摘要
目的 探讨睡眠因子1（Slfn1）对内皮祖细胞（EPC）迁移的影响及其机制.方法 实验分为大鼠Slfn1腺病毒载体组（Ad-Slfn1组）、腺病毒阴性对照组（Ad-对照组）、大鼠发夹状RNA干扰Slfn1组（ShRNA-Slfn1组）、发夹状RNA阴性对照组（ShRNA-对照组）、空白对照组（未给予任何处理的内皮祖细胞）.分离培养大鼠骨髓源性EPC,用构建好的大鼠Slfn1腺病毒载体、发夹状RNA干扰Slfn1质粒及相应的对照质粒分别转染EPC,采用逆转录聚合酶链反应和蛋白印迹法检测细胞Slfn1、Cyclin D1的mRNA和蛋白表达水平,Boyden小室检测细胞迁移能力,流式细胞技术检测细胞周期.结果 （1）质粒转染效率的检测结果：Ad-Slfn1组EPC中Slfn1mRNA表达水平高于Ad-对照组（P＜0.05）,Slfn1蛋白表达水平亦高于Ad-对照组（P＜0.05）.而ShRNA-Slfn1组EPC中Slfn1mRNA表达水平低于ShRNA-对照组（P＜0.05）,Slfn1蛋白表达水平亦低于ShRNA-对照组（P＜0.05）.（2）EPC迁移能力的检测结果：Ad-Slfn1组EPC迁移数目少于Ad-对照组（P＜0.05）.而ShRNA-Slfn1组EPC迁移数目多于ShRNA-对照组（P＜0.05）.（3） EPC细胞周期分布情况的检测结果：Ad-Slfn1组EPC分布于G1期的数目多于Ad-对照组（P＜0.05）,而分布于S期的数目低于Ad-对照组（P＜0.05）.而ShRNA-Slfn1组EPC分布于G1期的数目少于ShRNA-对照组（P＜0.05）,分布于S期的数目则多于ShRNA-对照组（P＜0.05）.（4） Cyclin D1 mRNA和蛋白表达水平的检测结果：Ad-Slfn1组Cyclin D1mRNA表达低于Ad-对照组（P＜0.05）,Cyclin D1蛋白表达水平亦低于Ad-对照组（P＜0.05）.而ShRNA-Slfn1组mRNA表达水平高于ShRNA-对照组（P＜0.05）,Cyclin D1蛋白表达水平亦高于ShRNA-对照组（P＜0.05）.结论 Slfn1可通过下游靶点Cyclin D1负性调控内皮祖细胞的迁移能力.
Objective To explore the effect of Schlafen 1 （Slfn1） on the migration of endothelial progenitor cells （EPCs）.Methods Rat bone marrow derived EPCs were isolated and cultured.Ad-Slfn1,ShRNA-Slfn1,ShRNA-control and Ad-control were transfected into EPCs respectively.The mRNA expression of Slfn1 and Cyclin D1 was examined by reverse transcriptase-PCR,and their protein expression was detected by Western blot.The migration of EPCs was examined by a modified Boyden chamber assay.EPCs cell cycle was determined using flow cytometry analysis.Results Forty-eight hours after ShRNA-Slfn1 transfection,the mRNA and protein expression of Slfn1 in EPCs was significantly down-regulated compared to ShRNA-control EPCs （P ＜ 0.05）.Transfection of Ad-Slfn1 reversed these changes.Overexpression of Slfn1 reduced the migration capacity of EPCs while the silencing of Slfn1 by shRNA-Slfn1 increased the migration capacity of EPCs.In addition,cell cycle was arrested at G1 phase in Slfn1 overexpression group while transfection of shRNA-Slfn1 reversed these responses.Interestingly,the mRNA and protein expression of Cyclin D1 was significantly up-regulated after shRNA-Slfn1 transfection compared to ShRNA-control group （all P ＜ 0.05）,but overexpression of Slfn1 reversed these results,suggesting Cyclin D1 was involved in regulating EPCs cell cycle via Slfn1 signaling.Conclusions Slfn1 could reduce the migration capacity of EPCs via Cyclin D1 pathway.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2014年第11期951-956,共6页
Chinese Journal of Cardiology
基金
国家自然科学基金（81360034）
贵州省科技厅-贵州省人民医院联合基金[黔科合LS字（2011）024号]
