KR20090118704A - Composition comprising rankl for activating thymus regeneration - Google Patents

Abstract

PURPOSE: A composition for promoting thymus generation, containing a vector which expresses RANKL or RANKL is provided to promote the proliferation of thymus epithelial cell and thymus generation. CONSTITUTION: A composition for promoting thymus generation contains RANKL(receptor activator of nuclear factor(NF)-κB ligand) or its fragment with activity of enhancing function of thymus epithelial cells. The RANKL fragment is entire deletion or partial deletion intracellular domain and transmembrane domain.

Description

Composition Comprising RANKL for Activating Thymus Regeneration

The present invention relates to a thymic regeneration promoting composition comprising RANKL or a vector expressing RANKL having an activity for enhancing the function of thymic epithelial cells and a method for promoting thyroid regeneration by administering the composition to an individual.

The thyroid gland, the central organ of the immune system, is a complex epithelial tissue in which thyroid cell development occurs depending on the sequential action of morphological and visually different stromal cell compartments that constitute the microenvironment of the thyroid gland. Specific combinations of intercellular interactions in these microenvironments, cytokines and chemokines, induce thymic progenitor cells to differentiate into functional T cells through differentiation programs. Thyroid epithelial cells, which constitute a major part of the stromal cells of the thymus, provide crucial signals for the generation and selection of T cells in the thymus.

It was demonstrated that chest gland retraction was induced in experimental animals that had progressive regeneration following removal of the stimulus (Yoon S, et al., 1997; 12: 401-13; Milicevic NM, et al., J Comp Pathol 1984; 94 Lee HW, et al., Histochem Cell Biol 2005; 123: 491-500. Cyclophosphamide (CY) has shown selective inhibitory effects in various lymphoid cell populations and subpopulations (Turk JL and Poulter LW., Clin Exp Immunol 1972; 10: 285-96; Ozer H, et al. J Exp Med 1982; 155: 276-90; Zhu LP, et al., J Clin Invest 1987; 79: 1082-90). Application of CY is a long-established method for inducing thymic retraction and thymic regeneration (Yoon S, et al., 1997; 12: 401-13; Milicevic NM, et al., J Comp Pathol 1984 94: 197-202 Lee HW, et al., Histochem Cell Biol 2005; 123: 491-500). In addition, CY, a type of agent that induces acute thymic regression, is not only successfully used to study regenerative processes, but also allows changes in the prenatal development to reflect and better understand the postnatal developmental process. It can also be a very useful tool for the study of the process of thymocytopoiesis (Milicevic NM and Milicevic Z, Int Rev Cytol 2004; 235: 1-52).

RANKL (receptor activator of nuclear factor (NF) -κB ligand) is a type II membrane protein consisting of 316 amino acids with a molecular weight of 35 kDa and is a member of the TNF family (Yasuda). H, et al., Proc Natl Acad Sci USA 1998; 95: 3597-602). With an important role of RANKL in bone metabolism (Simonet WS, et al., Cell 1997; 89: 309-19), RANKL is involved in a variety of immune systems, as in other members of the TNF ligand and TNF receptor superfamily. (Coi JW, et al., Exp Mol Med 2005; 37: 78-85; Park H, et al., Exp Mol Med 2005; 37: 524-32). RANKL has been identified as a determinant of lymphocyte development and lymph node organogenesis (Anderson DM, et al., Nature 1997; 390: 175-9; Kong YY, et al., Immunol Cell Biol 1999; 77: 188-93; Kong YY, et al., Nature 1999; 397: 315-23). In addition, expression of RANKL was observed in early thymic cell progenitor cells (Anderson DM, et al., Nature 1997; 390: 175-9), and the fact that RANKL-deficient mice show a defect in early differentiation of T lymphocytes is RANKL. It suggests that it is a novel regulator of early thyroid cell development in this pre-TCR expression stage (Kong YY et al., Immunol Cell Biol 1999; 77: 188-93; Kong YY, et al., Nature 1999; 397: 315 -23). In previous studies, we found strongly upregulated expression of RANKL in thymic epithelial cells during thymic regeneration (Lee HW, et al., Histochem Cell Biol 2005; 123: 491-500). In addition, CD4 ⁺ CD3 ^- of ^- (inducer CD4 ⁺ CD3 cells) of the RANKL-RANK signal thymus medulla (thymic medulla) controls the generation of the expression Aire- thymic epithelial cells and central tolerance (central tolerance) in inducer cells It has recently been shown that the deficiency of RANK in thymic epithelial cells, which represents RANK as an important regulator, promotes the development of autoimmunity (Rossi SW et al., J Exp Med 2007; 204: 1267-72). And it has been known that RANKL-RANK signaling induces NF-κB activation through cyclin D1 in the proliferation of mammary epithelial cells (Cao Y et al., Cell 2001; 107: 763-75). In addition, RANKL has recently been reported to directly induce proliferation of mouse mammary epithelial cells through nuclear transfer of Id2, a positive regulator of cell cycle progression (Kim NS et al., Mol Cell Biol 2006; 26: 1002-13). .

