不同浓度葛根素对体外培养血管内皮细胞增殖的影响

Effects of different doses of puerarin on vascular endothelial cell proliferation in vitro

目的:观察不同浓度葛根素对体外培养血管内皮细胞增殖的影响。方法:取3只5d龄SD大鼠,分离心脏,在体外消化收集血管内皮细胞并进行培养,第3代细胞用于实验。除空白对照组外,血管内皮细胞分别于含0.01、0.1、1、10和100μmol/L葛根素的培养基中进行培养。24h后倒置显微镜下观察细胞形态并进行细胞计数,采用甲基噻唑基四唑比色法、免疫组织化学法和流式细胞仪检测血管内皮细胞的增殖情况。结果:与空白对照组比较,1、10和100μmol/L葛根素组血管内皮细胞细胞计数增加(P<0.05,P<0.01),增殖细胞核抗原阳性细胞比率及细胞分裂增殖指数增加(P<0.05,P<0.01)。结论:葛根素对体外培养的血管内皮细胞有明显的促进增殖作用,其作用与葛根素的剂量有关。

Objective： To explore the effects of different doses of puerarin on proliferation of cultured vascular endothelial cells in vitro. Methods： The hearts of three new-born SD rats 5 days old were mechanically minced and enzymatically digested with collagenase and trypsin, then vascular endothelial cells were counted, washed and resuspended in Dulbecco＇s minimum essential medium （DMEM） added with 20% heat inactivated fetal calf serum, then inoculated in 2% gelatin-coated tissue culture flasks. Vascular endothelial cells at passage 3 were used in the experiment. Except for the normal control group, the vascular endothelial cells were cultured with puerarin in various concentrations （0.01,0. 1, 1, 10 and 100 umol/L） for 24 hours, and the morphology and the number of the cultured endothelial cells were observed. Methyl thiazolyl tetrazolium （MTT） colorimetry was used to determine the proliferation of the cultured vascular endothelial cells. Flow cytometry （FCM） were used to detect the proliferation index （PI） of the vascular endothelial cells and the expression of proliferating cell nuclear antigen （PCNA） was detected by immunohistochemical method. Results： The cell morphology was normal under the inverted microscope in each group. The number and proliferation activities of vascular endothelial cells were significantly increased by 1, 10 and 100 umol/L puerarin compared with those of the blank control group, especially the 100 umol/L puerarin group, and there were no remarkable changes in with 0.1umol/L and 0.01 umol/L puerarin groups. The same results were seen in the positive rate of PCNA expression and PI.