分化抑制因子1促进血管内皮细胞增殖的机制研究  

Mechanism of differentiation inhibiting factor 1 in promoting proliferation of vascular endothelial cells

作　　者：孙荣距[1] 闫乐媛[1] 张建波[1] 果应菲[1] 袁晓玲[1] 党伟[1] 秦宇红[1] 张宪[1] 赵晓东[1] 

机构地区：[1]解放军总医院第一附属医院急救部,北京100048

出　　处：《解放军医学杂志》2010年第4期364-366,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金青年基金(30700869)

摘　　要：目的探讨分化抑制因子1(Id1)促进血管内皮细胞增殖的机制。方法构建p21启动子介导的报告基因p21-Luc,分别转染Eahy926细胞、Id1抑制(siId1细胞)或诱导表达(pcDNAId1)细胞、E2A抑制或诱导表达细胞,检测荧光素酶报告基因活性。提取Eahy926细胞总蛋白,分别加入Id1抗体、E12抗体和E47抗体,免疫共沉淀,以Eahy926细胞作为对照,Western blotting检测Id1、E12和E47的表达情况。分别转染siId1、pcDNAId1载体于Eahy926细胞,与正常Eahy926细胞同步化培养48h后,行细胞染色,用流式细胞仪检测细胞G1期的DNA含量。结果与Eahy926细胞比较,siId1细胞、pcDNAE2A细胞p21基因启动子介导的荧光素酶活性增强,p21蛋白表达增高(P<0.05,P<0.01);pcDNAId1细胞、siE2A细胞p21基因启动子荧光素酶活性降低,p21蛋白表达减弱(P<0.05)。免疫共沉淀结果显示,Id1免疫共沉淀细胞裂解物中同时检测到E47/E12蛋白,E47/E12沉淀物中也能检测到Id1。与正常Eahy926细胞比较,转染siId1的细胞G1期DNA含量增多,约为Eahy926细胞的3.3倍(P<0.05),细胞周期停滞,细胞增殖缓慢;而转染pcDNAE2A的细胞G1期DNA含量减少,约占正常的35.4%(P<0.05),细胞周期演进,细胞增殖加快。结论Id1可能通过与转录因子E47/E12作用,调节p21表达,进而促进血管内皮细胞增殖。Objective To explore the mechanism of differentiation inhibiting factor 1 in promoting proliferation of vascular endothelial cells （VECs）.Methods p21-luc,a luciferase reporter gene driven by p21 promoter,was constructed and transfected into Eahy926 cells,Id1 knock-down/over-expressing cell line （named siId1 cell/pcDNAId1 cell） and E2A knock-down/over-expression cells,respectively.Luciferase activity and the protein p21 were detected.Western blotting was performed,with Eahy926 cells as control,to detect the expression of Id1,E12 and E47 protein following the immunoprecipitation （IP） of Id1 with E12 and/or E47.DNA content was measured by flow cytometry after transfection with siId1 or pcDNA-Id1 and synchronization with normal Eahy926 cells for 48 hours.Results Compared with those in Eahy926 cells,Luciferase activity and p21 protein level increased remarkably in siId1 or pcDNA-E2A cells （P0.05,P0.01）,while decreased in pcDNA-Id1 or siE2A cells （P0.05）.IP results showed that E12 and E47 proteins could be detected in immunoprecipitates of Id1.Id1 could also be detected in precipitates of E47 or E12.DNA content in G1 phase of siId1 cells increased about 3.3 fold over that of normal Eahy926 cells （P0.05）.Cells showed a cycle arrest and slow proliferation.The DNA content in G1 phase of pcDNA-E2A cells was reduced to 35.4% of normal cells （P0.05） and with a progressive cell cycle and a promotion of cell proliferation.Conclusion Id1 may regulate p21 expression and promote the proliferation of VECs through interacting with transcription factor E2A （E12/E47）.

关 键 词：基因 Id1 基因 p21 转录因子E2A 细胞周期 细胞增殖 

分 类 号：R641.023[医药卫生—外科学]