摘要
目的：纯化大肠杆菌表达的重组人血管抑素（ｒｈＡＧＮ）并鉴定其抗血管生成生物活性。方法：通过超声破碎、包涵体洗涤、Ｓｅｐｈａｄｅｘ Ｇ－１００ 凝胶过滤层析等步骤初步纯化ｒｈＡＧＮ，Ｌｏｗｒｙ法测定蛋白浓度，ＳＤＳ－ＰＡＧＥ分析观察其纯度；应用 ＭＴＴ法观察在终浓度为 ０．１～３．０ ｍｇ／Ｌ范围内 ｒｈＡＧＮ对 １０ μｇ／Ｌ ＥＧＦ刺激的人真皮微血管内皮细胞系ＨＤＭＥＣ的增殖抑制活性；以ＰＢＳ为对照（ｎ＝５），应用原位鸡胚绒毛尿囊膜（ＣＡＭ）技术观察１００ μｇ／只（ｎ＝５）和２００ μｇ／只（ｎ＝５）ｒｈＡＧＮ作用７２ ｈ对３０ｎｇ ｂＦＧＦ 诱导的鸡胚ＣＡＭ毛细血管生成的抑制作用。结果：①ｒｈＡＧＮ电泳纯度达到 ９６％，总回收率为 ６１．７％；②ｒｈＡＧＮ对 ＨＤＭＥＣ细胞增殖的最大抑制剂量约为 ２．０ ｍｇ／Ｌ，抑制率约９２．３％．半数最大抑制剂量在１．０ ｍｇ／Ｌ附近；③１００ μｇ／只和２００ μｇ／只ｒｈＡＧＮ组鸡胚ＣＡＭ 载样滤纸片下毛细血管数目分别为７９±２３条和３７±１１条，两组间比较差异显著（Ｐ＜０．０５）；与对照组（１４５±３６条）相比，均有显著性差异（Ｐ＜０．０１）。结论：ｒｈＡＧＮ达到了较高的电泳纯度，
Objective: To purify the recombinant human angiostatin (rhAGN) produced by engineered E. coli and to determine its antiangiogenic activity. Methods: rhAGN was purified with ultrasonication and Sephadex G-100 gel filtration. Lowry' S method was used to determine the protein concentration. The purity of rhAGN was analyzed by SDS-PAGE and photodensitometry scanning. MTT method was applied in the determination of the effect of rhAGN on the proliferation of human dermal microvascular endothelial cell line HDMEC. Modified CAM technique was used to determine the inhibitory effect of rhAGN on angiogenesis in 6-day-old chicken CAM induced by 30ng bFGF. Results: (1) The purity of rhAGN was 96%, with total recovery of 61. 7%. (2) Within the concentration range of 0. 1 ～3. 0 mg/L, rhAGN inhibited the proliferation of HDMEC stimulated by 10 μg/L EGF in a dose-dependent style. The maximum inhibitory dose was approximately 2. 0 mg/L with the inhibition rate of 92. 3%, and the half- maximum inhibition dose was around 1. 0 mg/L; (3 ) When administration of rhAGN at 100 μg/embryo and 200 μg/embryo, the amount of capillary microvasculars of chicken CAM under the testing disc were 79 ± 23(n= 5 ) and 37 ± 11 (n = 5 ) respectively and both were significantly less than that of the PBS control group(145±36, n= 5) (P＜0. 01 ). Conclusion: rhAGN expressed by E. coli was highly purified. It was demonstrated that the purified rhAGN had the inhibitory effect on endothelial cell proliferation and had antiangiogenic activity.
出处
《广州医学院学报》
2001年第2期5-8,共4页
Academic Journal of Guangzhou Medical College
基金
国家自然科学基金(39670013)
广东省自然科学基金(960052)资助项目。
