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目的:观察高效液相色谱(HPLC)监测制备的左归丸含药血清对人脐静脉血管内皮细胞(HUVEC)增殖及血管生成体外三维模型的影响。方法:左归丸灌胃大鼠后,于不同时间点制备血清,HPLC监测下,选取药物成分峰值最大时间点的含药血清进行体外实验。噻唑蓝(MTT)法观察左归丸含药血清对HUVEC增殖的影响,I型鼠尾胶原表面培养法观察左归丸含药血清对三维模型管样结构的影响。酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测雌二醇E2(estradiol)、血管内皮生长因子VEGF(vascular endothelial growth factor)水平。结果:左归丸灌胃后2h可达最高药物吸收峰值,左归丸11g/kg(72h),左归丸33g/kg(24、48、72h)对HUVEC均有明显的增殖作用。左归丸11g/kg、左归丸33g/kg均可明显的增加内皮小管的面积、长度、管径及生成指数。左归丸11g/kg、左归丸33g/kg均可明显的提高大鼠血清E2、VEGF水平。结论:HPLC监测下制备的左归丸含药血清能够促进血管内皮细胞的增殖,增大小管的面积、长度、管径及成管指数,提高血清E2、VEGF水平,从而促进血管生成。
OBJECTIVE: To observe the effect of Zuogui pill-containing serum prepared by high performance liquid chromatography (HPLC) on proliferation and angiogenesis in vitro of three-dimensional models of human umbilical vein endothelial cells (HUVECs). Methods: Zuogui pill was intragastrically administered to rats and serum was prepared at different time points. Under the condition of HPLC monitoring, the drug-containing serum with the peak time of drug composition was selected for in vitro experiments. The effect of Zuogui pill-containing serum on the proliferation of HUVEC was observed by MTT assay. The effect of Zuogui pill-containing serum on the tube-like structure of three-dimensional model was observed by T-collagen surface method. Enzyme linked immunosorbent assay (ELISA) was used to detect estradiol and vascular endothelial growth factor (VEGF) levels. Results: The highest absorption peak of Zuogui pill was reached at 2h after oral administration. Zuogui pill 11g / kg (72h) and Zuogui pill 33g / kg (24,48,72h) had obvious proliferative effects on HUVEC. Zuogui Wan 11g / kg, Zuogui Wan 33g / kg can significantly increase the area of endothelial tubules, length, diameter and the formation of index. Zuogui pill 11g / kg, Zuogui pill 33g / kg can significantly improve serum E2, VEGF levels. CONCLUSION: Zuogui pill-containing serum prepared by HPLC can promote the proliferation of vascular endothelial cells and increase the area, length, diameter and tube formation index of the tubules, increase the levels of serum E2 and VEGF, and promote angiogenesis.