JPH0799977A - Antisense oligodeoxynucleotide for fibrogenesis inducing cytokine and use thereof - Google Patents

Abstract

PURPOSE: To obtain a new anti-sense oligodeoxynucleotide which is complementarily bound to the mRNA of fibrogenic cytokine to inhibit the genetic expression thereof and which is useful as a scarring inhibitor for preventing the scarring of a wound portion.

CONSTITUTION: This new anti-sense oligodeoxynucleotide or its derivative can complementarily be bound to the mRNA of fibrogenic cytokine [for example, tumor necrosis factor-α(TNF-α)] to inhibit the genetic expression thereof. The new anti-sense oligodeoxynucleotide is obtained in a large amount by synthesizing the anti-sense oligodeoxynucleotide to the fibrogenic cytokine by use of an automatically DNA-synthesizing machine, ligating linker sequences to both the ends of the synthesized product, inserting the product into a proper plasmid, transducing the recombined plasmid into a host cell, culturing the transformant and subsequently recovering the anti-sense oligodeoxynucleotide.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to fibrogenesis-inducing (fibro
genic) novel antisense oligodeoxynucleotides against cytokines. In particular, the present invention relates to a novel antisense oligodeoxynucleotide capable of suppressing gene formation of a fibrogenesis-inducing cytokine secreted in large amounts from a wound site and preventing scar formation at the wound site.

[0002]

2. Description of the Related Art Immune reaction, inflammatory reaction and tissue regeneration reaction
The (tissue repair response) function protects the host from the dangerous surrounding environment. However, when such a reaction does not occur gently or excessively occurs, such a reaction may damage the host rather than protect the host. As an example of a biological process in which such reactions are involved, scars are formed in the healing process of wounds as one of the tissue regeneration reactions to trauma, surgery, burns, etc. It can significantly impede tissue growth, impair function and impair the appearance of the host.

Wound healing processes include acute and chronic inflammation, cell migration, angiogenesis and extracellular matrix (ECM).
It is a very elaborate tissue reaction in which accumulation and the like occur sequentially.
When a wound occurs, the blood vessels surrounding the wound tissue are damaged, causing local bleeding at the wound site and coagulation. If fibrinogen present in this clot forms a fibrin gel, plasma proteins such as fibronectin will enter the gel. In addition to this, inflammatory cells, fibroblasts and angiogenic cells enter the gel to melt the gel, while fibronectin causes components of ECM such as collagen and proteoglycan to surround the wound tissue. Accumulate in. By such a process, the fibrin matrix originally present in the tissue is replaced by the granulation tissue, a scar is formed in that place over time, and the keratinocytes migrate with the accumulation of ECM and the epithelial membrane moves. To prevent water loss and bacterial invasion. Such a series of processes related to wound healing include immune cells, inflammatory cells and mesenchymal cells of the tissue at the injury site and transforming growth factor β (TGF-β), platelet-derived growth factor (PDGF), fibroblast. It is carried out by the interaction of various cytokines such as cell growth factor (FGF) and fibroblast activator (FAF) with ECM such as collagen, fibronectin, tenascin and proteoglycan.

As described above, it is clear that TGF-β and PDGF play the most important role among various cytokines that promote the proliferation of fibroblasts at the wound site and increase the accumulation of ECM. became. TGF-
β not only significantly increases fibroblast proliferation,
It is known that PDGF increases gene expression of ECM such as collagen and fibronectin, and PDGF has little effect on collagen synthesis, but significantly promotes proliferation of fibroblasts.

Furthermore, according to a recent report, fetal wound (f
In the case of (etal wounds), it was revealed that not only scars were not formed during the wound healing process, but there was less inflammatory reaction, and the secretion of cytokines was less than that of adult wounds (see: White BJ
tby, DJ) and MW Ferguson, 991, Immunohistochemical localization of growth factors in fetal wound healing (Immunohisto-
chemical localization of growth factors infetal wo
und healing), Developmental Biology
(Dev. Biol.) 147: 207-215]. However, the above-referenced document also mentions that TG, even in the case of fetal wounds.
Injecting F-β into the tissue surrounding the wound creates a scar,
It has been reported that glomerulonephritis is induced.

