化痰祛瘀方含药血清对肝癌细胞血管内皮生长因子的调控作用研究

Regulation of Vascular Endothelial Growth Factor Expression in Hepatoma Carcinoma Cells with Serum Containing Phlegm-resolving and Stasis-removing Herbal Medicine

目的:观察化痰祛瘀方含药血清对肝癌细胞血管内皮生长因子(VEGF)表达的影响。方法:健康SD大鼠15只,随机分为3组,大剂量组:化痰祛瘀方大剂量含药血清组,灌服化痰祛瘀方,给药剂量24g/kg(相当于临床等效剂量2倍);小剂量组:化痰祛瘀方小剂量含药血清组,灌服化痰祛瘀方,给药剂量12g/kg;正常血清组:灌服相应体积的生理盐水,共计5mL。通过MTT法检测化痰祛瘀方含药血清对肝癌细胞株增殖的抑制作用;通过Western bolt技术、免疫荧光技术观察VEGF蛋白表达的变化。结果:化痰祛瘀方含药血清能明显抑制人肝癌细胞株HCCLM3的增殖活力,不同剂量时(0g/kg、12g/kg、24g/kg)抑制率分别为1.60%、36.64%、63.92%,差异均有显著性意义(P<0.01);化痰祛瘀方含药血清能使肝癌细胞内的VEGF蛋白表达量下降,且随着化痰祛瘀方含药血清剂量增高(12g/kg、24g/kg),VEGF的蛋白表达呈下降趋势;免疫荧光试验显示,大剂量化痰祛瘀含药血清(24g/kg)可以完全抑制VEGF蛋白的表达。结论:化痰祛瘀方含药血清通过抑制VEGF的表达从而抑制肝癌细胞的生长。

Objective. To study the effect of serum containing phlegm-resolving and stasis-removing herbal medicine on the expression of vascular endotherial growth factor （VEGF） in hepatoma carcinoma cells. Methods- Fifteen health SD rats were equally randomized into 3 groups, blank control group （given 5 mL of normal saline）, and large- and small-dose herbal medicine groups （in the dosages of 24, 12g/kg, respectively）. Hepatoma carcinoma cell line HCCLM3 was co-cultured with serum from rats of the corresponding groups, and then the proliferation of hepatoma carcinoma cells was detected by M-l-r, and the VEGF protein expression was examined by Western blot and immunofluorescence technique. Results. Serum con- taining phlegm-resolving and stasis-removing herbal medicine significantly inhibited the proliferation activity of HCCLM3, and the inhibitive rate of serum containing 0g/kg, 12g/kg, 24g/kg of herbal medicine was 1.60%, 36.64%, 63.92%, respectively, the difference being statistically significant among the three groups（P 〈 0.01）. Serum containing herbal medicine had an effect on decreasing VEGF protein expression level, and the effect was dose-dependant. The results of immunofluorescence test showed that serum containing large-dose herbal medicine completely inhibited VEGF protein expression. Conclusion. Serum containing phlegm-resolving and stasis-removing herbal medicine can suppress the growth of hepatoma carcinoma cells through inhibiting the expression of VEGF.