【作者】 欧阳曙明；

【导师】 杨业金；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 目的探讨雷公藤内酯醇(Triptolide,TPL)对人非小细胞肺癌A549细胞增殖的抑制作用及促A549细胞凋亡的分子机制,为雷公藤内酯醇治疗非小细胞肺癌提供一定的实验依据。方法1、以A549细胞为细胞模型,采用MTT法测定不同浓度不同时间TPL对A549细胞的增殖抑制作用。2、采用琼脂糖凝胶电泳法(DNA ladder法)、吖啶橙/溴乙啶(AO/EB)荧光染色法检测A549凋亡。3、采用Caspase-3活性检测法分析TPL促A549细胞凋亡机制。4、采用逆转录-聚合酶链式反应(RT-PCR)法检测A549细胞Survivin mRNA的水平,分析其凋亡调控途径。结果1、在浓度6.25-100ng/ml、时间12-48h检测范围内,TPL对A549细胞增殖抑制作用同对照组相比具有显著差异(p<0.05)。2、DNA ladder法检测到DNA梯状条带,证明A549细胞凋亡的发生;AO/EB荧光染色法检测到凋亡细胞特异性形态学改变,且48h内凋亡率随着时间延长而增加(p<0.05)。3、TPL激活Caspase-3,48h内Caspase-3活性较对照组有显著提高(p<0.05),12h处活性最大;TPL浓度6.25-100ng/ml内,Caspase-3活性升高,并具有浓度依赖性(p<0.05)。4、TPL显著抑制A549细胞Survivin mRNA水平(p<0.05)。5、Survivin mRNA水平与Caspase-3活性呈负相关。结论低浓度TPL能有效抑制A549细胞的增殖,这种抑制作用与TPL激活Caspase-3,降低Survivin mRNA水平从而促进细胞凋亡有关。更多还原

【Abstract】 Objective To investigate the proliferation inhibitory effect, the apoptosis-inducing activity of triptolide on A549 cells and take an initial approach on the mechanism of the apoptosis.Methods Non-small cell lung cancer cell line A549 was used in this study. The effects of triptolide on cell proliferation were observed in vitro by MTT method. The apoptosis was evaluated by DNA ladder and AO/EB fluorescent staining. Measurement of Caspase-3 activity and Survivin mRNA lever was taken to explaine the initial mechanism of apoptosis.Results Triptolide exhibited a marked antiproferative effect on A549 cells within the concentration from 6.25 to 100ng/ml and the time from 12 to 48 hours(p<0.05). The apoptotic ratio was time-dependant in 48 hours(p<0.05). The activity of Caspase-3 activity had a significant increase compared to the control group in 48 hours(p<0.05)and reached a maxlmum after 12 hours treating of triptolide. Likewise, Caspase-3 activity had a direct correlation with the concentration of triptolide during 6.25 to 100ng/m(lp<0.05). However, the lever of Survivin mRNA had a significant decrease compared to the control group(p<0.05). Furthmore, it had a negative correlation with the Caspase-3 activity .Conclusions Triptolide can inhibit the proliferation on A549 cells effectively at a low concentration. The effect may be correlated with the apoptosis-inducing activity through the Caspase-3 activation and down-regulation Survivin mRNA from triptolide.更多还原