中国糖尿病杂志
中國糖尿病雜誌
중국당뇨병잡지
CHINESE JOURNAL OF DIABETES
2015年
2期
158-164
,共7页
何泉%徐盈%唐新华%刘衡%朱宝生%蒋绿芝
何泉%徐盈%唐新華%劉衡%硃寶生%蔣綠芝
하천%서영%당신화%류형%주보생%장록지
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
基因%血管生成素-2%內質網應激%RNA榦擾%錶達載體%短髮夾RNA%細胞凋亡%人臍靜脈內皮細胞
기인%혈관생성소-2%내질망응격%RNA간우%표체재체%단발협RNA%세포조망%인제정맥내피세포
Gene%Angiopoietin-2 ( Ang-2 )%Endoplasmic reticulum stress ( ERS )%RNA interference%Expression vector%Short hairpin RNA (shRNA)%Apoptosis%Human umbilical vein endothelial cells (HUVECs)
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。
目的：構建人血管生成素‐2（Ang‐2）短髮夾RNA（shRNA）真覈錶達質粒，觀察其在內質網應激（ERS）狀態下對人臍靜脈內皮細胞（HUVECs）增殖和凋亡的影響，探討Ang‐2基因在 ERS狀態下介導血管內皮細胞凋亡的作用和機製。方法採用DNA重組技術根據人Ang‐2 mRNA序列設計併閤成2條shRNA寡覈苷痠片段，構建錶達質粒併通過脂質體介導轉染 HUVECs ，對 Ang‐2基因進行RNA沉默，採用RT‐PCR和Western blot分彆檢測Ang‐2在ERS狀態下mRNA及蛋白質的錶達。結果經雙酶切和測序鑒定證實，2箇shRNA錶達載體均構建成功。與空白對照、陰性對照組相比，Ang2‐1‐shRNA、Ang2‐2‐shRNA組HUVECs中Ang‐2基因mRNA及蛋白錶達水平均降低，其中，Ang2‐1‐shR‐NA榦擾效果最彊。Ang‐2 mRNA和蛋白的抑製率分彆達79.29％、68.21％。沉默 Ang‐2基因可增加ERS狀態下HUVECs細胞活力及抑製其凋亡。結論成功構建靶嚮Ang‐2基因的shRNA真覈錶達載體，Ang‐2基因沉默對ERS狀態下HUVECs凋亡有抑製作用。Ang‐2基因可抑製血管內皮細胞體外增殖，增彊ERS狀態下HUVECs凋亡。
목적：구건인혈관생성소‐2（Ang‐2）단발협RNA（shRNA）진핵표체질립，관찰기재내질망응격（ERS）상태하대인제정맥내피세포（HUVECs）증식화조망적영향，탐토Ang‐2기인재 ERS상태하개도혈관내피세포조망적작용화궤제。방법채용DNA중조기술근거인Ang‐2 mRNA서렬설계병합성2조shRNA과핵감산편단，구건표체질립병통과지질체개도전염 HUVECs ，대 Ang‐2기인진행RNA침묵，채용RT‐PCR화Western blot분별검측Ang‐2재ERS상태하mRNA급단백질적표체。결과경쌍매절화측서감정증실，2개shRNA표체재체균구건성공。여공백대조、음성대조조상비，Ang2‐1‐shRNA、Ang2‐2‐shRNA조HUVECs중Ang‐2기인mRNA급단백표체수평균강저，기중，Ang2‐1‐shR‐NA간우효과최강。Ang‐2 mRNA화단백적억제솔분별체79.29％、68.21％。침묵 Ang‐2기인가증가ERS상태하HUVECs세포활력급억제기조망。결론성공구건파향Ang‐2기인적shRNA진핵표체재체，Ang‐2기인침묵대ERS상태하HUVECs조망유억제작용。Ang‐2기인가억제혈관내피세포체외증식，증강ERS상태하HUVECs조망。
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.