[0006] As the relationship between such scar formation and the presence of cytokines became clear, it was proposed that the formation of scar tissue can be suppressed by regulating cytokines. For example, Shah M
During the wound healing process of (Shah M.) et al.
Due to the fact that F-β is physiologically excessively secreted, TG was generated around the wound after the artificial wound formation in the rat.
As a result of the injection of the neutralizing antibody (NA) against F-β, not only the tensile strength (tension strength) of the tissue does not change even after healing at the wound site treated with the neutralizing antibody, but the neutralizing antibody- It was possible to observe that the skin tissue findings were closer to normal compared to the untreated control wounds.
[Shah M., Forman Diem (Fo
reman DM.), Fergson M. W. Jay (F
erguson MWJ.), 1992, Controlling scarring in adult wounds by neutralizing antibodies against transforming growth factor-β.
tralizing antibody to transforming growth factor
β), Lancet 339: 213-214]. In addition, in the present experiment, the wound treated with the neutralizing antibody was healed without forming scars, whereas the wound site of the neutralizing antibody-untreated control group was compared to the wound treated with the neutralizing antibody. Therefore, not only more inflammatory cells were observed, but also the fact that ECM such as collagen and fibronectin was accumulated in a larger amount was confirmed. However, such a neutralizing antibody is very expensive, unstable, and has problems in storage, and thus it has been difficult to put it into practical use.

In addition, the border double (Border
WA) et al., TG as a kind of proteoglycan ECM
It is able to bind to the receptor for F-β and therefore T
Decorin (d) that can act as an inhibitor against GF-β
As a result of administering ecorin) to an experimental animal model of glomerulonephritis, it was confirmed that decorin can prevent glomerulonephritis by suppressing fibrinoid degeneration [Border WA, Rousrati. E. (Rouslahti E.), 1992, Transforming growth factor-β in disease: Transforming growth factor-β in dise
ase: The dark side of tissue repair), Journal of Clinical Investigation (J. Cli
n. Invest.), 90: 1-7]. However, proteinaceous drugs such as decorin have a macromolecular structure and are therefore difficult to penetrate into cells, and therefore can be used only by oral administration. Therefore, it is difficult for an effective amount of drug to reach the intended wound site.

In addition to the above methods, there is a method which is the most traditional and generally uses synthetic drugs such as steroids. Not only is it ineffective, but it also has the drawback of showing side effects of typical steroid drugs.

[0009]

Based on such prior art studies, the present inventor has been able to suppress or block the action of fibrogenesis-inducing cytokines already produced in the tissues around the wound. By intensively researching the means by which scar formation can be prevented at the wound site by suppressing the formation of fibrogenic cytokines themselves, rather than by preventing scar formation, the result is: It was confirmed that such an object could be achieved by using a specific antisense oligodeoxynucleotide against mRNA of fibrogenesis-inducing cytokine, and the present invention was completed.

[0010]

The present invention provides a novel antisense oligodeoxynucleotide against mRNA of fibrogenesis-inducing cytokine which is secreted in large amounts at the wound site and stimulates fibrinoid degeneration of tissues. is there. Furthermore, the present invention provides a scar comprising an antisense oligodeoxynucleotide directed against a fibrogenic cytokine in an amount sufficient to suppress gene expression of a fibrogenic cytokine and, consequently, scar formation at the wound site. A formation inhibitor composition is provided. Furthermore, the present invention also provides a method for producing a novel antisense oligodeoxynucleotide against a large amount of fibrogenesis-inducing cytokine.

In particular, the present invention substantially inhibits the formation of scars at the wound site of an animal by binding to a sequence complementary to the mRNA of fibrogenic cytokine to suppress the expression of the gene of fibrogenic cytokine. The present invention relates to a novel antisense oligodeoxynucleotide that can be completely or completely prevented.

The antisense oligodeoxynucleotide against the mRNA of the fibrogenesis-inducing cytokine of the present invention, when applied to the tissue surrounding the wound, binds to the complementary sequence of the mRNA to form a hybrid, thereby forming the fibrogenesis-inducing cytokine. Suppress the gene expression of. By doing so, the production of fibrogenesis-inducing cytokines around the wound can be suppressed, and thus scar formation can be prevented.