Because T lymphocytes are the most important and central cell in all immune responses, the thymus gland is of great significance as the central organ of the immune system to protect the body from various diseases. As aging progresses, the function of several organs decreases, and the earliest contraction of the organs in our body is the thymus gland, and as a result, the main cause of the decrease in immunity in the elderly and lower resistance to various diseases such as infections and tumors. This can be As aging progresses, the mammary gland undergoes physiological degeneration, thereby reducing the production of T cells and replacing parenchyma with adipose tissue.

In addition to physiological thymus degeneration that occurs with aging, non-physiological thymus degeneration can be caused by various causes regardless of age. In particular, nonphysiological thymic retraction, which may occur before physiological contraction of the thyroid gland, ie, pediatric, adolescents, and the elderly, occurs abnormally, causing the body's immunity to be abnormally reduced to increase or be susceptible to various diseases. This can be a very serious problem because it can reduce the ability to cure the disease. There are a number of factors that can lead to non-physiological thymic retraction, such as severe stress, chemotherapy, radiation, corticosteroids, corticosteroids, sex hormones, steroid-based drugs, microbial origin or other toxic factors. And a myriad of factors that can be relatively easily exposed to many diseases, especially tumors, infections, chronic wasting diseases, and the like, can cause nonphysiological degeneration of the thymus. In these conditions, immunity is suppressed as the thyroid cells die in large quantities and the thymus gland size is drastically reduced, thus making the disease susceptible.

Thyroid epithelial cells are characterized by (1) surface molecules of thymic epithelial cells interacting with thymic cells and (2) secreting many biologically active factors, including many types of cytokines such as IL-7 and GM-CSF. Provides a critical microenvironment for proliferation, maturation, differentiation and selection (Le PT, et al., J Immunol 1988; 141: 1211-7; Aspinall R, et al., Ann NY Acad Sci 2004; 1019: 116-22 ).

IL-7 is a cytokine predominantly produced in thymic epithelial cells and plays an important role in the survival and proliferation of thymic cells (Aspinall R, et al., Ann N YAcad Sci 2004; 1019: 116-22). IL-7 has also been shown to have the potential to stimulate T cell rebuilding and thymic regeneration (Alpdogan O, et al., Blood 2001; 98: 2256-65; Mackall CL, et al., Blood 2001; 97: 1491 -7). GM-CSF is produced locally by different cellular elements such as thymic epithelial cells in the thymus (Le PT, et al., J Immunol 1988). Human GM-CSF has been reported to promote proliferation and differentiation of thymic cells (Yasuda Y, et al., Immunology 2002; 106: 212-21).

To prevent and treat a variety of diseases, including cancer, diabetes, AIDS, infections, autoimmune diseases, and other chronic wasting diseases, understand the molecular mechanisms that control thyroid regeneration and normalize immunity when thymus repression is suppressed. Developing methods to improve is of clinical importance, but the molecular mechanisms involved in thymic regeneration are largely unknown. Therefore, there is an urgent need for research to develop a method for restoring immunity by preventing the progression of thymus retraction due to physiological or non-physiological causes and inducing or promoting thyroid regeneration, reconstitution, and reactivation.

For the regeneration of degenerated thyroid gland, it is important to activate the function of thyroid epithelial cells, which provide a crucial microenvironment for the proliferation, maturation, differentiation and selection of thyroid cells. As a new and effective approach to development, we seek to identify factors that enhance the function of thymic epithelial cells.

Therefore, in the present invention, it was confirmed that RANKL acts on thyroid epithelial cells to improve, enhance and activate functions. RANKL was used as an active ingredient for regeneration of thymus.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention relates to a thymic regeneration promoting composition comprising RANKL or a fragment thereof having an activity of enhancing the function of thymic epithelial cells.

The active ingredient used in the thymic regeneration promoting composition of the present invention is a mechanism that increases the proliferation of thymic epithelial cells and the interaction between thymic cell-chest epithelial cells and increases the expression of various thyroid cell formation promoting / regulatory factors. RANKL has the activity to promote.

In the present invention, the terms 'regeneration', 'reactivation' and 'reconstitution' are used interchangeably in a similar sense, meaning that the function of the retracted thyroid gland is improved and restored to the active state.

RANKL used in the compositions of the present invention may be derived from not only humans, but also all animals having RANKL, or proteins corresponding to the function of RANKL, such as horses, sheep, pigs, goats, camels, nutrition, dogs, and the like. .

It may also be an active fragment having not only a protein consisting of the entire amino acid sequence, but also some amino acid sequences which are biologically active. As long as RANKL has thymic regeneration activity, the form of RANKL fragment is not particularly limited, but is preferably a whole region or a partial region of the transmembrane domain, and a whole region or a partial region of the intracellular domain. This is a fragment of deleted RANKL. More preferably, it is a fragment consisting only of the RANKL extracellular domain, in which the entire region of the transmembrane domain and the entire region of the intracellular domain are deleted. It may also be in the form of shorter fragments derived from the extracellular domain, containing only the essential moieties exhibiting RANKL activity of the invention.

