血管生成抑素endostatin的表达、纯化及活性研究

Study on cloning, expression and biological activity of vascular endothelial cell growth inhibitor: endostatin

目的 :将从人胎肝组织克隆的血管生成抑素 endostatin基因进行原核表达、纯化 ,检测重组 endostatin的生物学活性。 方法 :利用原核表达载体 p BV 2 2 0在大肠杆菌 DH5α中表达 endostatin,利用肝素 Sepharose亲和层析及 Sephacryl S- 2 0 0分子筛纯化 ;通过体外内皮细胞 (ECV30 4)增殖实验及体内鸡胚尿囊膜 (CAM)新生血管实验检测其抑制活性。 结果 :Endo-statin在 DH5α中的表达率为 2 8.5 % ,纯化后纯度可达 90 .5 %。 Endostatin可明显抑制体外培养的内皮细胞增殖 ,IC5 0 为 72μg/ m l;2 0μg/ ml时使细胞于 48h发生明显凋亡 ;2 0 0μg/ ml的 endostatin可使 CAM新生血管化率下降 30 %。结论 :研究结果表明 endostatin对内皮细胞具有明显抑制作用 。

Objective: To investigate the biological activity of recombinant vascular endothelial growth inhibitor, endostatin from Chinese fetus hepatocytes. Methods: Endostatin was expressed in E.coli with pBV220 expression vector and purified with heparin Sepharose CL 6B and Sephacryl S 200. The biological activity of endostatin was detected with endothelial cells proliferation test in vitro and CAM vascular inhibition test. Results: The expression rate of endostatin in recombinant E.coli was 28.5% and its purity reached 90.5% after purification. Purified endostatin showed significant inhibitory effect on endothelial cells, which IC 50 was 72 μg/ml. At the concentration of 20 μg/ml, endostatin induced apoptosis of endothelial cells within 48 h. Endostatin also inhibited angiogenesis in CAM. At the concentration of 200 μg/ml, the vascular index was degraded by 30%. Conclusion: The results suggest that endostatin is an effective inhibitor of endothelial cells and has potential therapeutic usage on angiogenic disease and cancer.