The antisense oligodeoxynucleotide in the present invention is a fibrogenesis-inducing cytokine such as transforming growth factor-β (TGF-β),
Platelet-derived growth factor (PDGF), fibroblast growth factor
(FGF), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin- (I
L-6) and the like for mRNA of an active substance,
In particular, TG which most strongly induces fibroblast proliferation and connective tissue production among these fibrogenesis-inducing cytokines
Against F-β and PDGF mRNA.

The typical antisense oligodeoxynucleotides in the present invention are illustrated below.

Representative antisense oligodeoxynucleotides for TGF-β mRNA: 5'-CCGAGAGCGCGAACAGGGC-3 ';5'-GGTGTGTGGTGGGGAGGG-3';5'-GGCTGGGGGTCACCCC-3';5'-AGAGAGATCCGTCTC-3';5'-CCCGGAGGGGCGCAT-3';5'-GGCAAAAGGTTAGGAG-3';5'-GAAAGCTGAGGCTCC-3';5'-GAGAAGGGCGCAGGTG-3';5'-GTGGAGGGGAGGCTT-3';

Antisense oligodeoxynucleotide against TNF-α mRNA: 5'-AGCTTTCAGGTGCTCAT-3 ';5'-GGTGTCCTTTCCAGG-3';5'-TAGCTGGTCCTCTGC-3';5'-CCTGCCTGGCAGCTT-3';5'- GGTGGCGCCTGCCACGAT-3 '.

Antisense oligodeoxynucleotides against IL-6 mRNA: 5'-TGTGGAGAAGGAGTTTCAT-3 '.

Antisense oligodeoxynucleotides against PDGF mRNA: 5'-CCAGCAGGCGATTCAT-3 ';5'-AACGCGTAGATCGAG-3';5'-ATCAGGCGCTCAGGC-3';5'-GACCAGACGCAGGTA-3';5'-ACAGGGCGGCGCGG. 3 '.

The antisense oligodeoxynucleotide in the present invention can be produced by a conventional chemical nucleotide synthesis method, but for convenience of production, it is desirably synthesized using a computerized automatic DNA synthesizer. All bases in the synthetic process are protected by a protecting group that is not toxic to cells to prevent nuclease degradation. In this case, phosphorothioate is most preferably used as the protective group. However, when the antisense oligodeoxynucleotide is applied to the skin, even if the base sequence is modified, there is no great difference in the drug efficacy, and therefore the base can be synthesized and used in an unprotected state. However, since the unmodified sequence is very sensitive to the natural endonucleases in tissues, antisense oligodeoxynucleotides modified with phosphorothioate groups, which are relatively highly resistant to endonucleases, will not It is preferably used for prolonging the therapeutic effect period in. Thus, antisense oligodeoxynucleotides to the fibrogenesis-inducing cytokines of the present invention include both unmodified forms and modified (ie, protected) derivatives. The thus-synthesized antisense oligodeoxynucleotide was bound to both ends with appropriate linkers, and then this nucleotide was transferred into an appropriate vector plasmid to obtain a recombinant plasmid.
The recombinant plasmid is transformed into a suitable host bacterium. By culturing the obtained transformant under appropriate conditions, a large amount of antisense oligodeoxynucleotide is expressed and then digested with a restriction enzyme to isolate only the target antisense oligodeoxynucleotide purely. Therefore, it is possible to obtain a large amount.

The plasmids preferably used in the above method are, for example, pSPT18, pSPT19,
pBluescript, pGEM-3 and the like. Particularly preferably, the plasmid pBluescript is used. A preferred host microorganism for transformation with a recombinant plasmid consisting of such a plasmid and antisense oligodeoxynucleotide is E. coli HB101.
Is.

As described above, the antisense oligodeoxynucleotide produced in this manner can suppress the production of fibrogenesis-inducing cytokine known to be secreted in large amounts around the wound. It can be used as a scar formation inhibitor for a wound site.

Therefore, the present invention further relates to the use of the above novel antisense oligodeoxynucleotide as a scar formation inhibitor in a warm-blooded mammal including human.

When used as an anti-scarring agent, the antisense oligodeoxynucleotides of the present invention can be used alone for one fibrogenic cytokine, but if desired, two or more kinds of fibers can be used. Antisense oligodeoxynucleotides against formation-inducing cytokines can also be mixed and used. For example, if an antisense oligodeoxynucleotide against TGF-β is mixed with an antisense oligodeoxynucleotide against TNF-α and used,
A faster wound healing effect can be obtained.