Provided by the present invention, RANKL having a thymic regeneration promoting effect or a fragment thereof may be in monomeric or multimeric form. The multimer may be formed by various methods conventionally provided in the art, and the method of forming the multimer is not particularly limited. For example, the multimers may include a variety of sequences that induce multimer formation, specifically, isoleucine zipper (ILZ) sequences that induce trimer forms, or surfactant protien-D (SP-D) that induces dodecamer formation. It can also manufacture. Alternatively, the multimer may be prepared by a method of linking a polypeptide prepared with a monomer into two or more multimers using a linker or the like.

In addition, RANKL or fragments thereof used in the compositions of the present invention include amino acid sequence variants as well as wild type amino acid sequences derived from humans and animals. A sequence variant refers to a protein in which the natural amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). In addition, RANKL or a fragment thereof may be in a form to be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.

Such variants or modifications include proteins having modifications that increase or decrease functional equivalents or physicochemical properties with activities substantially equivalent to natural proteins. Preferably the variant is a modified physicochemical property of the protein. For example, physical factors such as temperature, moisture, pH, electrolytes, reducing sugars, pressurization, drying, freezing, interfacial tension, light rays, freezing and thawing, high concentration conditions, acids, alkalis, neutral salts, organic solvents, metal ions It is a variant with increased structural stability to the external environment of chemical factors such as redox, proteases and the like.

The RANKL or fragment thereof of the present invention can be obtained by isolation from a source of natural origin, by chemical synthesis, or by introducing a recombinant expression vector encoding RANKL or a fragment thereof into a suitable host, expressing it, and isolating therefrom. have.

In addition, the composition of the present invention may further comprise one or more components in addition to RANKL or a fragment thereof, existing therapeutic components of each disease, components that increase the immune response, and the like. Specifically, components that increase the immune response are cytokines including interferon (INF), tumor necrosis factor (TNF), interleukin (IL), colony-stimulating factor (CSF) and growth factor (GF). IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-15, IL- 16, IL-18, IL-23, IL-24, erythropoietin, GM-CSF, MSF, FLT-3 ligand, EGF, fibroblast growth factor (FGF), aFGF (FGF-1), bFGF (FGF -2), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, insulin-like growth factor-1 (IGF-1), IGF-2, pulsed endothelial growth factor (VEGF), IFN- Cytokines such as γ, IFN-α, IFN-β, TGF-α, TGF-β1, TGF-β2, SDF-1, TARC, TECK, etc. can be exemplified, but are not limited thereto. In addition, the components that increase the immune response is chemokine, BCA-1 / BLC-1, BRAK / Kec, CXCL16, CXCR3, ENA-78 / LIX, Eotaxin-1, Eotaxin-2 / MPIF-2, Exodus-2 / SLC, Pratalcaine / Nurotaxin, GROalpha / MGSA, HCC-1, I-TAC, Limpotactin / ATAC / SCM, MCP-1 / MCAF, MCP-3, MCP-4, MDC / STCP-1, ABCD- 1, MIP-1a, MIP-1b, MIP-2a / GROb, MIP-3a / Exodus / LARC, MIP-3b / Exodus-3 / ELC, MIP-4 / PARC / DC-CK1, PF-4, RANTES, etc. It can be illustrated, but is not limited thereto.

The RANKL or fragment thereof of the present invention may be in the form of a salt. Proteins can form salts by the addition of acids, for example inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (acetic acid, halo acetic acid such as trifluoroacetic acid, propionic acid, Maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid, etc.), acidic polysaccharides (hyaluronic acid, chondroitin sulfate, arginine acid, etc.), Or organic sulfonic acids (methane sulfonic acid, p-toluene sulfonic acid, etc.) including sulfonic acid sugar esters such as chondroitin sulfate.

In addition, the RANKL or fragment thereof of the present invention is a biocompatible polymer such as polyethylene glycol, polypropylene glycol (PPG), ethylene glycol-propylene glycol copolymer (co-poly (ethylene / propylene) ) glycol), polyoxyethylene (POE: polyoxyethane), polyurethane, polyphosphazene, polysaccharide, polysaccharide, dextran, polyvinyl alcohol, polyvinyl fatigue Livinyl (polyvinyl pyrrolidones), polyvinyl ethyl ether polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, It may be modified with heparin or the like to improve stability and metabolic rate in vivo.

RANKL or a fragment thereof used to promote thyroid regeneration of the present invention may be provided in the form of a protein capable of expressing RANKL or a fragment thereof in a cell for use in gene therapy or vaccine.

In another aspect, the invention relates to a thymic regeneration promoting composition comprising a vector expressing RANKL or a fragment thereof.

In the present invention, "vector" means a means for introducing a nucleic acid sequence (eg, DNA, RNA, etc.) encoding RANKL or a fragment thereof into a host cell, and may have the form of a virus (viral particle).

In the vector of the present invention, a nucleic acid sequence encoding RANKL or a fragment thereof is functionally linked to an expression control sequence. For example, a vector includes signal or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and can be prepared in various ways depending on the purpose. . The expression vector also includes a selectable marker and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA. Vectors of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.

