KR100885072B1 - Oligopeptide which binds specifically to chondrocytes - Google Patents

Abstract

The present invention relates to oligopeptides that specifically attach to chondrocytes, and more particularly to oligopeptides described in SEQ ID NOs: 1 to 3 , which specifically attach to chondrocytes selected by screening using a peptide library. And it relates to a method of specifically attaching to chondrocytes using the oligopeptide. Peptides specifically attached to chondrocytes of the present invention can be usefully used as a linker between chondrocytes and biomolecules or selectively separate biocompatible chondrocytes.

Description

Oligopeptide which binds specifically to chondrocytes             

1 Is a graph analyzing the nucleotide sequence of the oligopeptide described in SEQ ID NO: 1 of the present invention specifically attached to chondrocytes,

Figure 2 is a graph analyzing the nucleotide sequence of the oligopeptide described in SEQ ID NO: 2 of the present invention specifically attached to chondrocytes.

The present invention relates to oligopeptides that specifically attach to chondrocytes, and more particularly to oligopeptides described in SEQ ID NOs: 1 to 3 , which specifically attach to chondrocytes selected by screening using a peptide library. And it relates to a method of specifically attaching to chondrocytes using the oligopeptide.

All multicellular organisms, including humans, are a collection of tissue cells with a variety of functions and forms. These tissue cells have undergone numerous evolutionary processes and have evolved into uniquely shaped body tissues that exhibit their unique functions. Therefore, they have a very complicated structure and function, which is very vulnerable to damage from external stimulation or disease.

The human body attempts to recover disease or trauma damage to a normal state by an immune response, which can be seen as a phenomenon of replacing damaged tissue cells, which are the basic units of the body, with normal cells. The drugs or physiotherapy we often receive help to repair the body's self-healing, and if the body's damage is not repaired by itself, surgery can remove the damaged tissue and further damage the remaining normal tissue. Sometimes this does not happen. This concept of classical surgical treatment is actively changing in modern times with attempts to replace or regenerate damaged areas with normal tissues or organs. As a result, medical engineering technology has rapidly developed. However, the development of modern technology society has caused various accidents and illnesses to human beings, and the treatment of them has enormous expenses and the incidence of onset has become a serious social problem in recent years. For example, in the United States, an advanced nation of health technology, more than 100,000 patients need treatment by organ transplantation every year because of diseases and injuries of the liver, pancreas, skin, bones, and cardiovascular system. Donations have been made, and out of the approximately 36,000 people with waiting conditions, the number of patients who have died in the atmosphere in the last five years has reached 10,000. In addition, the cost of treatment, nursing care, subsidies and productivity is estimated to reach $ 400 billion annually. In Korea, more than 10,000 transplants are performed every year. However, the absolute shortage of organs is facing a severe supply shortage, unlike other countries.

The demand for donor organs to replace parts of the damaged body is increasing day by day, but the shortage of organs with no room for improvement is still causing social problems such as cancer trading. Therefore, there is a need to develop artificial tissues having life functions by artificially manipulating somatic cells constituting the human body or by artificially processing the organs up to large organs. The transplantation of tissues or organs is based on the development of immunosuppressive agents in addition to autograft, but allograft is used.However, it is far from supplying restricted organs and completely preventing the sequelae of immune response after transplantation. . In addition, the possibility of infecting a deadly disease, such as acquired immunodeficiency syndrome (AIDS) or infection, from an organ for transplantation cannot be overlooked. In addition, methods of obtaining organs such as heart, liver, lungs, and kidneys from genetically modified animals such as pigs and monkeys, as well as recently developed somatic cell cloning and stem cell culture techniques, can be used to damage patients. The direction of regenerating body tissue cells in vitro has been sought, but problems such as ethical issues, compatibility with the immune system, and organ formation of tissue cells derived from hepatocytes remain challenges.

Biomaterials used as extracellular matrix of artificial tissue are largely divided into natural polymer and synthetic polymer. As the natural polymer, collagen, fibronectin, gelatin, chitosan, alginic acid, hyaluronic acid, etc. are widely used. Poly lactic acid, poly glycolic acid (PGA), PLGA and its like copolymers with poly ε-caprolactone, polyanhydrides and polyorthoesters ).

