The Experimental Study Of Inhibition On Angiogenesis In Walker-256 Hepatoma In Rats By VEGF Antisense Oligonucleotides Loaded Nanoparticles

The formation of a blood supply(angiogenesis) is critical to the growth of solid tumors,including hepatoma, safe and effective anti-angiogenesis methods especially gene therapy are needed to determine whether control of angiogenesis would be therapeutic. The application of gene therapy in clinic is more and more regarded. Inputting the target gene to the cell is the basis to achieve the extrinsic gene exerting the biologic effect. Therefore, to study the carrier of gene becomes the key tache of whether the gene therapy can be successful.In recent years,nanoparticles come to us. Knubbly cell can produce more than 20 sorts of blood vessel creative gene, thereinto, VEGF is the presently discovered growth gene with the strongest and the most special effect to induce the knub blood vessel formed. The effect of many other genes to activate the new blood vessel formed is also entirely or partly via inducing VEGF to express for achievement. Thus, interdicting the effect of the VEGF is sufficient to restrain the most creative effect of the blood vessel. Recently, it is proved that VEGF-ASODN is capable to restrainthe expression of diversiform knub cell and the creation of knub blood vessel.We choose the antisense oligodeoxynuclieotide of VEGF165 mRNA as therapeutic gene,the polylactic-co-glycolic acid(PLGA) as gene carrier, prepared the PLGA nanoparticles of VEGF-ASODN with w/o/w double emulsion-solvent evaporation method. At the basis of unitary factor experimentation review, the study optimized the prescription and the preparation techniques of the nanoparticles by the orthogonal method, observed the shape, size and the distribution of the diameter of the nanoparticles, and studied the release character and the freeze desiccation techniques. So that to explore if the nanoparticles can be the carrier of the liver knub therapeutic gene after medicine via hepatic artery.Human umbilical vein endothelial cell (HUVEC) proliferation and migration ,two important events in the process of angiogenesis, were determined after influenced with VEGF-ASODN loaded PLGA nanoparticles.Using the modified method,rats were implanted with Walker-256 tumor tissues in the deep aspect of the left lobe of the liver to establish the planted Walker-256 hepatoma model. We examined the therapeutic effectiveness by using VEGF-ASODN loaded PLGA nanoparticles on planted rat models via hepatic artery infusion.Four treatment groups were compared:1)normal saline (A group);2)nothing loaded PLGA (Bgroup);3)AS-ODN(C group); 4) VEGF-ASODN loaded PLGA nanoparticles(D group).The vasculature and arterialcollaterals around the tumors were demonstrated with plane and enhanced spiral CT imaging and count of microvessel density(MVD)of CD34 immunohistochemistry staining.First, VEGF-ASODN loaded PLGA nanoparticles were prepared with w/o/w double emulsion-solvent evaporation method . The shape of VEGF-ASODN loaded PLGA nanoparticles is roundness, size is well-distributed, the mean diameter is 150nm, the encapsulation ratio reaches to 72%, the VEGF-ASODN loading in the nanoparticles is 0.84%, and the release is slow for at least 21 days, the drug release progress mostly is PLGA material corruption solely.The results indicated that the preparing method of VEGF-ASODN loaded PLGA nanoparticles is simple, and repeatability is fine.Second, Human umbilical vein endothelial cell (HUVEC ) proliferation and migration test suggest that the inhibition by VEGF-ASODN loaded PLGA nanoparticles is far more effective than by VEGF-ASODN only.Third, the modified method of implanted with Walker-256 tumor tissues is a ideal method,the rate of success is high to 86.7% (52/60rats) .Fourth, on the helical CT imaging,tumors were hypodensity without enhancement all time in the D group,compared with peri- enhancement ingroup C,and extremely enhancement in A,B groups; The survival of rats in group C and group D prolonged significantly than that of group A and B, especially in group D; Microscopically, the morphological features in group A was si...