The antisense oligodeoxynucleotide of the present invention is in the form of a pharmaceutically acceptable preparation together with a conventional pharmaceutically acceptable carrier, for example, injection, spray, ointment, cream and the like. It can be used by systemic administration, or by local application to the wound site. Conventional carriers that are pharmaceutically acceptable for use in such formulations include distilled water for injection, phosphate buffer,
Physiological saline is included, and for propellants, ethanol, propylene glycol, glycerin or propellant (p
ropellant) and the like, and for topical preparations such as ointments and creams, polyethylene glycol, liquid paraffin, carnauba wax and the like are included.

The amount of the antisense oligodeoxynucleotide used for the fibrogenesis-inducing cytokine of the present invention varies depending on the range and degree of the wound site and the condition of the application target, but it is generally 3 to 5 times a day, every time. Approximately 10 μM is used, most preferably 50 μM per day. Preferably, the antisense oligodeoxynucleotide in the present invention is topically administered to the skin in the form of a cream, ointment, lotion or the like, or is administered by subcutaneous injection.

Further, the antisense oligodeoxynucleotide in the present invention acts only in the gene expression process of the fibrogenesis-inducing cytokine involved in scar formation due to fibrinoid degeneration of tissue, and has no particular side effect or toxicity. As mentioned above for the purpose of treatment, which is not caused, a very small amount of about 10 μM is used.
The antisense oligodeoxynucleotide of the present invention does not substantially cause side effects including drug allergy and photosensitivity reaction.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0028]

【Example】

Example 1 5'-CCGAGAGCGCG, which is an antisense oligodeoxynucleotide against TGF-β mRNA
Synthesis of AACAGGGC-3 ′ A bottle containing acetonitrile for washing the cartridge, dichloroacetic acid for removing the protecting group of the base, and monodisperse plastic beads for removing water was connected, and a base C ( Cytosine), and a DN adjusted to introduce a predetermined amount of bases A (adenine), G (guanine) and C (cytosine) in a predetermined order.
Gene Assembler Special, an automatic synthesizer
(GENE ASSEMBLER SPECIAL: Phar
macia, LKB) was operated at room temperature for 2.5 hours, and the target antisense oligodeoxynucleotide against TGF-β, 5′-CCGAGAGCGCGAGACA
GGGC-3 'was synthesized.

After ligating a linker sequence GGGCC providing a cleavage site for ApaI to both ends of the antisense oligodeoxynucleotide synthesized above,
The obtained nucleotide sequence is used as the plasmid pBluescript.
To the ApaI cleavage site to obtain a recombinant expression plasmid. The resulting recombinant plasmid was introduced into the host strain E. coli HB101 to obtain a transformant. The transformant E. coli HB101 was transformed into LB medium (Luria-
1 liter of Luria-Bertani broth; 10 g of Bacto-tryptone, 5 g of Bacto-yeast extract, 5 g of NaCl, 1 g.
The target antisense oligodeoxynucleotide was expressed by culturing in N NaOH (1 ml) at 37 ° C. for about 16 hours. The isolated plasmid was digested with the restriction enzyme ApaI to obtain a pure antisense oligodeoxynucleotide having the desired sequence. Using a spectrophotometer, 2
The absorbance of the product was measured at 60 nm. The yield of the desired product was 92%. It was confirmed by the nucleotide sequence analysis and electrophoresis (“FIG. 1”) of the produced antisense oligodeoxynucleotide that the desired title antisense sequence was obtained.

Example 2 5'-AGCTTTCAGTG, an antisense oligodeoxynucleotide against TNF-α mRNA
Synthesis of CTCAT-3 ′ A bottle containing acetonitrile for washing the cartridge, dichloroacetic acid for removing the protecting group of the base, and monodisperse plastic beads for removing water was connected, and a predetermined amount of the base A ( Adenine), G (guanine), C
(Cytosine) and T (thymine), which are automatic DNA synthesizers regulated so that they can be introduced in a predetermined order,
Assembler Special (GENE ASSEMBLE
R SPECIAL; Pharmacia, LKB) at room temperature 2.
After operating for 5 hours, 5'-AG, an antisense oligodeoxynucleotide against the target TNF-α
CTTTCAGTGCTCAT-3 ′ was synthesized.

