8-硝基白杨素诱导人肝癌HepG2细胞分化研究

摘要

目的：探讨传统中药蜂胶的有效成分白杨素的人工半合成类似物8-溴-7-甲氧基白杨素，即8-硝基白杨素（BrMC）是否具有诱导人肝癌细胞分化作用。方法体外培养人肝癌HepG2细胞，分别加入1.0μg/mL BrMC和1.0μg/mL全反式维甲酸（ATRA），同时设溶媒对照组和空白对照组。瑞氏-姬姆萨染色法检测 BrMC 诱导 HepG2细胞形态与核质比的变化；酶促反应试剂盒检测BrMC 和 ATRA 诱导 HepG2细胞中碱性磷酸酶（ALP）和γ-谷氨酰转肽酶（γ-GT）的活性；放射免疫法检测甲胎蛋白（AFP）的质量分数；Diamondstone分光光度法（速率法）检测酪氨酸转氨酶（TAT）的活性。结果1.0μg/mL BrMC处理的细胞核质比远低于溶媒对照组，差异有统计学意义（P＜0.01）。1.0μg/mL BrMC、ATRA组处理24、48、96 h的HepG2细胞液中AFP质量分数，γ-GT、ALP、TAT活性分别与溶媒对照组比较，差异均有统计学意义（P＜0.01）。结论 BrMC能降低HepG2细胞核质比、减少HepG2细胞的AFP 分泌、有效地活化HepG2细胞TAT、ALP；且BrMC和ATRA均能抑制HepG2细胞γ-GT活性。BrMC通过以上作用诱导人肝癌细胞分化。%Objective To evaluate whether the artificial semisynthetic analogue 8-bromo-7-methoxychrysin (BrMC), i. e., 8-nitrochrysin as an effective component of traditional Chinese medicine propolis,having the ability for inducing the differen-tiation of human hepatocarcinoma cells. Methods The human hepatocarcinoma HepG2 cells were cultured in vitro. The Wright s-Giemse mixed coloring method was adopted to detect the changes of the cellular morphology and the nuclear-cytoplasmic ratio of Hep G2 cells induced by BrMc;the changes of microtubules microfilament protein array of HepG2 cells induced by BrMC and all trans retinoid acid(ATRA) were observed by Coomassie brilliant blue staining;the contents of alkaline phosphatase(ALP) and gamma-glutamyl transpeptidase(γ-GT) in HepG2 cells were detected by the enzyme catalyzed reaction;the radioimmunoassay(RIA) was used to detect the mass fraction of alpha fetal protein(AFP);the content of tyrosine transaminase(TAT) was measured by the Dia-mondstone spectrophotometry. Results The cytoplasmic ratio by 1.0 ug/mL BrMC handled was far lower than that of solvent con trol group,the difference was statistically significant(P<0.01).1.0μg/mL BrMC and ATRA handled 24,48,96 hours,the mass fraction of AFP,activity ofγ-GT,ALP,TAT compared with the solvent control group respectively,the difference were statistically significant(P<0.01). Conclusion BrMC induces human hepatocarcinoma cellular differentiation by lowing the nucleus-cytoplas-mic ratio,decreasing the secretion of AFP,effectively activating TAT and ALP in HepG2 cells;moreover BrMc and ATRA inhibit-ing the activity ofγ-GT.