Among these polymers, biodegradable polymers widely used in the field of tissue engineering generally refer to polymers that gradually lose their shape and weight as chemical degradation occurs in vivo, and use them to prepare carriers for tissue cells and together with the cells. When transplanted into the body, the biodegradable polymers are gradually degraded and disappeared as cells adapt to body tissue and form tissues, thereby reducing the inconvenience of removing the polymers through reoperation and reducing the chronic immune response by foreign body insertion. You can expect the effect. Of these, natural biodegradable polymers are difficult to artificially control the rate of biodegradation, whereas polymers prepared through organic synthesis have the advantage of controlling the biodegradation rate by adjusting their composition.

Chondrocytes are cells that can promote the growth of nerve cells, but can be used to promote nerve regeneration, but have not yet developed a substance that specifically binds to chondrocytes. Therefore, in order to specifically attach the polymer material used in the tissue engineering to the chondrocytes, a substance that specifically attaches to the chondrocytes is screened and applied to the polymer material to specifically attach the polymer material to the chondrocytes. It is urgent to develop a way to do this.

Thus, the present inventors screened an oligopeptide that specifically attaches to chondrocytes through screening using a peptide library, and the oligopeptide that specifically attaches to the chondrocytes is selectively separated from the biocompatible chondrocytes and the chondrocytes. The present invention has been completed by revealing that it can be usefully used as a linker between biomolecules.

It is an object of the present invention to provide an oligopeptide that specifically attaches to chondrocytes.

In order to achieve the above object, the present invention provides an oligopeptide that specifically attaches to chondrocytes.                     

The present invention also provides a method of specifically requesting chondrocytes using the oligopeptide.

Hereinafter, the present invention will be described in detail.

The present invention provides oligopeptides that specifically attach to chondrocytes.

Oligopeptides of the invention are oligopeptides selected from the group set forth in SEQ ID NO: 1 to SEQ ID NO: 3 , and specifically attach to chondrocytes. The oligopeptides are selected through screening using a peptide library, and can be usefully used as selective separation of biocompatible chondrocytes and as a connecting material between chondrocytes and biomolecules. In addition, chondrocytes are cells capable of promoting the growth of nerve cells, so that they can promote nerve regeneration.

The present invention also provides a method of specifically attaching a polymer material used for tissue engineering to chondrocytes using an oligopeptide specifically attached to chondrocytes.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Peptide Library Expressed in Escherichia Coli

We screened random amino acid sequences in the form of fusion proteins that can be expressed at the active site of thioredoxin to screen random amino acid sites that attach to chondrocytes. Specifically, a random amino acid sequence was inserted into the pFLITRIX vector containing the active site of thioredoxin so that the protein fused with thioredoxin can be exposed to the cell surface. The FLITRIX peptide expressed by the above method is regulated by the PL promoter and is inhibited by the bacteriophage λ cI repressor. The bacteriophage-derived cI inhibitor plays a role of strongly inhibiting the expression of the protein and inhibits the expression of the FLITRIX peptide under the control of the PL promoter. However, when tryptophan is added to the medium, a complex of tryptophan-trp inhibitors is formed, and the complex of tryptophan-trp inhibitors binds to the trp operator. Binding to the trp operator inhibits the expression of the cI inhibitor and induces the expression of the FLITRIX peptide under the control of the cI inhibitor.

Example 2 Isolation and Culture of Human Chondrocytes

The inventors have isolated human articular chondrocytes from the cartilage of the femur followed by arthroplasty for osteonecrosis without hip joint. Whole-thickness cartilage was ground, digested with 0.2% protease type XIV (Sigma, St. Louis, MO) at 37 ° C. for 1 hour and treated with 0.2% collagenase type IA (Sigma) for 3 hours. After digestion, undigested cartilage sections were removed using a 70 μm nylon sieve. The isolated chondrocytes were washed, plated in a tissue culture dish at a concentration of ³ × 10 ⁴ cells / cm 2, 10% fetal bovine serum (FBS), 100 units / ml penicillin-streptomycin (Sigma) and 25 μg / ml L − Incubated in DMEM (Dulbecco's modified Eagle's medium) containing ascorbic acid (Sigma) as a monolayer at 37 ° C., 5% CO ₂ in humid conditions. The medium was changed twice a week. Cells were passaged at 80-90% confluency using 0.05% trypsin-EDTA (Gibco BRL).