Vectors of the invention include plasmid vectors, cosmid vectors, viral vectors and the like. Preferably, the virus vector is a human immunodeficiency virus HIV (Murine leukemia virus) MLV (Avian sarcoma / leukosis virus), SNV (Spleen necrosis virus), RSV (Rous sarcoma virus), MMTV (Mouse mammary tumor virus) Retroviruses such as (Retrovirus), Adenovirus (Adenovirus), Adeno-associated virus (Adeno-associated virus), Herpes simplex virus (Herpes simplex virus) and the like, but are not limited to these examples. The composition of the present invention is more preferably provided as a virus (viral particle).

Vectors having nucleic acid sequences encoding RANKL or fragments thereof are known in the art, such as direct injection into naked DNA in the form of vectors, or cationic polymers such as liposomes, DOTMA or DOTAP. And the like can be delivered intracellularly to an individual for therapeutic or prophylactic purposes.

A thymic regeneration promoting composition comprising a vector expressing (i) RANKL or a fragment thereof or (ii) RANKL or a fragment thereof of the invention is a pharmaceutically acceptable carrier, for example, according to the mode of administration and formulation. One or more components selected from buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, binders, lubricants, disintegrants, excipients, dispersants, suspending agents, pigments and flavorings and the like.

Since the composition of the present invention has the activity of restoring, restoring, improving and normalizing immunity by directly regenerating and reactivating the functions of the thymus and T cells, physiological causes such as aging or stress, anticancer chemicals, radiation, All thyroid contractions may or may not be caused by non-physiological causes such as neocortical hormones, adrenal cortical stimulating hormones, sex hormones, steroid drugs, microbial origin or other toxic factors and diseases such as tumors, infections and chronic wasting diseases. It can be used as a new preventive and therapeutic agent to prevent the progression of the thymus retraction and promote the regeneration of the thymus.

In another aspect, the present invention provides a method for administering to a subject a thyroid regeneration promoting composition comprising (i) RANKL or a fragment thereof or (ii) a vector expressing RANKL or a fragment thereof having activity to enhance the function of thymic epithelial cells. A method for promoting thymic regeneration comprising a step.

The composition for promoting thymus regeneration of the present invention may be administered to all the individuals whose thyroid contraction is caused or may be induced by physiological causes or physiological causes for the purpose of treatment or prevention. Eggplant includes both humans with an immune system and animals such as horses, sheep, pigs, goats, camels, antelopes, and dogs.

Formulated in a suitable formulation with a pharmaceutical carrier to be administered in a variety of routes, for example oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, topical, intranasal, pulmonary, rectal May be administered.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the disease type, severity, age, sex, drug of the individual. Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

RANKL upregulates transcription of anti-apoptotic genes such as Bcl-2 and Bcl-xL in thymic epithelial cells, but at the same time downregulates Bax, a pre-apoptotic gene, and promotes proliferation of thyroid epithelial cells. Rather, it increases the expression of cell adhesion molecules ICAM-1 and VCAM-1 to enhance adhesion between thyroid cells and thyroid epithelial cells, thereby increasing the interaction between thyroid cells and thyroid epithelial cells and promoting thyroid cell formation in thyroid epithelial cells. Increased expression of the regulatory factors IL-7, GM-CSF. RANKL also directly promoted thymic regeneration.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

< Example  1: Materials and Methods>

1-1: Cell Line and Cell Culture

Mouse thyroid subcortical or thymic endothelial cells (427.1; SNEC; thymic subcapsular cortex or thymic nurseepithelial cells), cortical reticulum epithelial cells (1308.1; CREC; deep cortex or cortical reticular epithelial cells) and stromal epithelial cells (6.1.7; Regeneration, maintenance and functional properties of medullary epithelial cells (MEC) are described by Faas et al. Eur J Immunol 1993; 23: 1201-14. The cell lines were DMEM (Dulbecco's modified Eagle's medium) containing 10% (v / v) fetal bovine serum (FBS, GIBCO BRL) and 2 mM glutamine (Sigma, St. Louis, MO, USA). (GIBCO BRL, Grand Island, NY, USA).

1-2: animal, experimental thyroid regeneration model and RANKL In vivo treatment of

Male C57BL / 6 mice were purchased from Daehan Biolink (Seoul, Korea) and maintained at sterile, moisture-controlled breeding facilities at 24 ° C., 12 h day / night conditions. Mice were used between 8 and 10 weeks of age and had a single intraperitoneal dose of CY (450 mg / kg, as described in previous studies (Yoon et al. , 1997, 2003; Lee et al. , 2005, 2007)). Sigma) was administered. RANKL was administered intraperitoneally at a dose of 10 μg per mouse. RANKL was administered 8 hours after CY treatment and continued daily administration for 3 days. Finally, mice were sacrificed 24 hours after RANKL.

