明胶酶谱实验方法的优化  

An Improved Gelatin Zymography Method

作　　者：章淑平[1,2] 骆建民[2] 王俐[1] 

机构地区：[1]汕头大学医学院第一附属医院,广东汕头515041 [2]汕头大学医学院病理生理教研室,广东汕头515041

出　　处：《生物技术通讯》2014年第1期102-104,共3页Letters in Biotechnology

基　　金：国家自然科学基金(81272115)

摘　　要：目的:优化明胶酶谱实验方法,并检测不同浓度凝血酶刺激人皮肤成纤维细胞分泌的明胶酶B(MMP-9)活性的变化。方法:用含明胶的聚丙烯酰胺凝胶电泳分开上清中各蛋白条带,经2.5%Triton X-100洗脱、明胶酶缓冲液孵育、考马斯亮蓝染色液染色及脱色液脱色后,经光密度扫描记录,应用ImageJ软件进行密度计量分析。结果:凝血酶刺激人皮肤成纤维细胞分泌MMP-9具有浓度依赖性。结论:利用优化的明胶酶谱实验方法可做出高质量的明胶酶谱图片,对检测明胶酶活性有重要意义。Objective： To determine the MMP-9 activity in human primary dermal fibroblast（HDF） stimulated by thrombin with the modified gelatin zymography. Methods： The culture medium of HDF was collected stimulat ed by different dosage of thrombin. The medium was separated by native polyacrylamide gel containing gelatin and incubated with substrate buffer. The MMP-9 digests its substrate of gelatin and shows clear in gel by staining with Coomassie blue. The clear bands were measured and quatified with Scionlmage program software. Results： Thrombin provoking the expression of MMP-9 from primary cultured HDF is in a concentration-dependent man ner. Conclusion： The results indicated that the improved method of gelatin zymography can acquire the high quali ty images, which is effective to detect metal protease activity.

关 键 词：明胶酶谱 基质金属蛋白酶 聚丙烯酰胺凝胶 

分 类 号：R965[医药卫生—药理学]