arresten在CHO细胞的表达及其生物学效应

目的 初步探讨血管生成抑制因子arresten在中国仓鼠卵巢细胞（CHO）中的表达及其生物学效应。方法 用脂质体转染法将真核表达载体pSecTag-arresten导入CHO,抗生素Zeocin筛选获得阳性克隆。RT—PCR检测arresten在mRNA水平的表达,Western blot检测arresten在蛋白水平的表达。将人脐静脉内皮细胞（huvec）分为加DMEM培养组、加SFX无血清培养基培养组及加含arreten的上清培养液组,TransweH小室检测arresten对huvec细胞迁移的影响,Mamgel胶体外管腔生成试验检测arresten对huvec细胞血管形成能力的影响。结果 RT—PCR和Western blot检测显示,arresten在mRNA水平与蛋白水平均有表达。迁移抑制曲线显示,含arresten组的迁移抑制率明显高于对照组（P〈0．05）。加DMEM培养组、加SFX无血清培养基培养组和加含arresten的上清培养液组细胞形成的血管腔分别为16±2、13±1和7±2个,加含arresten的上清培养液组显著低于其他两组（P〈0．05）。提示arresten对huvec细胞的迁移和血管形成具有抑制作用。结论成功构建arresten的真核表达体系,并发现arresten因子可抑制huvec细胞迁移和管腔形成,这可能是其抑制血管生成的途径之一。

Expression and biological activities of arresten in CHO cells

Objective To explore the eukaryotic expression of arresten in CHO cells and to investigate its basic biological activities.Methods CHO cells were divided into three groups:transfected pSecTag-arresten group,transfected pSecTag group and control group without transfection.PSecTag-arresten was transfected into CHO cells by Lipofectamine 2000 method.The arresten mRNA in CHO cells was assayed by RT-PCR.The protein expression of arresten gene was examined by Western-Blot.The cells expressing arresten were screened out by Zeocin.The effect of arresten on huvec cell migration and anchoring to three- dimensional vascular structures was measured.Results The result of RT-PCR and Western-blot showed that arresten gene has been successfully transfected into CHO cells and expressed in those cells.Arrssten inhibited huvec cell migration and anchoring to three-dimensional vascular structures.Conclusion CHO cells expressing arresten have been obtained successfully.Arresten can inhibit huvec cell migration and anchoring to three-dimensional vascular structures,indicating that it might be one of its anti-angioganetic approaches.