1-3: recombination RANKL  And OPG  Preparation of Protein

Recombinant RANKL and OPG proteins were prepared using the pET-32b expression system (Novagen, Madison, WI, USA). To amplify the fragment encoding the extracellular domain of RANKL and the fragment encoding the full-length open reading frame (ORF) of OPG, RANKL-specific of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4 RT-PCR was performed using total RNA isolated from mouse thymus with primers. The amplified PCR product was cloned into pGEM T easy vector (Promega, Madison, Wis., USA). DNA fragments of RANKL and OPG digested with Hin dIII and Xho I were inserted into the pET-32b expression vector and transformed with E. coli strain BL21 (DE3). Overexpression of RANKL and OPG proteins was induced by the addition of 1 mM IPTG and isolated by affinity chromatography with Ni-NTA agarose resin (Qiagen, Valencia, CA, USA).

The sequence of the primer used is as follows.

RANKL sense primer, 5'-AAGCTTATGTTCCATGTTTCTTTTAGAT-3 '(SEQ ID NO: 1)

RANKL antisense primer, 5'-CTCGAGTTTATACTGCCCTTCAAAATT-3 '(SEQ ID NO: 2)

OPG sense primer, 5'-AAGCTTATGAACAAGTGGCTGTGCTGCG-3 '(SEQ ID NO: 3)

OPG antisense primer, 5'-CTCGAGTTATAAGCAGCTTATTTTCACG-3 '(SEQ ID NO: 4)

1-4: Cell Proliferation Assay ( cell proliferation assay )

Thymic epithelial cell lines were plated in 96-well microplates at 2 × 10 ³ cells / well in DMEM containing 10% FBS and transferred to medium containing 1% FBS. . The cell lines were then treated with thioredoxin (0.5 μg / ml), RANKL (0.5 μg / ml), and a mixture of RANKL (0.5 μg / ml) and OPG (2 μg / ml), respectively, 1, Incubation was for 2, 3, 4 and 6 days. Soluble OPG protein has been used as a decay receptor to inhibit the effects of RANKL on proliferation of thymic epithelial cells. Cell proliferation was analyzed using Cell Proliferation Reagent WST-1 (Roche, Mannheim, Germany).

1-5: Thyroid cells  detach

Small sections of the thymus were made into thyroid capsules and slowly stirred at 4 ° C. for 40 minutes using a magnetic stirrer in RPMI-1640 (GIBCO BRL). The supernatant 70-μm mesh was passed through three times and centrifuged. Cell pellets were resuspended in ACK Lysis solution and thymic cell supernatant was washed with HBSS (Hanks' balanced salt solution, GIBCO BRL) buffer and the number of surviving cells stained with trypan blue before hemocyto Counting was done with a hemocytometer.

1-6: Thyroid epithelial cells Thyroid cells  Adhesion

Analysis of thymic cell adhesion to thymic epithelial cells was based on the method described by Barda-Saad M, et al., Exp Hematol 1996; 24: 386-91. Briefly, freshly isolated thymic cells from mice were seeded onto thymic epithelial cell lines in 6-well microplates in an amount of 1.5 × 10 ⁷ cells / well in DMEM containing 10% FBS, RANKL (0.5 μg / ml) and a mixture of RANKL (0.5 μg / ml) and OPG (2 μg / ml) were incubated for 4 hours. After light washing to remove non-adhered thyroid cells, thyroid cells attached to thyroid epithelial cells were collected by treatment with 0.5 mM EDTA (Sigma) in PBS solution, stained with trypan blue, and counted using a hemocytometer. .

1-7: Reverse transcription - Polymerase Chain Reaction ( RT - PCR ) analysis

Total RNA from each sample was isolated using Easy-Blue RNA Extraction Reagent (Intron, Seoul, Korea). First strand cDNA was obtained with a reverse transcription system (Gibco BRL) and cDNA was used as a template for PCR amplification using gene-specific primers. Primers used for RT-PCR are described in Table 1. PCR amplification was carried out in 25 or 30 cycles for 60 seconds at 94 ℃, 60 seconds at 57 ℃, 60 seconds at 72 ℃, the amplified product was analyzed by electrophoresis. Band intensities of the PCR products were measured using an image analysis program (MetaMorph, Universal Imaging Corp., Downingtown, PA, USA).

1-8: Immunocytochemistry

For immunofluorescence, 12 mm diameter glass coverslips (Paul Marienfeld, Lauda-Konigshofen, Germany) coated with 50 μg / ml poly-l-lysine (Sigma) in mouse thyroid epithelial cell lines ) And fixed with cold 4% paraformaldehyde under 0.1 M phosphate buffer for 10 minutes. Next, the fixative was removed by washing with cold PBS three times for 5 minutes, and the cells were then incubated with 0.5% Triton X-100 under PBS solution for 5 minutes. The cells were washed again with cold PBS and incubated with 2% bovine serum albumin (Sigma) for 60 minutes at room temperature. Discard excess solution and dilute cells to 1: 100 goat polyclonal anti-ICAM-1 antibody (sc-1511, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 1 It was incubated at 4 ° C. for 16-18 hours with a goat polyclonal anti-VCAM-1 antibody (sc-1504, Santa Cruz Biotechnology) diluted to 100: 100. After incubation with primary antibody, cells were washed three times for 5 minutes with cold PBS and F (ab ′) ₂ fragments of affinity-isolated donkey anti-goat FITC-conjugated antibody diluted 1: 100 ( Jackson ImmunoResearch Laboratories, West Grove, Pa., USA). It was then washed with cold PBS and fixed with glass slides using Vectashield (Vector Laboratories, Burlingame, Calif., USA). Staining of the control group includes (1) omitting the primary antibody from the reaction sequence or solid to non-immune donkey serum, and (2) omitting the secondary antibody from the reaction sequence. Labeled cells were observed with an Olympus BX50 microscope with fluorescent epi-illumination. The micrographs were captured digitally at an 1,360 x 1,024 pixel resolution with an Olympus DP70 digital camera.

