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目的:研究北美香柏叶的乙醇提取物对非小细胞肺癌A549细胞的抗肿瘤及抗增殖作用。方法:噻唑蓝法检验不同剂量北美香柏叶的乙醇提取物对细胞活性的影响。确定半数最大抑制浓度为282μg/mL,另外选择两个浓度188μg/mL和376μg/mL进行剂量依赖性检测。进行溴脱氧尿苷结合实验和细胞迁移实验检测药物的抗肿瘤细胞增殖活性。膜联蛋白V-异硫氰酸荧光黄-碘化丙啶双染色后采用荧光激活细胞分类分析仪对细胞早期凋亡进行检测。Hoechst 33258及吖啶橙-溴化乙啶荧光染色法进行DNA片段分析。间接酶联免疫吸附法分析Bax-Bcl2的调节和表达情况。逆转录聚合酶链反应检测caspase3基因表达情况,其活性和蛋白水平的表达则使用间接酶联免疫吸附法和蛋白印迹法进行检测。结果:A549的细胞活性在暴露于北美香柏叶的乙醇提取物24h后呈剂量依赖性下降。脱氧尿苷结合实验和细胞迁移实验表明细胞的增殖活性与暴露于药物的时间有时间依赖性关系。11.72%的细胞在双染色后呈阳性反应,表明药物引起了细胞的早期凋亡。药物作用24h后DNA片段彗星尾的出现以及Hoechst 33258荧光染色的增加提示显著的DNA缺口出现以及染色质凝聚。Bax的上调及Bcl2的下调表明了细胞凋亡的出现。逆转录聚合酶链反应、间接酶联免疫吸附法以及蛋白印迹法的检测结果表明caspase3的活性随着抗聚(腺苷二磷酸-核糖)聚合酶的表达的增加而增加。结论:北美香柏叶的乙醇提取物能够促进A549细胞凋亡并抑制其增殖活性。
OBJECTIVE: To study the antitumor and anti-proliferative effects of ethanol extract from Cedarum scoparium in non-small cell lung cancer A549 cells. Methods: The effects of ethanol extract of Cortex Fragrans on cell viability were tested by thiazolyl blue method. The maximum inhibitory concentration was determined to be 282 μg / mL and two additional concentrations of 188 μg / mL and 376 μg / mL were selected for dose-dependent assays. The bromodeoxyuridine binding assay and cell migration assay were used to detect the anti-tumor cell proliferative activity of the drug. Annexin V-isothiocyanate fluorescent yellow - propidium iodide double staining using fluorescence activated cell sorting analyzer for early detection of cell apoptosis. Hoechst 33258 and acridine orange - ethidium bromide staining for DNA fragment analysis. Indirect enzyme-linked immunosorbent assay Bax-Bcl2 regulation and expression. Caspase3 gene expression was detected by reverse transcription polymerase chain reaction (RT-PCR), and its activity and protein expression were detected by indirect enzyme-linked immunosorbent assay and Western blotting. RESULTS: The cell viability of A549 decreased in a dose-dependent manner after exposure to ethanol extracts of Cedrus de la Frondosides for 24 h. Deoxy-uridine binding experiments and cell migration experiments showed that the cell proliferation activity and time-dependent exposure to drugs in a time-dependent manner. 11.72% of the cells were positive after double staining, indicating that the drug caused early apoptosis of cells. The appearance of DNA tail and the increase of Hoechst 33258 fluorescence after 24 h of drug treatment indicate significant DNA gap and chromatin condensation. The up-regulation of Bax and the down-regulation of Bcl2 indicate the appearance of apoptosis. RT-PCR, indirect enzyme-linked immunosorbent assay and Western blotting showed that the activity of caspase 3 increased with the increase of anti-poly (ADP-ribose) polymerase expression. Conclusion: ethanol extract of cedar of North America can promote A549 cell apoptosis and inhibit its proliferation activity.