摘要
目的:探讨Krüppel样因子4(Krüppel-like factor4,KLF4)对热应激所致Raw264.7巨噬细胞凋亡的影响。方法:构建pcDNA3.1-KLF4真核表达质粒,采用脂质体转染法建立KLF4过表达Raw264.7巨噬细胞株,用免疫印迹法检测KLF4的过表达,采用热应激(42℃)处理稳定表达小鼠KLF4基因的Raw264.7巨噬细胞1h,37℃恢复12h,采用流式细胞术、Hoechest33258染色及DNA琼脂糖凝胶电泳检测细胞凋亡。结果:获得了KLF4过表达的Raw264.7细胞株,流式细胞术结果显示,转空载体组和KLF4过表达组细胞凋亡百分率较热应激前均升高,但与转空载体组相比较KLF4过表达组升高更明显;荧光染色可见细胞出现核固缩、凋亡小体等凋亡形态学改变;KLF4过表达细胞琼脂糖凝胶电泳能检测到明显的DNA"梯状条带"。结论:KLF4可以促进热应激所致Raw264.7巨噬细胞凋亡。
Objective To observe the effect of Krppel-like factor 4（KLF4）overexpression on heat stress-induced apoptosis of Raw264.7 macrophages.Methods The fragment containing full length mouse KLF4 cDNA coding sequence was inserted into the pcDNA3.1 vector and Raw264.7 macrophages were transfected with pcDNA3.1-KLF4 plasmids using lipofectamine.The expression of KLF4 was examined by Western blot in the Raw264.7 macrophages stably transfected with pcDNA3.1-KLF4 plasmids.Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-KLF4 were exposed to heat stress（42 ℃）for 1h and recovered at 37 ℃ for 12 h.Flow cytometry,Hoechest 33258 staining assay,and DNA ladder assays were performed to assess the apoptosis.Results The KLF4 overexpressed Raw264.7 macrophages were established.After the heat stress,flow cytometry showed that apoptotic cells increased significantly in KLF4 overexpressed cells compared with the vector control;Hoechest 33258 staining was characterized with classical changes including apoptotic body,and nuclear condensation.Agarose gel electrophoresis showed that ＂DNA ladder＂ could be observed clearly.Conclusion KLF4 overexpression can increase heat stress-induced apoptosis of Raw264.7 macrophages.
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2007年第6期1002-1006,共5页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(30400169
30470851)~~