1-9: Adult Thyroid Culture Adult thymic organ culture : ATOC )

Thymus was obtained from mice 3 days after CY treatment. The thymus was cut into small pieces and soaked in HEPES-buffered RPMI with 10% FBS (GIBCO BRL). Thymus pieces were incubated in 0.45 μm filters (Millipore, Bedford, Mass., USA) and placed on top of Gelfoam sponges (Upjohn, Kalamazoo, Mass., USA). Each gelfoam sponge comprises 2 mM glutamine, 1x nonnessential amino acids, 5x 10 ^-5 M 2β-ME, and 20% heat-inactivated FBS The cells were hydrated overnight in 6-well plates containing HEPES-buffered DMEM with sodium pyruvate. Thymus sections were incubated in a 37 ° C. incubator with 5% CO ₂ in air. HEPES-buffered DMEM was supplemented on day 7 and maintained for at least 14 days. Cultured thymic sections were cut to a thickness of 5 μm in Reichert cryostat, and slides were H & E stained.

1-10: Statistical Analysis

Data is expressed as mean ± standard error (SEM) of each condition. The results were statistically analyzed with two-tailed Student's t- test ( t- test). Statistical significance was obtained when P <0.05.

< Example  2: RANKL Silver mouse Thyroid epithelial cells  Multiplication Facilitation >

Before examining the effects of RANKL on the activity of thyroid epithelial cells, the presence of RANKL and RANKL transcripts was confirmed by RT-PCR analysis in three types of mouse thyroid epithelial cell lines (FIG. 1). RANKL markedly promoted proliferation of SNEC, CREC and MEC (FIG. 2). The effect of RANKL on proliferation of thymic epithelial cells was completely blocked when co-culture of RANKL and OPG (Fig. 2). RANKL affected thymic epithelial cells in a dose- and time-dependent manner. The thioredoxin control had no effect on the proliferation of thymic epithelial cells like the non-administered control (FIG. 2).

< Example  3: RANKL Silver mouse In thyroid epithelial cells Bcl -2 and Bcl - xL Upregulates expression of Bax Down-regulates expression of>

To investigate the effect of RANKL on the expression of Bcl-2, Bcl-xL and Bax mRNA in three types of mouse thyroid epithelial cell lines, these cells were treated with H ₂ O ₂ to induce apoptosis, followed by RANKL treatment. Incubate for 1 or 2 days. RT-PCR analysis showed that after treatment of thymic epithelial cells with RANKL, the expression of Bcl-2 and Bcl-xL mRNAs was significantly upregulated on both day 1 and day 2 in all three cell lines, but Bax mRNA was upregulated under the same conditions. Markedly decreased (FIG. 3).

< Example  4: RANKL Silver mouse Thyroid epithelial cells Thyroid  Enhance adhesion and mouse In thyroid epithelial cells ICAM -1 and VCAM Increases expression of -1

All three types of thyroid epithelial cells treated with RANKL significantly increased the number of attached thyroid cells (FIG. 4). When RANKL and OPG were simultaneously treated, the effect of RANKL to attach thymic cells to thymic epithelial cells was completely inhibited (FIG. 4). These results demonstrate that RANKL promotes the attachment of thymic cells to thymic epithelial cells. The effect of RANKL on ICAM-1 and VCAM-1 mRNA expression was analyzed by RT-PCR in three types of mouse thyroid epithelial cell lines. ICAM-1 mRNA expression was upregulated in three types of thyroid epithelial cells 6-24 hours after RANKL treatment (FIG. 5A). VCAM-1 mRNA expression was upregulated in CREC and MEC between 6 and 24 hours after RANKL treatment, but no difference was observed in SNEC under the same conditions (FIG. 5A). The upregulatory effects of RANKL on the expression of ICAM-1 and VCAM-1 mRNA in these thymic epithelial cell lines were completely blocked when RANKL and OPG were simultaneously treated (FIG. 5B). In addition, this upregulatory effect of RANKL on mRNA expression of ICAM-1 and VCAM-1 in thymic epithelial cell lines was confirmed at the protein level by immunofluorescence staining. Increased ICAM-1 and VCAM-1 protein expression in thymic epithelial cells after RANKL treatment supports the results of RT-PCR analysis (FIG. 6). These results suggest that RANKL upregulates expression of ICAM-1 and VCAM-1 to enhance the attachment of thymic cells to mouse thyroid epithelial cells.

