重楼皂苷Ⅰ对人黑素瘤A375细胞增殖和凋亡的影响  

Effects of polyphyllin I on the proliferation and apoptosis of human melanoma cell line A375

作　　者：龙剑文 罗晶[3] 尹绪文 卫静 贺琪[3] 李恒[3] 石全[3] 皮先明[3] 

机构地区：[1]湖北中医药大学第一临床学院皮肤科,武汉430061 [2]湖北省中医院皮肤科,武汉430061 [3]湖北省中医院皮肤科

出　　处：《中华皮肤科杂志》2017年第12期883-888,共6页Chinese Journal of Dermatology

基　　金：湖北省卫生计生委中西医结合科研计划项目（2013Z-B05）

摘　　要：目的 研究重楼皂苷Ⅰ对人黑素瘤A375细胞增殖和凋亡的影响及相关机制。方法 采用CCK8法测定0（对照组）、1.5、3.0、6.0 mg/L重楼皂苷Ⅰ对正常人黑素细胞和A375细胞增殖的影响；Hoechst33258荧光染色法观察细胞凋亡形态；流式细胞仪检测A375细胞周期变化及细胞凋亡水平；荧光染料（DCFH？DA）测定A375细胞内活性氧的变化；罗丹明123染色测定线粒体膜电位变化；利用分光光度法检测重楼皂苷Ⅰ处理后的A375细胞内ATP和上清液中乳酸、葡萄糖含量；Western印迹法检测A375细胞内Bcl？2、Bcl？2相关X蛋白（Bax）、活化的半胱氨酸天冬氨酸蛋白水解酶3、细胞周期蛋白D1、丙酮酸激酶同工酶2（PKM2）表达。多组均数比较采用单因素方差分析，组间多重比较采用SNK？q检验。结果 在工作浓度内重楼皂苷Ⅰ对正常人黑素细胞增殖无明显影响，但能明显抑制 A375 细胞的增殖（P 〈 0.01）；荧光显微镜下发现，重楼皂苷Ⅰ处理后的 A375 细胞出现明显的凋亡形态。0（对照组）、1.5、3.0、6.0 mg/L重楼皂苷Ⅰ分别处理A375细胞后，随重楼皂苷Ⅰ浓度增加，细胞凋亡率（分别为4.25% ± 1.27%、10.03% ± 1.49%、36.62% ± 1.97%、44.11% ± 2.47%）逐渐升高（F = 665.7，P 〈 0.01），G0/G1期细胞比例（分别为54.13% ± 2.57%、67.35% ± 3.79%、74.39% ± 3.29%、82.29% ± 3.99%）亦渐增多（F = 71.81，P 〈 0.01）；细胞内活性氧呈明显上升趋势，细胞线粒体膜电位呈明显下降趋势（P 〈 0.01）；细胞内ATP含量下降（P 〈 0.01），培养液中乳酸含量下降，葡萄糖含量上升（P 〈 0.01）；细胞Bax、cleaved？caspase？3蛋白表达增多，但Cyclin D1、Bcl？2和PKM2蛋白表达下降（P 〈 0.01）。结论 重楼皂苷Ⅰ可能通过激活细胞内活性氧的产生，引起线粒体膜电位下降，诱导A375细胞凋亡，并通过抑制PKM2、细胞周期蛋白D1表达，使细胞阻滞�Objective To investigate effects of polyphylinⅠon the proliferation and apoptosis of human melanoma cell line A375, and to explore their mechanisms. Methods Normal human melanocytes isolated from healthy human foreskin were divided into 6 groups to be treated with 0, 1.5, 3.0, 6.0, 9.0, 12.0 mg/L polyphyllinⅠrespectively. A375 melanoma cells were divided into 4 groups, i.e., control group, 1.5-, 3.0-, 6.0-mg/L polyphyllinⅠgroups, to be treated with 0, 1.5, 3.0, 6.0 mg/L polyphyllinⅠ, respectively. Cell counting kit-8 （CCK8） assay was performed to evaluate the effect of polyphyllinⅠ on the proliferation of normal human melanocytes and A375 cells. Hoechst 33258 fluorescent staining was conducted to observe the morphology of apoptotic cells, flow cytometry to estimate cell cycle phase distribution and apoptosis rate, dichloro-dihydro-fluorescein diacetate （DCFH-DA） fluorescent probe assay to detect the level of reactive oxygen species（ROS）, rhodamine-123 staining to evaluate changes of mitochondrial membrane potential, spectrophotography to detect the level of ATP in A375 cells, as well as levels of lactic acid and glucose in the culture supernatant of A375 cells, and Western blot analysis to determine the protein of Bcl-2, Bcl-2-related X protein （Bax）, cleaved-caspase-3, cyclin D1 and pyruvate kinase isozyme type M2 （PKM2）. Statistical analysis was carried out by using one-way analysis of variance （ANOVA） for comparisons among groups and Student-Newman-Keuls-q （SNK-q） test for multiple comparisons. Results CCK8 assay showed that the treatment with polyphyllinⅠat concentrations of 1.5, 3.0, 6.0 mg/L for 48 hours had no effects on the proliferation of normal human melanocytes, but significantly inhibited the proliferation of A375 cells. The survival rate of A375 cells was significantly lower in the 1.5-, 3.0-, 6.0-mg/L polyphyllinⅠgroups than in the control group （P 〈 0.01）. After the treatment with polyphyllinⅠ, distinct apoptotic morphology of A375 cells was observ

关 键 词：黑素瘤 细胞系 肿瘤 细胞凋亡 活性氧 细胞增殖 重楼皂苷Ⅰ 

分 类 号：R285[医药卫生—中药学]