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‐2 mRNA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‐shR‐NA干扰效果最强。Ang‐2 mRNA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。%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. RT‐PCR 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‐2 mRNA 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‐2 gene 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.
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