瘤背石磺多糖对人宫颈癌细胞的体外抑制作用

Inhibitory effect of Onchidium reevesii polysaccharides on Hela cells in vitro

摘要:

体外试验研究瘤背石磺多糖(Onchidium reevesii polysaccharides, OSP)和硫酸酯化修饰后瘤背石磺多糖(Sulfated Onchidium reevesii polysaccharides, S-OSP)对人宫颈癌Hela细胞生长的影响,并以阳性药物顺铂(0.5、0.75、1.5 μg/mL)作为对照。将不同质量浓度(50、75、150 μg/mL)的OSP和S-OSP作用于Hela细胞,用CCK-8法分析Hela细胞在12、24、48和72 h的增殖情况,用流式细胞仪分析细胞周期,并应用qRT-PCR检测相关凋亡基因的表达变化。结果表明,OSP和S-OSP均能降低Hela细胞活性并诱导其凋亡,而且S-OSP比OSP更能显著抑制Hela细胞增殖活性,表明硫酸酯化修饰改变了多糖结构进而影响多糖抗肿瘤活性。作用48 h后,3种质量浓度OSP组Hela细胞生长抑制率分别为60%、67%和71%;S-OSP组Hela细胞生长抑制率分别为74%、77%和84%;顺铂组Hela细胞生长抑制率分别为82%、86%和90%。流式细胞术检测显示OSP和S-OSP作用24 h后,细胞早期和晚期凋亡的比率显著,G0/G1期细胞比例增多,S期细胞比例不断降低;实时荧光定量表明促凋亡基因Caspase-3的表达量随药物质量浓度的提高显著增加,抗凋亡基因Bcl-2和Bcl-xL的表达量随药物质量浓度的提高无明显变化,说明OSP和S-OSP可通过调节Caspase信号通路诱导Hela细胞凋亡。

Abstract:

In vitro experiments were conducted to study the effects of Onchidium reevesii polysaccharides and sulfated Onchidium reevesii polysaccharides on the growth of Hela human cervical cancer cells, and with positive drug cisplatin (0.5, 0.75, 1.5 μg/mL) as control. Different mass concentrations (50, 75, 150 μg/mL) of OSP and S-OSP were applied to Hela cells, and we used CCK-8 method to analyze the proliferation of Hela cells at 12, 24, 48 and 72 h, and analyzed the cell cycle with flow cytometry, and used qRT-PCR to detect the expression changes of related apoptosis genes. The results show that both OSP and S-OSP can reduce the viability of Hela cells and induce their apoptosis, moreover, S-OSP can more significantly inhibit the proliferation activity of Hela cells than OSP, It shows that the sulfation modification changes the structure of the polysaccharide and affects the anti-tumor activity of the polysaccharide. After 48 hours of treatment, the growth inhibition rates of Hela cells in the three concentrations of OSP group were 60%, 67%, and 71%, respectively; the growth inhibition rates of Hela cells in the S-OSP group were 74%, 77%, and 84%, respectively; the growth inhibition rates of Hela cells in the cisplatin group were 82%, 86%, and 90%, respectively. Flow cytometry showed that after OSP and S-OSP had acted for 24 h, the ratio of early and late apoptosis of cells was significant, and increased proportion of cells in G0/G1 phase, and the proportion of S-phase cells continued to decrease; Real-time fluorescence quantification showed that the expression of pro-apoptotic gene Caspase-3 increased significantly with the increase of drug mass concentration, and the expression of anti-apoptotic genes Bcl-2 and Bcl-xL did not change significantly with the increase of drug mass concentration. It shows that OSP and S-OSP can induce Hela cell apoptosis by regulating the Caspase signaling pathway.