米哚妥林对急性白血病细胞株HL-60细胞抑制效应的研究

Inhibitory Effect of PKC412 Against Human Acute Leukemia Cell Line HL-60 Cells

目的:探讨蛋白激酶C(PKC)抑制剂米哚妥林(PKC412)对HL-60细胞株增殖和凋亡的影响及作用机制。方法:采用CCK-8法检测不同浓度PKC412对HL-60细胞增殖的影响;瑞士吉姆萨染色法鉴定PKC412对HL-60细胞凋亡的影响;qRT-PCR检测BCL-2、P53基因的mRNA表达,Western blot检测BCL-2、P53蛋白的表达;以尾静脉注射HL-60细胞构建急性白血病小鼠模型,以PKC41231.25nmol/kg尾静脉给药,生理盐水组作为对照组,研究PKC412在小鼠体内对HL-60细胞的抑制;ELISA法检测PKC412对肿瘤小鼠细胞因子TNF-α和TGF-β分泌的影响。结果:PKC412抑制HL-60细胞株细胞增殖,呈剂量依赖性(r=0.9973),其中IC50=0.31μmol/L;并能诱导HL-60细胞凋亡。PKC412作用HL-60细胞株48 h后,与对照组比较BCL-2基因mRNA表达下调(0.417±0.044 vs 0.933±0.033,t=9.347,P<0.001),P53基因mRNA表达上调(1.533±0.145 vs 1.050±0.161,t=2.231,P>0.05);BCL-2蛋白表达量下降,P53蛋白表达量升高。PKC412在体内可抑制HL-60肿瘤细胞生长,给药后小鼠生存率为50%,与对照组比较体重增加(18.02±0.403 g vs 16.44±0.562 g,t=2.272,P=0.0356)。PKC412组血清及脾细胞中TNF-α和TGF-β细胞因子较对照组分泌明显减少(P<0.05)。结论:PKC412能通过抑制BCL-2基因表达水平诱导HL-60细胞凋亡,体内给药可抑制肿瘤的生长。

Objective:To explore the effects and mechanisms of PKC412 inhibitor on proliferation and apoptosis of HL-60 cell line.Methods:CCK-8 assay was used to detect the effect of PKC412 on the proliferation of HL-60 cells at different concentrations;Wright-Giemsa staining was used to estimated the effect of PKC412 on the apoptosis of HL-60 cells;the mRNA expression of BCL-2 and P53 genes was detected by qRT-PCR,the expression of BCL-2 and P53 proteins was detected by Western blot.HL-60 cells were injected into mouse caudal vein to construct acute myeloid leukemia model,PKC412 was administered to tail vein for 31.25 nmol/kg,normal saline was injected into the same site of the mice as control group,and the inhibitory effect of PKC412 on HL-60 cells in mice was observed.ELISA assay was used to detect the effect of PKC412 on the inflammatory factors of TNF-α and TGF-β in tumor mice.Results:PKC412 could inhibit the proliferation of HL-60 cell,which was in a dose dependent manner(r=0.9973)(IC50 was0.31 μmol/L),and induce apoptosis of HL-60 cells.After HL-60 cell was treated by PKC412 for 48 h the expression of BCL-2 gene was down regulated(0.417±0.044 vs 0.933±0.033,t=9.347,P<0.001),the expression of P53 gene was up regulated(1.533±0.145 vs 1.050±0.161,t=2.231,P>0.05) as compared with control group.And the expression of BCL-2 protein was decreased,while the expression of P53 protein was increased.PKC412 could inhibited the growth of HL-60 tumor cells in vivo,the survival rate of mice after administration was 50% and the weight was increased as compared with that in control group(18.02±0.403 g vs 16.44±0.562 g,t=2.272,P=0.0356).The secretion of TNF-α and TGF-β cytokine in serum and spleen cells in PKC412 group was significantly lower than that in control group(P <0.05).Conclusion:PKC412 can induce apoptosis of HL-60 cells by inhibiting the expression level of BCL-2 gene,PKC412 administration in vivo can inhibit the growth of the tumors.