水溶性乌梢蛇总蛋白的提取方法及细胞毒性检测  

Total soluble protein extraction from Zaocys Dhumnades and its cytotoxicity detection

作　　者：吴贺勇[1] 李娟[2] 李亚玲[2] 

机构地区：[1]南方医科大学北医院中西医结合科,广东省佛山市528311 [2]南方医科大学南方医院风湿科,广东省广州市510515

出　　处：《中国组织工程研究与临床康复》2010年第50期9403-9406,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金面上项目(30572457)~~

摘　　要：背景:寻找分析乌梢蛇的具体活性成分,需对乌梢蛇总蛋白进行提取和分析,但目前有关乌梢蛇蛋白的提取方法甚少。目的:建立提取水溶性乌梢蛇总蛋白的方法,检验水溶性乌梢蛇总蛋白体外细胞毒性。方法:应用酶裂解与盐析相结合的方法提取乌梢蛇总蛋白,采用Bradford蛋白浓度测定试剂盒检测盐析前后所得的蛋白浓度,使用SDS-PAGE分离蛋白,应用图像分析软件分析QuantityOne4.4比较分析其区带差异。分别用盐析后所得的不同浓度的蛋白(0.5,5,50,150,450和900mg/L)和蛋白提取液作用于成纤维样滑膜细胞,四甲基偶氮唑蓝法测定各组成纤维样滑膜细胞增殖率。结果与结论:水溶性总蛋白浓度与盐析总蛋白浓度比较差异无显著性意义(P>0.05);两种方法所提取的水溶性乌梢蛇总蛋白差异无显著性;提取液组和盐析后所得的蛋白质的150和450mg/L组均可以抑制成纤维样滑膜细胞的增殖;而空白对照组和酶裂解与盐析后所得蛋白的0.5,5和50mg/L对细胞的增殖作用无影响。提示酶裂解和盐析结合的方法可提取高质量低毒的水溶性乌梢蛇总蛋白,所提取蛋白包含了乌梢蛇的特征性蛋白,适合于体外细胞模型的研究。BACKGROUND：Total soluble protein of Zaocys Dhumnades should be extracted and analyzed for searching active component.However,methods for extracting total protein from Zaocys Dhumnades are few.OBJECTIVE：To establish the method for extracting total soluble protein from Zaocys Dhumnades and observing its in vitro cytotoxicity.METHODS：Schizolysis enzyme combined with salting-out method were used to extract the total soluble protein of Zaocys Dhumnades,and then the protein level was measured by Bradford and separated by SDS-PAGE.The band sizes and their differences were analyzed with quantity one 4.4.The liquid for extracting protein and different concentrations（0.5,5,50,150,450 and 900 mg/L） of total soluble protein of Zaocys Dhumnades purified by（NH4）2SO4,were used to culture fibroblast-like synoviocytes.The cell proliferation was analyzed by MTT.RESULTS AND CONCLUSION：There was no significant differences between extraction amount of total soluble protein without and with being purified by（NH4）2SO4（P 0.05）.The liquid for extracting protein and total soluble protein of Zaocys Dhumnades in concentrations of 150,450 mg/L significantly inhibited fibroblast-like synoviocytes proliferation;while total soluble protein of Zaocys Dhumnades in concentrations of 0.5,5 and 50 mg/L had no effect on the fibroblast-like synoviocytes proliferation in the control group.The findings demonstrated that,proteins of Zaocys Dhumnade can be extracted effectively with low cytotoxicity using schizolysis enzyme combined with salting-out method,which suitable for in vitro cell study.

关 键 词：乌梢蛇总蛋白 提取 MTT法 细胞毒性 类风湿性关节炎 

分 类 号：R318[医药卫生—生物医学工程]