靶向血管生成素-2基因短发夹RNA真核表达载体的构建及内质网应激状态下对人脐静脉内皮细胞凋亡影响的观察

	靶向血管生成素-2基因短发夹RNA真核表达载体的构建及内质网应激状态下对人脐静脉内皮细胞凋亡影响的观察
	何泉 1; 徐盈[*]1; 唐新华 2; 刘衡 3; 朱宝生 2; 蒋绿芝 4
刊名	中国糖尿病杂志
	2015
卷号	23 期号:2 页码:158-164
关键词	基因血管生成素-2 内质网应激 RNA干扰表达载体短发夹RNA 细胞凋亡人脐静脉内皮细胞
其他题名	Construction of shRNA eukaryotic expression vectors targeting angiopoietin-2 gene and effects of angiopoietin-2 gene knockdown on apoptosis in endoplasmic reticulum stress-induced human umbilical vein endothelial cell
通讯作者	xuying13669757616@163.com
合作状况	其它
中文摘要	目的构建人血管生成素-2(Ang-2)短发夹RNA(shRNA)真核表达质粒,观察其在内质网应激(ERS)状态下对人脐静脉内皮细胞(HUVECs)增殖和凋亡的影响,探讨Ang-2基因在ERS状态下介导血管内皮细胞凋亡的作用和机制。方法采用DNA重组技术根据人Ang-2mRNA序列设计并合成2条shRNA寡核苷酸片段,构建表达质粒并通过脂质体介导转染HUVECs,对Ang-2基因进行RNA沉默,采用RT-PCR和Western blot分别检测Ang-2在ERS状态下mRNA及蛋白质的表达。结果经双酶切和测序鉴定证实,2个shRNA表达载体均构建成功。与空白对照、阴性对照组相比,Ang2-1-shRNA、Ang2-2-shRNA组HUVECs中Ang-2基因mRNA及蛋白表达水平均降低,其中,Ang2-1-shRNA干扰效果最强。Ang-2mRNA和蛋白的抑制率分别达79.29%、68.21%。沉默Ang-2基因可增加ERS状态下HUVECs细胞活力及抑制其凋亡。结论成功构建靶向Ang-2基因的shRNA真核表达载体,Ang-2基因沉默对ERS状态下HUVECs凋亡有抑制作用。Ang-2基因可抑制血管内皮细胞体外增殖,增强ERS状态下HUVECs凋亡。
英文摘要	Objective To construct short hairpin RNA（shRNA）eukaryotic expression plasmid aiming to induce human angiopoietin-2（Ang-2）gene silencing,and to observe its effect on proliferation and apoptosis of human umbilical vein endothelial cells（HUVECs）in the endoplasmic reticulum stress（ERS）,finally to study the role and the mechanism of Ang-2 gene in the ERS-mediated apoptosis of vascular endothelial cells. Methods Two shRNA oligonucleotides according to the sequence of human Ang-2 mRNA were designed and synthesized by recombinant DNA techniques,and used to construct the expression plasmid,which were mediated by liposome to transfect HUVECs for Ang-2 gene silencing.RTPCR and western blot were employed to detect the mRNA and protein expression of Ang-2 under the ERS respectively. Results Two shRNA expression vectors were constructed successfully and identified by double-enzyme digestion and sequencing.Compared with the blank control group and the negative control group,in the Ang2-1-shRNA and Ang2-2-shRNA group the HUVECs with mRNA and protein level decreased significantly,especially by Ang2-1-shRNA with the strongest interference effect.The inhibition rate of Ang-2mRNA and protein reached 78.32% and 68.21%,respectively.The MTT assay and flow cytometry results showed that under the ERS state,cell viability of HUVECs has been improved,apoptosis has been depressed obviously by Ang-2 gene silencing. Conclusion The eukaryotic expression vector targeting shRNA Ang-2gene is constructed successfully.Ang-2 gene silencing inhibits the apoptosis of HUVECs under the condition of ERS.Ang-2 gene can inhibit the proliferation of vascular endothelial cells in vitro,and enhance the apoptosis of HUVECs under ERS.
收录类别	其他
资助信息	云南省教育厅科学研究基金项目(2014CO12Y)
语种	中文
公开日期	2015-03-25
内容类型	期刊论文
源URL	[http://159.226.149.42:8088/handle/152453/8285]
专题	昆明动物研究所_结构生物信息学
作者单位	1.云南省第一人民医院急诊科,昆明650032 2.云南省第一人民医院遗传诊断中心,昆明650032 3.中国科学院昆明动物研究所;昆明650032 4.昆明医科大学第二附属医院内分泌科,昆明650032
推荐引用方式 GB/T 7714	何泉,徐盈[*],唐新华,等. 靶向血管生成素-2基因短发夹RNA真核表达载体的构建及内质网应激状态下对人脐静脉内皮细胞凋亡影响的观察[J]. 中国糖尿病杂志,2015,23(2):158-164.
APA	何泉,徐盈[*],唐新华,刘衡,朱宝生,&蒋绿芝.(2015).靶向血管生成素-2基因短发夹RNA真核表达载体的构建及内质网应激状态下对人脐静脉内皮细胞凋亡影响的观察.中国糖尿病杂志,23(2),158-164.
MLA	何泉,et al."靶向血管生成素-2基因短发夹RNA真核表达载体的构建及内质网应激状态下对人脐静脉内皮细胞凋亡影响的观察".中国糖尿病杂志 23.2(2015):158-164.