藻蓝蛋白诱导喉癌HEP-2细胞凋亡的实验研究

Effects of phycocyanin on apoptosis of human laryngeal cancer HEP-2 cells

目的:探讨藻蓝蛋白对人喉癌HEP-2细胞凋亡的影响,并初步探讨其机制。方法:不同浓度藻蓝蛋白处理HEP-2细胞,分别以MTT、倒置显微镜、扫描电镜、透射电镜、流式细胞术检测藻蓝蛋白对HEP-2细胞活力及凋亡的影响;DCFH-DA标记的流式细胞术检测细胞内的活性氧水平;分光光度法检测细胞内caspase-3、-8、-9的活性;RT-PCR、Western blot检测细胞凋亡相关基因的表达。结果:MTT结果显示藻蓝蛋白能够抑制喉癌HEP-2的细胞活力,且呈时间和剂量依赖性;倒置显微镜、电镜、流式细胞术的一系列定性化和定量化实验证实藻蓝蛋白可显著诱导HEP-2细胞的凋亡;藻蓝蛋白处理后细胞内ROS水平升高,caspase-3、-8、-9被激活;RT-PCR结果表明,藻蓝蛋白作用后Bax、Fas、P53、caspase-3和caspase-9表达显著上调(P<0.05),Bcl-2表达显著下调(P<0.05);Western blot结果和RT-PCR结果一致。结论:藻蓝蛋白能够诱导HEP-2细胞的凋亡,其机制可能与细胞内ROS水平升高,上调Bax、Fas和P53 mRNA,下调Bcl-2 mRNA的表达,从而促进凋亡信号的转导最终导致细胞凋亡有关。

AIM： To investigate the effect of phycocyanin on the apoptosis of human laryngeal cancer HEP-2cells and to explore the inhibitory mechanism of phycocyanin to tumor. METHODS： Highly purified phycocyanin was extracted from spirulina. The effects of phycocyanin at different concentrations on the growth of human laryngeal cancer HEP-2 cells were detected by MTT assay. In addition,the cell structures were observed under electron microscope. The cell apoptosis was analyzed by flow cytometry. The production of reactive oxygen species（ ROS） was measured by flow cytometry.Enzymatic activities of caspase-3,-8 and-9 were measured by chemical colorimatry. The expression of Bax,Bcl-2,Fas,P53,caspase-3 and caspase-9 at mRNA and protein levels was determined by RT-PCR and Western blot. RESULTS：MTT test confirmed that phycocyanin inhibited the cell activity of HEP-2 cells with time and dose dependent manners. The result of electron microscope observation and flow cytometry indicated that phycocyanin induced the apoptosis of HEP-2cells. The intracellular content of ROS was increased. The activities of caspase-3,-8 and-9 were increased. RT-PCR showed that the mRNA expression of Bax,Fas,P53,caspase-3,caspase-9 was increased and Bcl-2 was decreased. The results of Western blot were consistent with the results of RT-PCR. CONCLUSION： Phycocyanin might induce apoptosis of HEP-2 cells by down-regulating Bcl-2,up-regulating Bax,Fas and P53,and the transduction of apoptotic signals in the human laryngeal cancer cells.