< Example  5: RANKL Silver mouse In thyroid epithelial cells IL -7 lesson GM - CSF mRNA Up-regulates expression of>

The effect of RANKL on the expression of IL-7 and GM-CSF mRNA was evaluated by RT-PCR analysis in three types of mouse thyroid cell lines. IL-7 and GM-CSF mRNA expression gradually increased in all three cell types during 6-24 hours after RANKL treatment (FIG. 7A). The effects of RANKL stimulating the expression of IL-7 and GM-CSF mRNA in these thymic epithelial cell lines were completely inhibited when co-culture of RANKL with OPG (FIG. 7B).

< Example  6: RANKL Have thymus play in in vivo and ex vivo Promote >

Significant increases in the size and weight of the thymus were observed in the RANKL-treated group and rapid regeneration of the thymus after RANKL treatment was clearly visually compared with the saline-treated controls (FIGS. 8A and B). Then, using the ATOC system to study the development of T cells in ex vivo, the in vivo stimulatory effects of RANKL on mammary gland regeneration may be due to RANKL acting directly on the mammary gland, or on the indirect or systemic effects of RANKL. It was intended to determine whether mediated by. In ATOC experiments, the cytoplasm of the thymus was significantly increased in the group treated with RANKL (FIG. 8C), which means that RANKL has the ability to exert a direct formation promoting effect on the thymus to promote mammary gland regeneration.

The mammary gland regeneration promoting composition comprising the RANKL of the present invention or a fragment thereof may be used for physiological causes such as aging or stress, anticancer chemicals, irradiation, corticosteroids, corticosteroids, sex hormones, steroid-based drugs, microorganisms, or the like. Non-physiological factors such as various toxic factors and diseases such as tumors, infections and chronic wasting diseases can prevent the progression of thymus retraction and promote regeneration and T cell reconstruction of the thymus. As new prophylactic and therapeutic agents, it is expected to be used in medical and veterinary fields.

Figure 1 shows the treatment of RANKL (0.5 μg / ml) in three types of mouse thyroid epithelial cell lines (SNEC, CREC and MEC), and after 6, 12, 24 and 48 hours of incubation, the genes of (A) RANKL and GAPDH Expression was examined by RT-PCR analysis (B) the intensity of each band was measured by scanning densitometry (mean ± SD) is the result represented by the mean ± standard deviation (mean ± SD). Data is expressed as the ratio of RANKL mRNA normalized to GAPDH mRNA. ^{* P} <0.05 was compared with the corresponding control values that are measured at t -test.

FIG. 2 shows the effects of RANKL on the proliferation of thymic epithelial cell lines in three types of mouse thyroid epithelial cell lines SNEC, CREC and MEC. , And a mixture of RANKL (0.5 μg / ml) and OPG (2 μg / ml), respectively, were incubated for 4 days. Cell proliferation rates were performed by WST-1 analysis and the data were expressed as mean ± standard deviation (mean ± SD). ^* P <0.01 was compared with the corresponding control value measured by t- test.

FIG. 3 shows treatment of RANKL (0.5 μg / ml) and H ₂ O ₂ (100 μM) in three types of mouse thyroid epithelial cell lines (SNEC, CREC and MEC), followed by one or two days of culture, followed by Bcl − 2, Bcl-xL and Bax expression is analyzed. Apoptosis was induced by treatment with H ₂ O ₂ . The control group was treated with 100 μM of H ₂ O ₂ . (A) The gene expression of Bcl-2, Bcl-xL, Bax and GAPDH was examined by RT-PCR analysis. (B) The intensity of each band was measured by scanning densitometry and the mean ± standard deviation (mean ± SD). Represented by. Data is expressed as the ratio of Bcl-2, Bcl-xL and Bax mRNA normalized to GAPDH mRNA. ^{* P} <0.05 was compared with the corresponding control values that are measured at t -test.

4 shows seeding of thymic cells into the cultured thyroid epithelial layer and of thioredoxin (0.5 μg / ml), RANKL (0.5 μg / ml), and RANKL (0.5 μg / ml) and OPG (2 μg / ml). After treatment of each mixture and incubation for 4 hours, the result of quantitative analysis of thymic cell adhesion to mouse thyroid epithelial cell lines (SNEC, CREC and MEC). ^* P <0.05 and ^** P <0.01 were compared with corresponding control values measured by t- test.

5 shows (A) RANKL (0.5 μg / ml) in three types of mouse thyroid epithelial cell lines (SNEC, CREC and MEC), and (B) T after incubation for 6, 12, 24, and 48 hours. Oredoxin (0.5 μg / ml), RANKL (0.5 μg / ml), and a mixture of RANKL (0.5 μg / ml) and OPG (2 μg / ml), respectively, were treated with ICAM-1, Gene expression of VCAM-1 and GAPDH was examined by RT-PCR analysis. Data are expressed as mean ± SD. ^{* P} <0.05 was compared with the corresponding control values that are measured at t -test. Lane 1: untreated control, lane 2: RANKL-treated group, lane 3: mixed RANKL and OPG-treated group, 4: thioredoxin-treated group

FIG. 6 shows the results of analysis of ICAM-1 and VCAM-1 protein expression after treatment with RANKL (0.5 μg / ml) in three types of mouse thyroid epithelial cell lines (SNEC, CREC and MEC) and cultured for 24 hours. Representative diagrams of immunofluorescence staining are the results of ICAM-1 in CREC and VCAM-1 in MEC.

