人脐静脉内皮细胞增殖、迁移与和血生络方干预的影响

Effects of Hexue Shengluo recipe on the proliferation and migration of human umbilical vein endothelial cells

目的:和血生络方是在《内经》理论指导下,由人参养荣汤化裁而成,临床治疗缺血性心脏病效果良好。实验拟进一步观察和血生络方对人脐静脉内皮细胞增殖、迁移及细胞周期的影响,探讨其促血管生成作用。方法:实验于2006-11/2007-04在白求恩国际和平医院中心实验室进行。和血生络方所用中药黄芪、人参、熟地、当归、白芍、丹参、降香、红景天等购自石家庄乐仁堂药店。常规煎煮,离心去杂,浓缩为1kg/L的水煎液,分别通过1.20,0.45μm的微孔滤膜过滤,最后无菌条件下通过0.22μm的微孔滤膜过滤,密封分装,4℃冷藏备用。采用人脐静脉内皮细胞系ECV-304为模型。将ECV-304细胞贴壁培养24h后,分别换含0.1,0.5,1.0,2.0,4.0g/L和血生络方的RPMI-1640培养液培养,另设正常对照组。每组实验重复3次。采用四甲基偶氮唑盐法分别于药物作用24,48,72,96h检测人脐静脉内皮细胞增殖;流式细胞仪技术分析细胞DNA含量,计算细胞增殖指数;细胞划痕法检测细胞迁移。结果:①与正常对照组比较,和血生络方各浓度组(0.5～4.0mg/L)均可促进ECV-304细胞增殖(P<0.05,P<0.01),且呈剂量依赖性。②正常对照组及和血生络方固定剂量(2g/L)随作用时间的延长,内皮细胞逐渐增殖,72h达到峰值;不同时间点和血生络方组吸光度值均高于正常对照组(P<0.01)。③正常培养的ECV-304细胞周期中G0/G1期、S期及G2/M期细胞分布比例正常;和血生络方组(2g/L)G0/G1期细胞百分率较正常对照组下降(P<0.01),S期细胞百分率增高(P<0.01),而M期无明显变化。④正常对照组ECV-304细胞6h迁移率为17.49%,24h迁移率为41.32%,不同时间点和血生络方各浓度组(0.5～4.0g/L)细胞迁移率均高于正常对照组(P<0.05,P<0.01),且具有剂量依赖效应。结论:和血生络方能够促进人脐静脉内皮细胞增殖、迁移及DNA合成,提示具有促血管生成作用,可能是其防治缺血性心脏病的作用机制之一。

AIM： Hexue Shengluo recipe is revised from Renshen Yangrong Tang according to the Inner Canon of Yellow Emperor, and it has good effects in the treatment of ischemic heart disease. This study observes the effects of Hexue Shengluo recipe on the proliferation, migration and cell cycle of human umbilicus vein endothelial cells, and explores its activity of pro-angiogenesis. METHODS： Experiments were conducted at the Center Laboratory of the Bethune International Peace Hospital between November 2006 and April 2007. Hexue Shengluo recipe was composed of astragalus mongholicus, ginseng, prepared rhizome of rehmannia, angelica, paeoniae radix, salvia mUtiorrhiza, dalbergia odorifera and integripetal rhodiola herb, which were bought from the Le-ren-tang medicamentarius of Shijiazhuang, The Hexue Shengluo recipe was made into 1 kg/L liquid after boiled, centrifuged and filtrated with 1.20,0.45 μm filtering membrane, sterilely filtrated with 0.22 μm filtering membrane, and then sealed up and stored at 4 ℃ for use. Human umbilical vein endothelial cell line ECV-304 was used as the model. After 24-hour adherent culture, ECV-304 cells were treated with RPMI-1640 culture fluid containing 0.1, 0.5, 1.0, 2,0 and 4.0 g/L Chinese medicine as Hexue Shengluo recipe groups. A normal control group was normal control group. Every experiment was performed three times in each group. The proliferation of human umbilical vein endothelial cells was measured by MTT assay at hours 24, 48, 72 and 96. Flow cytometry was used to detect cell DNA content and calculate cell proliferation index. The cell migration was observed by scratch wound assay. RESULTS. （1）Compared with the normal control group, ECV-304 cell proliferation was significantly increased in Hexue Shengluo recipe groups （0.5-4.0 mg/L） in a dose-dependent manner （P 〈 0.05 or P 〈 0.01）. （2）The cell proliferation was promoted in the normal control group and the Hexue Shengluo recipe group （2 g/L）, reaching a peak at hour 72. Absorbance was higher in different time in the Hexue Shengluo recipe groups than the normal control group （P 〈 0.01）. （3）ECV-304 cells were commonly distributed in the G0/G1 phase, S phase and G2/M phase. The cell percentage in the Hexue Shengluo recipe group （2 g/L） in G0/G1 phase was reduced as compared with the normal control group （P 〈 0.01 ）, increased in the S phase （P 〈 0.01）, and no changes were found in the M phase. ④Cell migration rate was 17.49% at hour 6 and 41.32% at hour 24 in the normal control group. The cell migration rate was higher in the Hexue Shengluo recipe groups （0.5-4.0 g/L） than in the normal control group （P 〈 0.05 or P 〈 0.01） in a dose-dependent manner. CONCLUSION： Hexue Shengluo recipe can promote the proliferation, migration and DNA synthesis of human umbilical vein endothelial cells. It shows that the recipe has a pro-angiogenesis activity, and it may be one of the mechanisms in preventing and treating ischemic heart disease.