Adenovirus-mediated Rna Interference To Reduce Vegf And Its Receptors Kdr, Transcription Of Flt Inhibition Of Angiogenesis Research

Angiogenesis, the formation of new blood vessels is a critical process for the growth, invasion and spread of tumors, and allows tumors to expand beyond a few millimeters by providing oxygen, nutrients, and waste disposal at distances. Some clinical research reported that the growth and metathesis capability of tumor depend on the ability of angiogenesis. If we can efficiently inhibit the process of angiogenesis, then block the supplement of blood for tumor, thus achieve the goal to inhibit the growth, proliferation and metathesis of tumor.The expression of vascular endothelial growth factor (VEGF) and its receptor(VEGFR) is up-regulated in most malignant tumors. VEGF enhances the division and proliferation of endothelial cells to induces the formation of new blood vessels, and directly promotes the growth of tumor cells by autocrine or paracrine. In present study, we hypothesis that down-regulation of VEGF and VEGFR expression level can inhibit the angiogenesis of tumor, thus decrease the growth and invasion of tumor cells.RNA interference is a newly-developed technique to knock down the specific gene expression and has potential application in cancer gene therapy studies. Several vectors have been developed to deliver siRNA. Advantages such as the availability of high virus titer, infection of a broad spectrum of cell types and independence on active cell division, efficient delivery of siRNA both in vitro and in vivo make adenovirus the choice for siRNA delivery in cancer gene therapy studies. Here we therefore employed this technique to investigate the effects of knock down of VEGF and its two receptors FLT and KDR on the growth, invasion and angiogenesis of endothelial cells to confirm our hypothesis. our results are as follows.1. Used the homologous recombination technique we constructed a recombinant adenovirus AdH1-siRNA/VEGF targeting human VEGF and AdH1-siRNA/FLT, AdH1-siRNA/KDR targeting the two receptors, respectively. Reverse Transcription-PCR showed that this adenovirus could specifically decrease the VEGF, FLT and KDR expression of human endothelial cells (HUVEC). Compared with control group, the mRNA expression of VEGF, KDR and FLt decreased to 50%,52% and 40%, repectively. 2. AdH1-siRNA/VEGF , AdH1-siRNA/KDR, AdH1-siRNA/FLT can efficiently interfere the proliferation of endothelial cells, and the efficiency of AdH1-siRNA/FLT is the best. 9 days after infection of adenovirous, the average cell number of control group and vector group are 30.5×10~4/μl and 28×10~4/μl, but that of the groups respectively infected with AdH1-siRNA/VEGF, AdH1-siRNA/KDR and AdH1-siRNA/FLT are 21.5×10~4/μl,21×10~4/μl and 16,25×10~4/μl.3. This three kinds adenovirus also can efficiently interfere the tube formation of HUVEC on matrigel. Averagely, the tube number of every HPF is 10.75 in the control group, and 10.25 in the vector group. The interference ability of AdH1-siRNA/FLT is the most efficient. In the group treated with AdH1-siRNA/FLT, there are only 1or2 tubes in the HPF. Most of endothelial cells in the field cann't form the capillary-like structure.