In-vitro Knock Down of VEGFR2 Expression Using Specific siRNA in the Culture Medium
DOI:
https://doi.org/10.22100/jkh.v10i4.786Keywords:
VEGFR-2؛siRNA؛ نئوواسکولاریزاسیون؛ HUVEC؛ RT-PCRAbstract
Introduction:The use of siRNAforsilencinggene expressionis expandingtoday. Knock down ofvascular endothelial growth factor is one of theobjectives ofthistechnology. In this study we aimed to inhibit the expression of receptor type II of VEGF (VEGFR-2) using specific siRNA in the culture medium.
Methods: First, according to the target gene sequences, sequence of the specific siRNA were designed; blasted and built. Using RT-PCR, cDNA of HUVEC was synthesized and PCR with specific primers was reproduced. PEFGP-N1 expression vector was cloned with target gene and confirmed. Then Hela cells which has not any expression of the target genes were transfected whit cloned pEGFP-N1 vector using lipofectamin. GFP expression rate in the Hela cellsContaining initial vector and cloned vector in presence and absence of specific siRNA was assessed. Through evaluation of gene inhibition, decreasing of green fluorescence from GFP, RT-PCR was investigated.
Results: The results of the two used siRNA, indicate reduced gene expression 56% and 63% in comparison with the control group (P<0.05).
Conclusion: Transfection of specific VEGFR-2 siRNA using lipofectamin significantly inhibits expression of receptor. In fact,it cancut thesignal transduction pathwayand preventsthe creation ofnewblood vessels. Therefore, probably it can act as a new treatment factor for prevention or reduction of neovascularization.
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