hVEGF_(121)基因转染对人脐静脉内皮细胞生长的作用

Effects of hVEGF_(121 ) gene transfection on human umbilical vein endothelial cells growth

目的 :构建人血管内皮生长因子 (h VEGF) - 12 1的真核细胞复制表达质粒 ,将 h VEGF1 2 1 基因转染进人脐静脉内皮细胞 (human umbilical vein endothelial cell,HUVEC) ,通过旁分泌作用来加快其生长速度。 方法 :采用 RT- PCR方法 ,从人胚胎成纤维细胞中克隆 h VEGF1 2 1 前体蛋白全长 c DNA,构建重组真核表达质粒 pc DNA3- h VEGF1 2 1 ,用 Fu GENE6介导转染入HU VEC。收集转染的 HU VEC培养上清 ,用 EL ISA法定量检测 h VEGF蛋白的表达情况 ;绘制细胞生长曲线 ,检测 h VEGF1 2 1促进 HUVEC增殖的生物学活性。设转染 pc DNA3空质粒载体和不转染任何 DNA的 HU VEC做对照。 结果 :(1)成功构建了 pc DNA3- h VEGF1 2 1 真核表达质粒 ,经测序证明基因序列与 Gen Bank中核酸序列一致 ,且插入方向正确 ;(2 )转染细胞瞬时表达 h VEGF蛋白 ,于转染 1d后每 10 4 个细胞每天可分泌 h VEGF蛋白 5 9.95 pg,约为对照组的 10 0倍 ;之后表达水平先快后慢下降 ,于转染 7d后仍较对照组高近 1倍 ;(3)转染细胞生长速度明显加快 ,在转染 3d后细胞数与对照组相比即有显著性差异 (P<0 .0 1) ,细胞倍增时间从对照组的 7d以上缩短为 3d左右。结论 :成功构建了 pc DNA3- h VEGF1 2 1 真核表达质粒 ;该质粒能在 HU

Objective:To construct human vascular endothelial growth factor (hVEGF)-121 protein expression plasmid, and to transfect it into human umbilical vein endothelial cells (HUVEC) to speed up their growth in vitro through a paracrine way. Methods: hVEGF 121 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human embryonic fibroblast and was inserted into eukaryotic expression vector pcDNA3. The recombinant plasmid was tranfected into HUVEC by FuGENE 6 liposome. The culture supernatant of the transfected cells was collected, and the expression of hVEGF protein was tested by enzyme-linked immunosorbent assay (ELISA). The role of hVEGF 121 gene transfer in HUVEC proliferation was studied by cell-counting. pcDNA3 vector transfected and non-transfected HUVEC were used as the control groups. Results: pcDNA3-hVEGF 121 plasmid was constructed. After cell transfection, hVEGF protein was transiently expressed and peaked on 1 d at 59.95 pg/(10 4 cells·d), about 100 times that of the control group level; afterwards, the productions decreased quickly and then slowly, but still about 1 time higher than that of the control group level on 7 d. The transfected HUVEC proliferated more rapidly than that of the control groups on 3 d (P<0.01),with the cell doubling time shortened to about 3 d from over 7 d in the control group.Conclusion:Plasmid pcDNA3- hVEGF 121 has been constructed successfully, which can express active hVEGF 121 protein in HUVEC cultured in vitro,thus promoting HUVEC proliferation by a paracrine loop.