Example 3 Screening Using Peptide Library Expressed in Escherichia Coli

The present inventors transformed the pFLITRIX vector prepared by the method of Example 1 to E. coli and cultured in an IMC selection medium to which ampicillin was added. After incubation for 15 hours, tryptophan was added to incubate for 6 hours to induce the expression of the FLITRIX peptide. After blocking the culture plate in which the chondrocytes were fixed in advance, E. coli transformants induced expression above were attached. The above procedure was repeated to select peptide candidate groups that specifically attach to chondrocytes.

Example 4 Sequence Analysis of Peptides Using an Autobase Sequence Analyzer

The inventors performed sequencing by ABI PRISMTM Dye terminator cycle sequencing using an automatic base sequencer (ABI Prism 377, Perkin Elmer, USA). Specifically, the PCR amplification product was purified using only the PCR product using a QIAquick PCR purification kit (QIAGEN, USA). The PCR purified product was quantified using GeneQuant (Pharmacia, USA). PCR reaction for auto sequencing is as follows. 8.0 μl of terminator ready reaction mix (Perkin Elmer, USA), 5 μl of purified PCR product at 10-30 ng / μl concentration, 1 μl of PCR primer pair of 6.4 pmole and 6 μl of distilled water The volume of the reaction solution was adjusted to 20 μl. PCR reactions were performed using GeneAMP PCR system 9600 (Perkin Elmer, USA) to start hot start PCR at 96 ° C, 25 cycles of 10 seconds at 96 ° C, 5 seconds at 50 ° C, and 4 minutes at 60 ° C. After the cycle was quenched to 4 ℃. 1 μl of 3 M sodium acetate (pH 5.2) and 25 μl of 100% ethanol were added to each PCR amplification product, which was then allowed to stand on ice for 30 minutes to increase the precipitation of the PCR product, 4 ° C. and 15,000 rpm. PCR pellets were collected by centrifugation for 30 minutes at. After removing the supernatant, it was washed with 70% ethanol to remove salt and dried in air. PCR precipitated using 6 μl of sequencing loading buffer mixed with deionized formamide and 25 mM EDTA / 50 mg / ml blue dextran at a ratio of 5 to 1 The product was suspended. The suspension was denatured by standing at 90 ° C. for 2 minutes and then quenched on ice. To prepare a gel for auto sequencing, 18 g of urea was dissolved in 25 ml of distilled water, 5.625 ml of 40% acrylamide was added, and 0.5 g of mixed bed resin was added. It was added and then deionized by stirring well for 15 minutes. The gel solution was filtered through a 0.2 μM filter paper using a vacuum pump and then degassed for 5 minutes. To this was added 5 ml of 10 × TBE and filled with distilled water so that the final volume was 50 ml, to prepare 4.5% acrylamide / 6 M urea / 1 × TBE gel. The gel was placed on an automatic sequencing analyzer and loaded with the PCR product that was quenched for sequencing. After electrophoresis for 7 hours at 2 × Run, 1680 volts, the data were analyzed using ABI Prism Sequence Navigator and ABI Prism DNA sequencing software such as ABI Prism Factura.

By determining the DNA base sequence by the above method, the inventors determined the amino acid sequence of the active antibiotic peptide described in SEQ ID NO: 1 to SEQ ID NO: 3 specifically attaching to chondrocytes ( Fig. 1 and Fig. 2 ).

As described above, the peptide specifically attached to chondrocytes of the present invention can be used as a selective separation of biocompatible chondrocytes and as a connecting material between chondrocytes and biomolecules, and also useful for regeneration of nerve cells. have.

<110> JANG, JUN-HYEOG          CHUNG, CHONG PYOUNG          KIM, Hee Joong <120> Oligopeptide which binds specifically to chondrocytes <130> 2p-05-20 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> oligopeptide <400> 1 Arg Glu Asp   One <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> oligopeptide <400> 2 Gly Gly Xaa   One <210> 3 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> oligopeptide <400> 3 Gly Gly Arg   One

Claims (5)

Oligopeptides that specifically attach to chondrocytes having an amino acid sequence as set forth in SEQ ID NO: 1. delete delete delete delete

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