目的 研究δ阿片受体激动剂D-丙2,D-亮5脑啡肽(DADLE)对人肝癌HepG2细胞增殖的影响,并探讨其与蛋白激酶C(PKC)途径的关系.方法 分别以0.01、0.1、1.0和10 μmol/L的DADLE处理HepG2细胞,采用四甲基偶氮唑蓝(MTT)法检测细胞的增殖活性,采用逆转录聚合酶链反应(RT-PCR)和Western blot法检测PKC mRNA和p-PKC蛋白的表达.分别以DADLE+纳曲吲哚(NAL)或佛波酯(PMA)处理HepG2细胞,采用流式细胞仪观察细胞周期,MTT法检测细胞的增殖活性,Western blot法检测p-PKC蛋白的表达.以DADLE+顺铂处理HepG2细胞,MTI法检测HepG2细胞的生长抑制率.结果 不同浓度的DADLE通过抑制HepG2细胞中PKC mRNA和p-PKC蛋白的表达对HepG2细胞的增殖发挥抑制效应.流式细胞仪检测结果显示,与对照组相比,DADLE处理组HepG2细胞中S+G2/M期细胞的比例降低了3.94％(P＜0.01)；给予NAL或PMA处理后,S+G2/M 期细胞的比例分别增加了3.22％和3.63％ (P＜0.01).MTT法和Western blot法检测结果显示,与对照组相比,DADLE处理组HepG2细胞的A570nm值和p-PKC蛋白的表达显著下降(P＜0.01)；给予NAL或PMA处理后,HepG2细胞的A570nm值和p-PKC蛋白的表达明显升高(P＜0.01).DADLE+顺铂处理组HepG2细胞的生长抑制率为79.9％,显著高于DADLE处理组(25.2％)和顺铂处理组 (43.2％,P＜0.01).结论 DADLE激活δ阿片受体对人肝癌HepG2细胞的增殖具有抑制作用,其机制与PKC途径相关.DADLE可增强HepG2细胞对顺铂的敏感性.%Objective To investigate the effect of DADLE,a δ-opioid receptor agonist,on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway.Methods HepG2 cells were treated with DADLE at different doses (0.01,0.1,1.0 and 10 μmol/L).Cell viability was determined using methyl thiazolyl terazolium (MTT) assay.The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay.After treated separately with DADLE plusing NAL or PMA,the cell cycle of HepG2 cells was analyzed by flow cytometer.MTT was used to detect their proliferation capaeity and Western blot was used to examine the p-PKC expression, The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed.Resnlts DADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group,the percentage of S + G2/M cells in DADLE-treated group was lowered by 3.94％ ( P ＜ 0.01 ).Meanwhile,after treated with NAL and PMA,the percentage was elevated by 3.22％ and 3.63％,respectively ( P ＜ 0.01 ).The MTT and Western blot assays showed that compared with the control group,the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased ( P ＜ 0.01 ).After treatment with NAL and PMA,the values of A570 and p-PKC protein levels were elevated significantly (P ＜0.01 ).The growth inhibitory rate of DADLE + CDDP group was 79.9％,significantly lower than 25.2％ and 43.2％ of the DADLE and CDDP groups,respectively.Conclusions Activation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells.The underlying mechanism may be correlated with PKC pathway.DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.
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