蟾蜍灵作用24小时可诱导HL细胞凋亡 ③50 nmol/L 蟾蜍灵处理HL细胞6-24小时，Bcl蛋白表达水平明显下调，并出现23 kD的裂解片段，磷酸化水平逐渐降低 ④1-100 nmol/L 蟾蜍灵分别作用30分钟，对PKC总活性无影响，但可促使PKCβⅡ膜转位。结论：蟾蜍灵诱导HL细胞凋亡可能与Bcl表达降低、裂解及脱磷酸化有关。 【关键词】 白血病 蟾蜍灵 凋亡 Bcl蛋白激酶C Effect of Bufalin Abstract Previous study revealed that bufalin can inhibit proliferation, and induce apoptosis in some human cancer cell lines. However, the mechanism of its anticancer effect has not been fully understood. The present study was designed to investigate the effects of bufalininduced apoptosis on Bcl2 and PKC in human leukemic HL60 cells. The cell viability was determined by trypan blue dye exclusion. The apoptosis was detected by morphology, flow cytometry and DNA agarose gel electrophoresis. The expressions of Bcl2 and PKC were analyzed by Western blot, and activity of PKC was assayed by ［γ］ isotope incorporation method.The results showed as follows: (1) proliferation of HLs. (3) compared with control, treatment with bufalin at concentration of 50 nmol/L for 6-24 hours resulted in downregulation of protein expression, decrease of phosphorylation, and cleavage of Bcl2, simultaneously. (4) the activity of total PKC was unchanged when HL60 cells were exposed to 1-100 nmol/L bufalin for 30 minutes, but PKCβⅡ underwent translocation from cytosol to membrane. It is concluded that apoptosis induced by bufalin is associated with downregulation of protein expression, dephosphorylation, and cleavage of Bcl2 in HL60 cells. Key words leukemia bufalin apoptosis Bcl2 PKC 中药蟾酥在我国已广泛应用于恶性肿瘤的治疗，但其成分复杂，作用机理不明。蟾蜍灵是蟾酥的主要配基之一，近年来国外和我们的研究表明，蟾蜍灵可诱导人类白血病K562，THP和HL等细胞凋亡 ［1-3］，为进一步研究蟾蜍灵诱导白血病细胞凋亡的分子生物学机理，本实验采用蟾蜍灵作用HL细胞，研究其对Bcl表达、裂解及磷酸化的影响，并探讨PKC活性及其同工酶表达