| Osteosarcoma is the most common malignant bone tumor occurs in adolescents. Its incidence is about22.36%of all major malignant bone tumors. At present surgery and chemotherapy are the main treatments of ostoesarcoma. Five years survival rate are80%～90%. Metastasis rate is about80%of the patients at the time of diagnosis. It is almost impossible to make a complete resection. At present, high-dose chemotherapy is necessary and is still the main treatment for it, however, chemotherapy is also of serious side effect for the normal tissues. Although the concept of neoadjuvant chemotherapy has been accepted, the measures were carried on in groping way. There is a great difference in sensitivity to chemotherapy drugs between tumor tissues. Only a part of patients have a satisfactory effect. Therefore, to enhance the effect of targeting of the chemotherapy and Improve treatment for osteosarcoma chemotherapy has become an urgent problem.Targeted therapy can kill tumor cells specially which can avoid serious chemotherapy toxic effect for the normal tissues. Targeted therapy may be a harmless and efficient way to osteosarcoma therapy. Searching for targeting position and supporter is an important topic. In this experiment we try to screen a peptide that specially binding to human osteosarcoma cells. In future we hope the peptide can be used as a carrier which delivers drug to tumor tissues. This experiment may lay a foundation of targeted therapy for osteosarcoma. In order to get the mentioned specific binding peptide, an effective method needed. Phage display technology is the most effective one.Phage-display technology, established by Smith GP etc, is an effective molecular biologic tool which has been developed and widely used since1990s. The basic principle of this technology describes a selection technique in which a peptide or proteins expressed as a fusion protein in phage, resulting in showing of the fused protein on the virion surface, while the genetic code of fusion protein virion place to live. Its major characteristic is that genotype and phenotype were linked of a specific molecular in the same phage, combines the characteristics of antigens and the amplification of bacteriophage, and becomes an effective screening system. The simplest panning measure of this technology is practiced by incubating a library of phage-displayed peptides coated with the target in a plate, with the unbound phages washed away, and the specifically bound phages eluted. Then, the specifically bound phages are amplified and used for the next panning round. After3to4rounds, the specially binding phages are enriched. Phage display technique contains a lot of features, such as simplicity of operation and high throughput and so on. It has been widely used in a number of areas especially in life science. It becomes a powerful tool in searching the new targeting peptides.Cell contains a complex biological system; There are a large number of signal molecules on its membrane, which reflect the characteristics and functional status of cells. Theoretically these signal molecules can be purified for screening the specific binding peptides, but the purification is very difficult to achieve. It is easier to use whole native cells for selecting the different molecules on the cell membrane without the information of the receptors. Whole-cell screening usually includes the native conformation and biological activity of receptors which can simulate the natural protein molecules, and effectively find the specific proteins binding to the cell membrane. However, for the low integrity cell membrane antigen density, it is difficult to screen by a single chain antibodies. This screening method presents many disadvantages, such as high background, high unspecificity. We may make lose of specific ligands in the process of washing. In order to solve the problems mentioned and get very close to the in vivo environment as far as possible, we chose a very efficient screening method to screen the specific binding peptides of human osteosarcoma cells. The method was established by Giordano in2001, and termed as biopanning and rapid analysis of selective interactive ligands (BRASIL).This technique is based on differential centrifugation of the aqueous phage and cell suspension through a non-miscible organic in lower phase. We chose293T cells as control. We make an incubation of phage displayed peptide library and293T. After differential centrifugation; we will get non-binding phage from peptide library. Another incubation will be made between MG63cells and non-binding phage. After differential centrifugation, we will get the peptides binding to MG63cells only. Next, the cell/phage pellets are recovered by bacterial infection. Then next round will be carried on,BRASIL has many advantages compared to traditional ways. The key of the technology is special organic solvent (dibutyl phthalate:cyclohexane=9:1volume ratio). We can obtain peptides binding only to MG63cells after differential centrifugation twice. Target phage recovered by bacterial infection with huge amplification. The dosage putting into next round can be determined after we know the titer of phage. The chosen peptide will turn up after four round screening. As the method involves one centrifugation and does not require repeated washes, it is more convenient to recover phage from cell membranes than other cell-panning techniques, It is easily for us to control uncertain factors during the experiment and save us much time and labor. So the method is the most befitting one for the experiment.The phage display peptide library is based on a built-up library of random peptide7-mers insert into a minor coat protein of M13phage. The randomized sequence both sides is a pair of homocysteine. Under non-reducing conditions a disulfide cross-link will be formed by the cysteines spontaneously, resulting in the cyclization of displayed peptides contrasted to the linear peptides. Disulfide-constrained peptide libraries have also been proven to be useful in recognition antigen structure, development of peptide-based therapeutics and mirror-image ligands for D-amino acid targets. Human osteosarcoma cell MG-63was used as targeted cells with293T human embryonic kidney cells as a control for screening form Ph. D.7TM phage display peptide library with method BRASIL. Cells were collected and incubated with phage within1.5-ml centrifuge tube. Cells and media were kept on ice. The cell-phage suspension was gently transferred to the top of a non-miscible organic lower phase and centrifuged. Another incubation will be made between MG63cells and non-binding phage. Target phage recovered by bacterial infection with huge amplification. Then next round screening will be carried on till there is no obvious enrichment than last round. Then we took four rounds of screening to MG63cells. We selected50phage clones at random and amplified the objective segment by PCR method. All the segments were sequenced and transformed into amino acid sequence.There are24phage clones containing amino acid sequence. DNA sequencing indicates that all the segments contain the same amino acid sequence: TCGCTTACGAATTTGAGTAAG. The sequence:SLTNLSK.The enriched specially binding peptide was verified by cell enzyme linked immunosorbent assay (ELISA) with293T cells and Hela cells as control. The peptide has a preferable MG63cells targeting.We inoculated human osteosarcoma cells MG63in the medullary cavity of the femur of BALB/c nude mice, and had established the tumor-bearing animal models successfully. SLTNLSK was injected through tail vein. Ten minutes later tumor-bearing animal models were killed. As shown in immunohistochemical staining, there was apparent staining in the tumor tissue of nude mouse, while there was only a little bit of staining in liver, and no apparent staining in brain, lung and kidney. These data proved that the SLTNLSK phage can effectively target to the tumor tissue of nude mouse model.On the whole, we have screened the human osteosarcoma cells MG63with293T as control for four rounds by BRASIL phage display technique without knowing the target. DNA sequencing indicates that all the segments contain the same amino acid sequence:SLTNLSK. The enriched specially binding peptide was verified by cell enzyme linked immunosorbent assay (ELISA). Targeting of the peptide was tested by organ Immunohistochemistry with Osteosarcoma model. Taken together, we draw the following conclusions:1. We screened the peptide library specifically binding to the human osteosarcoma cells MG63by BRASIL phage display technique. After four rounds of screening, the phage libraries successfully gathered.2. We scraped the50phage clones at random and obtained the peptide sequence by PCR method. The sequence SLTNLSK had the highest frequency.3. The target peptide specially binding peptide was verified by cell enzyme linked immunosorbent assay (ELISA).4. We had successfully established the tumor-bearing animal models of osteosarcoma. Immunohistochemical staining result also proved that the TKPDKGY phage can specifically bind to the human osteoatrcoma cells of nude mouse. |
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