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目的研究一种萘酰亚胺-多胺缀合物对人肝癌HepG2细胞的杀伤作用,并探讨其作用机制。方法以体外培养的人肝癌HepG2细胞为研究对象,噻唑蓝法检测细胞毒性,AO/EB/Hochest 33342染色观察细胞凋亡形态,DCFH-DA检测HepG2细胞内ROS变化,Rh123染色检测线粒体膜电位变化,免疫荧光法检测caspase-9,PARP-1表达,PI染色分析细胞周期变化,流式细胞术分析细胞凋亡率。结果噻唑蓝法显示,该萘酰亚胺-多胺缀合物呈剂量依赖性抑制HepG2细胞增殖,48 h的IC_(50)为17.28μmol·L~(-1),24 h的IC_(50)为29.71μmol·L~(-1),10μmol·L~(-1)该萘酰亚胺-多胺缀合物作用48 h后:细胞内活性氧簇、caspase-9荧光强度均显著升高(P<0.01,n=20),Rh123、PARP-1荧光强度显著减低(P<0.01,n=20);细胞增殖阻滞在S期;25μmol·L~(-1)萘酰亚胺-多胺缀合物作用细胞48 h后,高内涵活细胞成像系统可见细胞核固缩呈圆珠状凋亡特征,流式细胞术显示细胞早期凋亡率为33.13%,晚期凋亡率为30.99%。结论该萘酰亚胺-多胺缀合物对HepG2细胞具有较强的促凋亡作用,其机制与升高细胞内ROS水平,降低线粒体膜电位,进而活化caspase-9,通过线粒体途径诱导细胞凋亡有关,另外,该萘酰亚胺-多胺缀合物还通过下调PARP-1表达而发挥促凋亡作用。
Objective To study the killing effect of a naphthalimide-polyamine conjugate on human hepatoma HepG2 cells and to explore its mechanism. Methods HepG2 cells were cultured in vitro. Cytotoxicity was detected by thiazolyl blue staining. AO / EB / Hochest 33342 staining was used to observe the morphological changes of apoptotic cells. DCFH-DA was used to detect the changes of ROS in HepG2 cells. Rh123 staining was used to detect the changes of mitochondrial membrane potential , The expression of caspase-9 and PARP-1 were detected by immunofluorescence, the cell cycle was analyzed by PI staining, and the apoptosis rate was analyzed by flow cytometry. The results of thiazolyl blue method showed that the naphthalimide-polyamine conjugate inhibited the proliferation of HepG2 cells in a dose-dependent manner. IC 50 of IC50 for 48 h was 17.28 μmol·L -1 and IC 50 for 24 h ) For 29.71μmol·L -1, 10μmol·L -1 for 48 h, the fluorescence intensity of intracellular reactive oxygen species and caspase-9 increased significantly (P <0.01, n = 20), the fluorescence intensity of Rh123 and PARP-1 was significantly reduced (P <0.01, n = 20); cell proliferation arrest was in S phase; 25μmol·L -1 naphthalimide After 48 h of polyamine conjugate, the apoptotic cell nuclei were observed in the highly connate living cell imaging system. Flow cytometry showed that the early apoptosis rate was 33.13% and the late apoptosis rate was 30.99 %. CONCLUSION: The naphthalimide-polyamine conjugate has a strong pro-apoptotic effect on HepG2 cells. Its mechanism is related to the increase of intracellular ROS level, mitochondrial membrane potential, and activation of caspase-9, and the induction of cells by mitochondrial pathway In addition, the naphthalimide-polyamine conjugates also exert a pro-apoptotic effect by downregulating PARP-1 expression.