7 shows (A) RANKL (0.5 μg / ml) in three types of mouse thyroid epithelial cell lines (SNEC, CREC and MEC), and (B) T after incubation for 6, 12, 24, and 48 hours. Oredoxine (0.5 μg / ml), RANKL (0.5 μg / ml), and a mixture of RANKL (0.5 μg / ml) and OPG (2 μg / ml), respectively, and incubated cells for 24 hours before IL-7, The mRNA expression of GM-CSF and GAPDH is analyzed. Data are expressed as mean ± SD. ^{* P} <0.05 was compared with the corresponding control values that are measured at t -test. Lane 1: untreated control, lane 2: RANKL-treated group, lane 3: mixed RANKL and OPG-treated group, 4: thioredoxin-treated group

FIG. 8 illustrates the effect of RANKL on regeneration of thymus after acute mouse thymus retraction induced by CY treatment in mature male C57BL / 6 mice. CY-treated mice were intraperitoneally treated with 10 μg of RANKL or saline daily for 3 days. One day after the last treatment, the size (A) and weight (B) of the thymus gland were measured in untreated control mice (normal), 3 days CY and saline-treated control mice (CY3d Control), and 3 days CY and It is the result measured in RANKL-treated group mouse (CY3d RANKL). (C) The results of ATOC experiments at the histological level, showing untreated control and 0.5 μg / ml RANKL-treated thymus after 10 days of ATOC with thymus at day 3 after CY treatment. Data are expressed as mean ± SD. Hematoxylin-Eosin was stained and magnification was x400. ^* P <0.05 compared with the corresponding saline-treated control values measured by t- test.

<110> Pusan National University Industry-University Cooperation Foundation <120> Composition Comprising RANKL for Activating Thymus Regeneration <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RANKL sense primer <400> 1 aagcttatgt tccatgtttc ttttagat 28 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> RANKL antisense primer <400> 2 ctcgagttta tactgccctt caaaatt 27 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> OPG sense primer <400> 3 aagcttatga acaagtggct gtgctgcg 28 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> OPG antisense primer <400> 4 ctcgagttat aagcagctta ttttcacg 28 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VCAM-1 sense primer <400> 5 cccaaggatc cagagattca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VCAM-1 antisense primer <400> 6 acgtcagaac aaccgaatcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 sense primer <400> 7 gagagtggac ccaactggaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 antisense primer <400> 8 ctttgggatg gtagctggaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-2 sense primer <400> 9 agtcgggact tgaagtgcca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-2 antisense primer <400> 10 ggtacatcat tgataatgca 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Bax sense primer <400> 11 ggtttcatcc aggatcgagc agg 23 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Bax antisense primer <400> 12 acaaagatgg tcacggtctg cc 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-xL sense primer <400> 13 ttggacaatg gactggttga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-xL antisense primer <400> 14 ctgctcaaag ctctgatacg 20 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-7 sense primer <400> 15 gccctgtcac atcatctgag tgcc 24 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-7 antisense primer <400> 16 caggaggcat ccaggaactt ctg 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GM-CSF sense primer <400> 17 gtcacccggc cttggaagca t 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GM-CSF antisense primer <400> 18 acagtccgtt tccggagttg g 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RANK sense primer <400> 19 aaaccttgga ccaactgcac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RANK antisense primer <400> 20 accatcttct cctcccgagt 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense primer <400> 21 caactccctc aagattgtca gc 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense primer <400> 22 gggagttgct gttgaagtca ca 22  

Claims (11)

A thymic regeneration promoting composition comprising RANKL or a fragment thereof having an activity of enhancing the function of thymic epithelial cells. The composition of claim 1, wherein the RANKL fragment is deleted in the whole or part of the transmembrane domain and in the whole or part of the intracellular domain in RANKL. The composition of claim 2, wherein the RANKL fragment is an extracellular domain of RANKL. The composition of claim 1 further comprising a cytokine, chemokine or both components. A thyroid regeneration promoting composition comprising a vector expressing RANKL or a fragment thereof having an activity of enhancing the function of thymic epithelial cells. The composition of claim 5, wherein the RANKL fragment is deleted in the whole or part of the transmembrane domain and in the whole or part of the intracellular domain in RANKL. The composition of claim 6, wherein the RANKL fragment is an extracellular domain of RANKL. The composition of claim 5, wherein the vector is a viral or nonviral vector. The composition of claim 8, wherein the viral vector is selected from the group consisting of retroviruses, adenoviruses, adeno-associated-viruses, herpes simplex virus, and lentivirus vectors. A method of promoting thymic regeneration comprising administering to a subject the composition of claim 1. The method of claim 10, wherein the subject is a physiological or nonphysiological mammary gland degeneration.

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