羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖(Sargassum Fusiforme Polysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western blot法测定Caspase-3酶原的变化; RT-PCR检测Caspase-3 mRNA表达。结果显示:SFPS作用lovo细胞24,36,48和72h的IC_(50)分别为375,355,178和60mg/L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征:细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5-300mg/L作用24h后,显示有凋亡细胞特有的DNA梯状条带;而500mg/L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。

STUDY ON THE APOPTOSIS AND ITS MECHANISM OF LOVO HUMAN COLORECTAL CANCER CELLS INDUCED BY SFPS

Human colorectal cancer (lovo) cells were chose to study the anti-tumor effects of SFPS and explore the significance of caspase 3 in the apoptosis of lovo cells induced by SFPS. Inhibition of the cell proliferation was measured by MTT assay. SFPS induced apoptosis of lovo cells was observed by electron microscopy, flow cytometry and DNA electrophoresis. Furthermore, the expressions of caspase 3 mRNA and pro-caspase 3 were tested by RT-PCR and Western blot. SFPS exhibited anti-proliferative activity in the dosage and time-dependent manner. After incubation for 24, 36, 48 and 72 h, the IC_(50) of SFPS on lovo cells was 375 mg/L, 355 mg/L, 178 mg/L and 60 mg/L, respectively. DNA ladders of lovo cells were showed on agarose gel electrophoresis and the fragments of DNA were integral of 180-200 bp 24 hours after SFPS treatment at the doses of 5 mg/L, 50 mg/L and 300 mg/L. However, mistiness DNA ladder or smear was found in lovo cells treated with 500 mg/L SFPS. When SFPS concentration was 1000 mg/l, DNA ladder disappeared and showed smear entirely. Morphological examination showed chromosomal condensation, karyotheca margination, cell shrinkage and the presence of apoptosis bodies with electron microscopy. With concentration dependent manner, the apoptosis and sub-G1 peaks were observed through flow cytometry, but the cell cycle didn't change obviously. Furthermore, the expression of pro-caspase 3 was decreased and the level of caspase 3 mRNA was increased in the time and dose- dependent manner. It suggests that SFPS may induce the apoptosis of lovo cells in vitro resulting in the inhibition of proliferation. And caspase 3 activation may participate in the processes of the apoptosis of lovo cells induced by SFPS.