The Establishing Of The Model Of Osteosarcoma In Nude Mice And The In Vivo Screening Of The Peptides Specifically Binding To The Blood Vessel Endothelium Of The Tumor

Osteosarcoma is the most common malignant bone tumor.Its incidence rate is about 22.36%of all of the primary malignant bone tumor.,Metastasis has occurred in 80～90%of the patients at the time of diagnosis.At present,high-dose chemotherapy is still the main treatment for it,but chemotherapy is also of serious toxic effect for the normal tissues.Therefore,to enhance the effect of targeting of the chemotherapy for osteosarcoma has become an urgent problem.Tumor angiogenesis is an important aspect of tumor development,because tumor needs the new vascular system to provide adequate nutrition to support its expansion.The researchers has being paid more and more attention on some specific molecules on the tumor endothelium,which are expected to play as the new drug targets.In order to get the above-mentioned specific binding ligands,an effective screening tool is needed. Phage display technology is one of the most effective one.Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s.It describes a selection technique in which a peptide or protein turns into fusion protein with a coat protein of a bacteriophage. The fused protein will be displayed on the surface of the bacteriophage,while the DNA encoding it resides within the virion.One of the most significant feature of phage-display technology is that it links phenotype with genotype successfully. According to this,the expression of a particular phenotype on phage is directly associated with the genetic information contained within the phage particle.The expression of the desired phenotype can be achieved by incorporating the relevant genetic material into the phage genome.Phage display has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence,allowing rapid identification of peptide ligands for a variety of target molecules(antibodies,enzymes,cell-surface receptors,etc.) by an in vitro selection process,which is called panning.The simplest panning form is as follows:first,we incubate a library of phage-displayed peptides with plates(or beads) coated with targets,then wash away the unbound phage,and elute the specifically-bound phage.The eluted phage will be amplified before taken through additional binding/amplification cycles to enrich the binding sequences.After 3 to 4 rounds,individual clones will be characterized by DNA sequencing.Random peptide libraries displayed on phage can be used in a number of applications,including epitope mapping,mapping protein-protein contacts,and identification of peptide mimics of non-peptide ligands.The Ph.D.-C7C Phage Display Peptide Library is based on a combinatorial library of random peptide 7-mers fused to a minor coat protein(pⅢ) of M13 phage. The randomized sequence is flanked by a pair of cysteine residues.Under nonreducing conditions the cysteines will form a disulfide cross-link spontaneously, and result in the cyclization of displayed peptides contrasted to the linear peptides. Disulfide-constrained peptide libraries have been proven to be useful in identification of structural epitopes,mirror-image ligands for D-amino acid targets,and development of peptide-based therapeutics. The in vivo phage display technique combining the phage display technology with animal models,is an more effective way to looking for the organ-specific binding peptides.This method can help us to find the target and confirm the domain in the natural environment-tissues or organs,by using the different character of antigen of the phage.In order to provide a new method and clue for the therapy and mechanism of osteosarcoma,we will screen the specific peptides binding to endothelial cell of tumor vasculture of nude mouse using the in vivo phage display technique.We inoculated murine osteosarcoma cells UMR-106 in the medullary cavity of the femur of BALB/c nude mice,and had established the tumor-bearing animal models successfully.Then we took four rounds of screening to endothelial cell of tumor vasculture of nude mouse.We selected 60 phage clones at random and amplified the objective segment by PCR method.All the segments were sequenced and transformed into amino acid sequence.We validated the phage(TKPDKGY) whose frequency is the highest in vivo experiment and immunohistochemical staining.The TKPDKGY phage recovered from tumor was3.67×10~8 pfu/g,which was 32.19 times higher than the amount recovered from brain,24.14 times higher than lung and 9.97 higher than kidney.So we drew an elementary conclution:the TKPDKGY phage which we got had a good effect on targeting to the endothelial cell of tumor vasculture of nude mouse.As shown in immunohistochemical staining,there was apparent staining in the tumor vasculture of nude mouse,while there was only a little bit of staining in lung and kidney,and no apparent staining in brain.These data proved that the TKPDKGY phage can effectively target to the endothelial cell of tumor vasculture of nude mouse.On the whole,we have screened the endothelial cell of tumor vasculture of nude mouse,for four rounds by in vivo phage display technique without knowing the target. According to the result of in vivo experiment and immunohistochemical staining,we could get conclusion that the TKPDKGY phage can effectively target to the endothelial cell of tumor vasculture of nude mouse.Taken together,we draw the following conclusions:1.We had successfully established the tumor-bearing animal models of osteosarcoma.2.We screened the peptide library specifically binding to the endothelial cell of tumor vasculture of nude mouse.After four rounds of screening,the phage libraries successfully gathered in the tumor vasculture of nude mouse.3.We scraped the 60 phage clones at random and obtained the peptide sequence by PCR method with three of which had the highest frequency.4.We validated the TKPDKGY phage whose frequency is the highest in the vivo experiment and found this phage clone can target to the tumor vasculture of nude mouse effectively.5.Immunohistochemical staining result also proved that the TKPDKGY phage can specifically bind to the endothelial cell of tumor vasculture of nude mouse.