中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2011年
4期
499-502
,共4页
王刚%张凯伦%蒋雄刚%刘成硅
王剛%張凱倫%蔣雄剛%劉成硅
왕강%장개륜%장웅강%류성규
血管内皮细胞生长因子%真核表达载体%细胞增殖%内皮化
血管內皮細胞生長因子%真覈錶達載體%細胞增殖%內皮化
혈관내피세포생장인자%진핵표체재체%세포증식%내피화
Vascular endothelial growth factor%Eukaryotic expression vector%Cellular proliferation%Endothelialization
目的 构建携带人血管内皮细胞生长因子165(VEGF165)基因的新型真核表达载体pIRES-EGFP-VEGF165,并通过转染人脐静脉内皮细胞株EA.hy926细胞,观察载体的体外表达和对血管内皮细胞增殖的影响.方法 将VEGF165目的 基因片段插入到真核表达载体pIRES-EGFP质粒中,按每孔加入质粒DNA 1.2μg介导体外瞬时转染人脐静脉内皮细胞株EA.hy926细胞,通过荧光实时定量逆转录-聚合酶链反应(RT-PCR,n=3)和Western blot(n=5)分别检测mRNA和蛋白水平的表达,并通过MTT法(n=5)检测转染pIRES-EGFP-VEGF165质粒对血管内皮细胞增殖的促进作用.结果 成功构建pIRES-EGFP-VEGF165真核表达载体,测序结果 与预期相符;荧光计数显示体外瞬时转染人脐静脉内皮细胞株EA.by926细胞的转染效率约为(11.2±2.5)%;荧光实时定量RT-PCR和Western blot证实转染pIRES-EGFP-VEGF165质粒组细胞在mRNA和蛋白水平表达的VEGF165均高于对照组(P＜0.01),MTT法检测表明转染pIRES-EGFP-VEGF165质粒组细胞增殖速度较对照组明显增加(P＜0.01).结论 成功构建pIRES-EGFP-VEGF165真核表达载体,并实现其在人脐静脉内皮细胞株EA.hy926细胞中的表达,而且证实可以促进血管内皮细胞的增殖.
目的 構建攜帶人血管內皮細胞生長因子165(VEGF165)基因的新型真覈錶達載體pIRES-EGFP-VEGF165,併通過轉染人臍靜脈內皮細胞株EA.hy926細胞,觀察載體的體外錶達和對血管內皮細胞增殖的影響.方法 將VEGF165目的 基因片段插入到真覈錶達載體pIRES-EGFP質粒中,按每孔加入質粒DNA 1.2μg介導體外瞬時轉染人臍靜脈內皮細胞株EA.hy926細胞,通過熒光實時定量逆轉錄-聚閤酶鏈反應(RT-PCR,n=3)和Western blot(n=5)分彆檢測mRNA和蛋白水平的錶達,併通過MTT法(n=5)檢測轉染pIRES-EGFP-VEGF165質粒對血管內皮細胞增殖的促進作用.結果 成功構建pIRES-EGFP-VEGF165真覈錶達載體,測序結果 與預期相符;熒光計數顯示體外瞬時轉染人臍靜脈內皮細胞株EA.by926細胞的轉染效率約為(11.2±2.5)%;熒光實時定量RT-PCR和Western blot證實轉染pIRES-EGFP-VEGF165質粒組細胞在mRNA和蛋白水平錶達的VEGF165均高于對照組(P＜0.01),MTT法檢測錶明轉染pIRES-EGFP-VEGF165質粒組細胞增殖速度較對照組明顯增加(P＜0.01).結論 成功構建pIRES-EGFP-VEGF165真覈錶達載體,併實現其在人臍靜脈內皮細胞株EA.hy926細胞中的錶達,而且證實可以促進血管內皮細胞的增殖.
목적 구건휴대인혈관내피세포생장인자165(VEGF165)기인적신형진핵표체재체pIRES-EGFP-VEGF165,병통과전염인제정맥내피세포주EA.hy926세포,관찰재체적체외표체화대혈관내피세포증식적영향.방법 장VEGF165목적 기인편단삽입도진핵표체재체pIRES-EGFP질립중,안매공가입질립DNA 1.2μg개도체외순시전염인제정맥내피세포주EA.hy926세포,통과형광실시정량역전록-취합매련반응(RT-PCR,n=3)화Western blot(n=5)분별검측mRNA화단백수평적표체,병통과MTT법(n=5)검측전염pIRES-EGFP-VEGF165질립대혈관내피세포증식적촉진작용.결과 성공구건pIRES-EGFP-VEGF165진핵표체재체,측서결과 여예기상부;형광계수현시체외순시전염인제정맥내피세포주EA.by926세포적전염효솔약위(11.2±2.5)%;형광실시정량RT-PCR화Western blot증실전염pIRES-EGFP-VEGF165질립조세포재mRNA화단백수평표체적VEGF165균고우대조조(P＜0.01),MTT법검측표명전염pIRES-EGFP-VEGF165질립조세포증식속도교대조조명현증가(P＜0.01).결론 성공구건pIRES-EGFP-VEGF165진핵표체재체,병실현기재인제정맥내피세포주EA.hy926세포중적표체,이차증실가이촉진혈관내피세포적증식.
Objective To construct a recombinant eukaryotic expression vector pIRES-VEGF165-tPA containing functional region of human vascular endothelial growth factor 165 (VEGF165) gene, and investigate its in vitro expression and effect on the proliferation of vascular endothelial cells in transfected human umbilical vein endothelial cell lines (EA. hy926). Methods The VEGF165 gene was inserted into the eukaryotic expression vector plRES-EGFP to construct the recombinant plasmid, which was transfected into human umbilical vein endothelial cell lines (EA. hy926) at 1.2 μg/well subsequently. The transcription and expression of VEGF165 gene were detected by reverse transcription-polymerase chain reaction ( RT-PCR, n = 3 ) and Western blotting ( n = 5 ) respectively. And the effect on the proliferation of vascular endothelial cells was evaluated by MTT assay (n =5). Results The sequence of pIRES-EGFP-VEGF165 approved that the gene of VEGF165 was inserted into the eukaryotic expression vector correctly. The transfection efficiency was about ( 11.2 ± 2. 5) %, detected by counting the positive cells of green fluorescence.The mRNA and protein levels of VEGF165 in the pIRES-EGFP-VEGF165 transfected group were significantly higher than those in the control groups respectively (P ＜ 0. 01 ). Also, MTT assay indicated the proliferation of endothelia cells was greatly improved in the pIRES-EGFP-VEGF165 transfected group. Conclusion The recombinant eukaryotic expression vector pIRES-EGFP-VEGF165 is successfully constructed and can be expressed in transfected EA. hy926 cells, and it is proved that transfection with this vector could improve the proliferation of vascular endothelial cells.
