Construction Of Truncated Platelet Factor 4 Lentiviral Vector And Study On Anti-angiogenesis Activity

Angiogenesis is a critical step for tumor growth and metastatic activity. Inhibition of angiogenesis can suppress tumor growth. Hence a therapeutic strategy with the target of tumor angiogenesis has developed into the focus fields of the tumor biology treatment. Platelet factor-4(PF4) is a kind of endogenous angiogenesis inhibitor, which inhibits endothelial cell migration, proliferation through multiple mechanisms, with the consequence of restraining the angiogenesis. The lentiviral vector is one of the most commonly used gene transduction system and plays an important role in gene therapy because it proposes several attributes such as sustained gene expression, high gene transduction efficiency and large insertion capacity. As the research moves along, the lentiviral vector has been applied to cancer gene therapy and much progress has been made. This subject constucts a Lentiviral vector carrying PF447-70-RGD gene, then transfect it into package cell 293 T to produce virus. And anti-angiogenesis biological activity of the lentiviral-mediated PF447-70-RGD is explored which paves the way for vascular targeting of cancer gene therapy.In this study, the gene sequences of the recombinant PF447-70-RGD was amplified from pcDNA3.1(+)-PF447-70-RGD vector preserved in laboratory and then was cloned into pLV-MCS-Bsd of lentiviral vector. So the lentiviral vector pLV-PF447-70-RGD was constructed successfully firstly. Then the recombinant lentiviral vector and three helper plasmids were transfected into 293 T cells with Lipofect2000 Reagent and the lentiviral stocks was titered by real-time PCR. The recombinant lentiviral titer reached 6.16×107IU/ml. A549 Cells were divided into blank group, control group and experimental group. Transfected pLV-PF447-70-RGD and pLV-MCS-Bsd cells were selected by Bsd to produce the stable recombinant lentivirus. The mRNA and protein expression level of recombinant peptides were analyzed via RT-PCR and Western blot. Finally, vitro experiments were conducted to detecte the growth inhibition effect of lentivirus infection and expressing products of stable-transfected A549 cell line and on HUVEC. MTT assay showed that growth inhibitory rate of expressing products of stable-transfected A549 to HUVEC was 36.7%. And recombinant lentiviral supernatant infection had the same results. Compared with the control group, the growth and tubular formation of HUVEC in experimental group were obviously inhibited.