Endostar体外稳定性研究

In vitro stability of Endostar

目的:比较重组人内皮抑素恩度TM(EndostarTM)和毕赤酵母表达的内皮抑素(endostatin)在不同温度及不同pH条件下的稳定性,通过检测对内皮细胞的增殖抑制比较Endostar在4℃和37℃下的生物活性。方法:将Endostar和endostatin于4℃保存或37℃放置96 h后进行不同条件的聚丙烯酰胺凝胶电泳,比较Endostar和endostatin之间以及它们在不同温度放置后的电泳行为,采用兔源多克隆抗体进行Western blot验证。同时检测在两种温度下的Endostar对于人脐静脉内皮细胞HUVEC细胞增殖的抑制作用。结果:在还原性电泳中Endostar和endostatin在20 kD左右处均只出现一条带。在3种酸性非还原电泳条件下,endostatin出现明显的"smear"现象,无法观察到主要条带,Endostar在酸性不连续非还原电泳条件下只出现一条带,而在两种连续非还原电泳中虽然也出现"smear"现象,但是主要条带仍然存在,Endostar和en-dostatin在37℃放置96 h后与它们保存在4℃的样品相比,在各种电泳条件下有相似的电泳行为,Western blot验证了相应电泳的结果。在两种温度下的Endostar对于HUVEC的增殖抑制作用基本相同。结论:Endostar和endostatin具有相似的热稳定性,但在酸性条件下Endostar较endostatin更为稳定。

Aim:To compare the stability of EndostarTM and endostatin under different temperatures and pH using polyacrylamide gel electrophoresis(PAGE) and Western blot and to compare the activity of Endostar kept at 4 ℃ and 37 ℃ by inhibition of endothelial cell proliferation.Methods:Endostar and endostatin expressed by Phicha pastoris were kept at 4 ℃,and 37 ℃ for 96 hours,respectively.The electrophoresis of the samples was detected by reduced and non-reduced PAGE.The results were further confirmed by Western blot w...