【作者】 李忠魁；

【导师】 刘艳平；

【作者基本信息】 中南大学 ， 细胞生物学， 2007， 硕士

【摘要】 肝癌是我国常见的恶性肿瘤之一，临床治疗难度大、疗效差。利用药物对细胞凋亡相关基因的调控来诱导细胞凋亡成为目前肿瘤治疗的研究热点。黄芩中的黄酮在体外抗肿瘤作用方面表现出活性，但目前国内外尚未有关于汉黄芩苷体外抗肿瘤作用的相关研究。本实验研究汉黄芩苷对体外培养的人Bel-7402细胞的生长抑制及凋亡的影响，旨在探讨汉黄芩苷抗肿瘤作用机制，为汉黄芩苷的药物开发及临床应用提供理论依据。通过使用不同浓度汉黄芩苷作用于体外培养的Bel-7402细胞，利用倒置显微镜观察细胞形态改变，收集细胞后，使用AO／EB双染色法对细胞进行染色，荧光显微镜下观察细胞死亡情况。我们发现汉黄芩苷可以明显抑制Bel-7402细胞生长，荧光显微镜下可见细胞凋亡。使用96孔培养板培养细胞，MTT试验后检测570nm吸光值，通过计算细胞生长抑制率进一步了解汉黄芩苷作用后细胞生长的抑制情况，得出4μM浓度以上的汉黄芩苷对Bel-7402细胞均有抑制作用，48小时半数抑制浓度约为8μM。通过琼脂糖凝胶电泳分析汉黄芩苷作用后的细胞DNA，与对照组相比药物处理组可见较明显的DNA梯状条带，证实处理组细胞中有典型的细胞凋亡发生。为研究汉黄芩苷抑制Bel-7402细胞并诱导其凋亡的机制，采用流式细胞仪分析凋亡情况及细胞周期的改变，发现细胞凋亡比率随汉黄芩苷作用浓度的增加而增加，相同浓度下随时间的延长G₀-G₁期细胞比率逐渐减小，S期、G₂期细胞比率明显增大，说明汉黄芩苷对细胞增长的抑制作用主要在G₂／M期，诱导细胞发生G₂／M周期阻滞。Western-blotting方法检测细胞中Bcl-2／Bax和P53的表达，发现Bcl-2表达有下调趋势，并随汉黄芩苷作用浓度的增加，抑制作用增强，，通过这些结果分析得出：汉黄芩苷可以抑制体外培养的Bel-7402细胞生长并诱导其凋亡的机制与抑制Bcl-2表达、上调Bax／Bcl-2比例相关，而与P53蛋白关系不大，作用途径可能与凋亡的线粒体通路关系密切。更多还原

【Abstract】 The cancer of the liver is one of the familiar malignant tumors of our country, difficult to be treatment in clinic. It has been a hotspot to control the gene related to the cell aopoptosis and induce the cell aopoptosis in research of tumor treatment recently. The anti-tumor function of the flavonoid in Radix scutellariaeis expresses actively in vivo and in vitro. But current research domestic about wogonoside in vitro is poor. Our studies of wogonoside inhibitor on human liver cancer cell line Bel-7402 aims at discussing the mechanism of wogonoside inhibitor of tumor and providing theories for pharmaprojecting medicine of wogonoside clinical application.We treated the cultured Bel-7402 cell with wogonoside on different density; observe the change of cell appearance through microscope observation; and the cell stained with AO/EB through the fluorescence microscope. We discover that the wogonoside can repress the cell Bel-7402 growth obviously, the fluorescence microscope show clearly cell apoptosis. We cultured cell in 96 bores board, examines the absorbance on 570nm after MTT experiment. Cells proliferation was inhibited by wogonoside density above 4μM. The 50%-inhibitory concentration (IC₅₀) was 8μM for 48hours respectively. DNA fragmentation was visualized by agarose gel electrophoresis, DNA Agarose gel electrophoresis of the treated cell revealed "ladder" pattern. All above shows that wogonoside can inhibit cell proliferation of Bel-7402 and induce cell apoptosis.For studding the mechanism that the wogonoside repress the cells Bel-7402s and induce it apoptosis, cell apoptosis rate and cell cycle arrest was detected by flow cytometry. The flow cytometry profiles revealed that wogonoside induced Bel-7402 cell G₂/M arresting because the reduce of G₀/G₁ rate and the increase of S and G₂/M in 60hrs. It also show that the rate of apoptosis cell was increase when the density was increased （4～20μM） during the same time. Expression of Bax/Bcl-2 and P53 was analysed through Western blotting. Wogonoside induced the down-expression of Bcl-2, and the increase of Bax/Bcl-2 ratio through Western blotting. It had no effect on expression of P53.In conclusion wogonoside can inhibit cell proliferation of Bel-7402 and induce cell apoptosis. It may be related with the increase of Bax/Bcl-2 ratio but not P53 through the mitochondria pathway.更多还原