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构建重组真核表达载体pc DNA3.1(+)-PF447-70-RGD,并检测靶向性重组血小板因子四(PF4)在真核细胞内的表达水平,探讨其体外抗血管生成活性。设计构建了靶向性重组PF4真核表达载体pc DNA3.1(+)-PF447-70-RGD,用脂质体法将其转染至中华仓鼠卵巢细胞系(CHO),用G418筛选稳系,采用RT-PCR和Western blot检测表达产物,MTT法测定CHO稳转体系培养上清对脐静脉内皮细胞(HUVEC)增殖的影响。成功构建出重组真核表达载体pc DNA3.1(+)-PF447-70-RGD,并获得稳转CHO细胞系,从基因水平和蛋白水平均可检测到目的基因的表达。MTT法显示,CHO稳系培养上清对HUVEC生长抑制率是50.8%,与对照组比,血管内皮细胞的增殖能力显著减小。靶向性重组PF4具有较强的体外抑制血管生成活性,该重组肽能够抑制HUVEC增殖。
The recombinant eukaryotic expression vector pcDNA3.1 (+) - PF447-70-RGD was constructed and the expression level of targeted recombinant platelet factor 4 (PF4) in eukaryotic cells was detected to investigate the anti-angiogenic activity in vitro. Targeted recombinant PF4 eukaryotic expression vector pcDNA3.1 (+) - PF447-70-RGD was designed and constructed and transfected into Chinese hamster ovary cell line (CHO) by lipofectamine. , And the expression products were detected by RT-PCR and Western blot. The proliferation of human umbilical vein endothelial cells (HUVECs) was detected by MTT assay. The recombinant eukaryotic expression vector pcDNA3.1 (+) - PF447-70-RGD was successfully constructed and transfected into CHO cell line. The expression of the target gene was detected both at the gene and protein levels. MTT assay showed that the growth inhibition rate of CHO stable culture supernatant on HUVEC was 50.8%, compared with the control group, the proliferation of vascular endothelial cells was significantly reduced. Targeted recombinant PF4 has a strong in vitro inhibitory angiogenic activity, which inhibits the proliferation of HUVECs.