柴胡皂苷D通过诱导C2－M期阻滞抑制结直肠癌 SW480细胞的增殖

摘要

Objective:To observe the anti -proliferation capacity of saikosaponin -D(SSD)on colorectal cancer cell and normal colorectal cell and further explore the underlying mechanisms.Methods:Using MTT,cell counting and clone formation assay to assess the anti -growth effects of SSD.Flow -cytometer was used to check apoptosis and cell cycle.RT -PCR and Western -blot were used to check the pivotal elements in the regulation of G2 -Mcell cycle arrest.Results:SSD significantly inhibited the proliferation of SW480 rather than FHC(P <0.05).It had no impact on cell apoptosis(P >0.05),but significantly induced G2 -Mcell cycle arrest as determined by Flow -cytomete(P<0.05).SSD down -regulated the expression of CCNA1,CCNA2,CCNB1 and CCNB2 in mRNA level by RT -PCR (P <0.05).In protein level,CCNA2,CCNB1 and p34 /cdc2 were down -regulated,p -H3S10 was up -regulated, while another pivotal G2 -Mregulation element p21 WAF1 /CIP1 was up -regulated.Conclusion:SSD inhibits the prolifer-ation of colorectal cancer SW480 cell by up -regulating p21 WAF1 /CIP1 to induce G2 -Mcell cycle arrest.%目的：观察柴胡皂苷 D（SSD）对于结直肠癌肿瘤细胞增殖的影响并初步探索其潜在的分子机制。方法：运用 MTT、细胞计数试验及克隆形成试验观察 SSD 对细胞增殖的影响。流式细胞仪检测 SSD 对细胞凋亡及周期分布情况的影响。RT －PCR 和 Western －blot 技术检测 G2－M周期阻滞关键调控因子在 SW480细胞中的表达。结果：MTT 试验发现 SSD 能够显著抑制结直肠癌肿瘤细胞 SW480的增殖，而对正常结直肠细胞FHC 的增殖无明显影响。细胞计数试验和克隆形成试验进一步验证了 SSD 对于 SW480细胞增殖的影响（P＜0．05）。流式细胞术检测发现 SSD 不影响细胞的凋亡（P ＞0．05），但却显著诱导 G2－M 周期阻滞（P ＜0．05）。SSD 在 mRNA 水平下调了 G2－M周期调控因子 CCNA 1、CCNA2、CCNB 1和 CCNB2的表达（P ＜0．05）；在蛋白水平，CCNA2、CCNB1和 p34／cdc2明显被下调，p －H3S10上调，p21 WAF1／CIP1被明显上调。结论：SSD 可以通过上调 p21 WAF1／CIP1的表达诱导 G2－M周期阻滞来抑制结直肠肿瘤细胞 SW480的增殖。