白头翁醇提物抑制血管生成的体外实验研究

Inhibitory effect of the alcohol extractive of pulsatillae chinensis regel on angiogenesis in vitro

目的探讨白头翁醇提物对体外血管生成的抑制作用及其可能机制。方法采用MTT法观察白头翁醇提物对人脐静脉血管内皮细胞(HUVEC)和人肠癌LoVo细胞增殖的影响;通过Transwell小室趋化实验、体外小管形成实验观察白头翁醇提物对HUVEC迁移、形成血管能力的影响;采用流式细胞仪检测白头翁醇提物对HUVEC的凋亡率及细胞周期的影响。结果 2～8μg/ml的白头翁醇提物作用48h对HUVEC的增殖抑制率为21.6%～72.0%,对LoVo细胞的增殖抑制率为13.2%～22.9%;体外小管形成实验发现,2～8μg/ml白头翁醇提物作用24h,HUVEC小管形成数目减少,且管腔不完整,与对照组比较差异有统计学意义(P<0.001)。经2～8μg/ml白头翁醇提物处理12h,HUVEC迁移数明显少于对照组(P<0.001)。2、4、8μg/ml白头翁醇提物作用于HUVEC细胞48h后,其凋亡率分别为9.52%、18.64%和20.07%,对照组为6.25%。8μg/ml白头翁醇提物作用于HUVEC细胞24h后,细胞周期停滞在G2/M期。结论白头翁醇提物在体外能有效抑制血管生成,其机制可能与抑制HUVEC增殖、迁移和小管形成,诱导HUVEC凋亡,抑制HUVEC有丝分裂有关。

Objective To investigate the inhibitory effect and mechanism of the alcohol extractive of pulsatillae chinensis re- gel on angiogenesis in vitro. Methods MTF assay was used to observe the effect of the alcohol extractive of pulsatillae chinensis regel on the proliferation of human umbilical vein endothelial cells （HUVECs） and human colorectal carcinoma LoVo cells. Transwell migra- tion assay and tube formation assay were used to observe the impact of the alcohol extractive of pulsatillae chinensis regel on cell migra- tion and tube forming ability of HUVECs. The apoptosis and cell cycle phases were examined by flow cytometery. Results The alco- hol extractive of pulsatillae chinensis regel （2-8t^g/ml） inhibited the proliferation of HUVECs （21.6%-72. 0% ） and LoVo cells （ 13.2% -22. 9% ） after 48h treatment. When HUVECs were incubated with 2-8μg/ml alcohol extractive of pulsatillae chinensis regel, the number of tubules was reduced and the lumen lost its integrity after 24h ; compared with the control group, the number of migrating HUVECs was decreased after 12h （ P 〈 0. 001 ）. After treatment with the alcohol extractive of pulsatillae chinensis regel at the dosage of 2,4,8μg/ml for 48h, the apoptotic rate of HUVEC were 9. 52%, 18.64% and 20.07%, respectively. The apoptotic rate was 6. 25% in the control group. After treatment with the alcohol extractive of pulsatillae chinensis regel at the dosage of 8μg/ml for 24h, the percentage of HUVEC was at G2/M phase. Conclusion The alcohol extractive of pulsatillae chinensis regel effectively suppresses angiogenesis in vitro, which may be through inhibiting cell proliferation, migration and tube-formation, inducing apoptosis and sup- pressing DNA synthesis of HUVECs.