Screening And Identification Of Peptides Bound Specifically To Human Prostate Carcinoma Cells

Prostate carcinoma(PC) is one of the most common carcinomas in urogenital system. Along with the prolonged life expectancy, the developed diagnostic techniques, and the changed life style, the incidence of prostate cancer is keeping increasing, and most patients are at developing stage when diagnosis are obtained. Forane metastasis of prostate cancer is the predominant reason causing death. Reports indicated that 30% patients had been found metastasis at the first diagnosis, and more than 80% patients would suffer bone metastasis during the course of disease. Thus, in a long future, the invasion and metastasis of tumor cells will be one of the momentous tasks for fundamental and clinical researches.In the medicine treatment process of tumors, the chemotherapy medicine not only acts on the tumors, bu also acts on the healthy tissues and organs after spreading in the bodies.As a result, while killing tumors,they brought bodies large side effects, and influenced the effect of therapy. But make use of the molecules specially bound to tumors can work the problem out. They can improve the delivery system of anti-cancer medicine. And we can realize the pertinency and the security of treatments only when we can improve the specificity of oriented medicine.Directed therapy means delivering medicine especially to tumors by some kind of carrier, releasing medicine locally to fully exert the special function, and to kill the tumor cells specifically. Traditional directed therapy was based on monoclonal antibody, which currently yet had limitation unable to overcome. Phage displayed random peptide libraries based on phage surface display technique could confirm the special peptides bounded to target cells. By this we could effectively improve the power of directed theray.From establishment to now, phage surface display technique had been a kind of powerful experiment technique, and was used widely in many fields. Its basic principle was inserting foreign DNA segment to protein coding gene Pâ…¢or pâ…§in the phage, to make the corresponding expression product fused into capsid protein as fusion protein, and to be shown on the surface of phage, and finally got the phage expressed special peptides by enrichment panning. This technique integrated phenotype and genotype of protein into filobactivirus, which was easy to be operated bio-chemically and hereditarily as a carrier. So the filtration and identification of protein's expression library became simpler. Phage pepetide library was a much important branch of phage display technique. And it was a simple and efficient technique to filtrate needed short peptides. This technique randomly assorted oligonucleotides coding certain amount of short peptides and integrated them into capsid protein gene of filobactivirus, to express short peptides on phage surface as fusion protein, and finally got a random peptide library.To provide a experiment basis of designing directed medicine aiming at sites on human prostate cancer cells, this study made human prostate cancer cell line PC3M cells target cells, made normal human prostate cells and human prostate cancer cell line PC3 cells adsorption cells, performed subtraction biopanning by phage 7 peptides library, and finally got several PC associated short peptides and PC high metastasis associated short peptides, which identified by cell-ELISA, immunocytochemistry technique and DNA sequencing. In this study, the PC associated short peptides'influence on PC3M spreading was investigated through tumor cell morphological observation. The influences on PC3M migration and invasion were measured by using Boyden chamber assay. The full DNA sequence represented by the PC high metastasis associated short peptides was Isolated by RT-PCR. The membrane protein on PC3M cells bound to PC high metastasis associated short peptides were examed by WesterBlot.The results are as following. We got several PC associated short peptides that specifically bound to PC3M cells rather than nomal human prostate cells after performing subtraction biopanning by phage 7 peptides library, which making PC3M cells target cells, and making normal human prostate cells adsorption cells. Some of the specific PC associated peptides inhibited PC3M cell spread, and showing a rising trend as the concentration growed. Boyden chamber assay showed that the PC associated peptides had inhibitory influence on migration and invasion of PC3M cell. We got several PC high metastasis associated short peptides that specifically bound to PC3M cells rather than PC3 cells after performing subtraction biopanning by phage 7 peptides library, which making PC3M cells target cells, and making normal human prostate cells and PC3 cells adsorption cells. The protein specifically bound to PC3M cells maybe existed in native condition by RT-PCR research. WesternBlot showed PC3M cell membrane protein could have several sites which bound to PC high metastasis associated short peptides.Through this research, we found that some of the PC associated short peptides, obtained through subtraction biopanning by phage random peptide library, could effectively influence tumor cell's biological behavior. This research provides experimental basis for structuring new drugs to target different sites on tumor cells, which supplies a new hope for clinical tumor therapeutic strategies. The characteristic and innovation in this research were that the PC associated short peptides got by subtraction biopanning had high efficiency on targeting the PC3M cells; the PC high metastasis associated short peptides were related to the high metastasis ability of PC3M.