After the linker sequence AA which provides a cleavage site for HindIII was ligated to both ends of the antisense oligodeoxynucleotide synthesized above, the obtained nucleotide sequence was used for HindI of the plasmid pBluescript.
It was transferred to the II cleavage site to obtain a recombinant expression plasmid. Then, the obtained recombinant plasmid was introduced into a host strain, E. coli HB101, to obtain a transformant. Transform the E. coli HB101 into LB medium 3
After culturing at 7 ° C. for about 16 hours, the desired antisense oligodeoxynucleotide was expressed. The isolated plasmid was digested with the restriction enzyme HindIII to obtain a pure antisense oligodeoxynucleotide having the desired sequence. The absorbance of the product was measured at 260 nm using a spectrophotometer. The yield of the desired product was 90%. It was confirmed by the nucleotide sequence analysis and electrophoresis of the produced antisense oligodeoxynucleotide that the desired title antisense sequence was obtained.

Example 3 5'-ATCAGGCGCTCA which is an antisense oligodeoxynucleotide against PDGF mRNA
Synthesis of GGG-3 ′ A bottle containing acetonitrile for washing the cartridge, dichloroacetic acid for removing the protecting group of the base, and monodisperse plastic beads for removing water was connected, and a predetermined amount of the base A ( Adenine), G (guanine), C
Gene Assembler Special (GENE ASSEMBLER), which is an automatic DNA synthesizer adjusted so that (cytosine) and T (thymine) are introduced in a predetermined order.
SPECIAL; Pharmacia, LKB) at room temperature for 2.5
5'-ATCA, which is an antisense oligodeoxynucleotide against PDGF of interest, is operated for a time.
GGCGCTCAGGG-3 'was synthesized.

At the both ends of the antisense oligodeoxynucleotide synthesized above, a linker sequence ATCG providing a cleavage site for ClaI was ligated, and the obtained nucleotide sequence was transferred to the ClaI cleavage site of the plasmid pBluescript. A recombinant expression plasmid was obtained.

Next, the obtained recombinant plasmid was introduced into the host strain E. coli HB101 to obtain a transformant. Obtained transformant E. coli HB10
1 was cultured in LB medium at 37 ° C. for about 16 hours to express the target antisense oligodeoxynucleotide. The isolated plasmid was digested with the restriction enzyme ClaI to obtain a pure antisense oligodeoxynucleotide having the desired sequence. The absorbance of the product was measured at 260 nm using a spectrophotometer. The yield of the desired product was 92%. It was confirmed by the nucleotide sequence analysis and electrophoresis (“FIG. 1”) of the produced antisense oligodeoxynucleotide that the desired title antisense sequence was obtained.

Example 4 Effect of antisense oligodeoxynucleotide against TGF-β on gene expression Antisense oligodeoxy against TGF-β, which is a typical fibrogenic cytokine in animals in which experimental wounds were induced. The effect of the administered antisense oligodeoxynucleotides on the expression of cytokines was evaluated as compared to a control group that received the nucleotides and did not.

1) After Balb / C mice were anesthetized with ether, the hair on the back was completely removed using a hair cutter and thioglycolic acid, and the hair was removed from the midline to the extremities at the same intervals. The healing was naturally induced without suturing by cutting the muscle up to just above the muscle. At this time, in one group of mice, the antisense oligodeoxynucleotide (5'-CCG) against TGF-β produced in Example 1 was added.
AGAGCGGCGAACAGGGGC-3 ′) (dissolved or suspended in an excipient suitable for topical administration to the skin) was administered topically at a concentration of 50 μM and treated with another group of animals not treated in this way. Mice were used as a control group, and in each group, the expression of fibrogenesis-inducing cytokine was measured as the course of wound healing.

Immediately after wound formation and 1, 2 of wound formation,
After 3, 5 and 10 weeks, the wound tissue was separated from each group of mice and cells were collected. The collected cells were washed with PBS containing no Ca ²⁺ , Mg ²⁺ and RNase, and then NP-40 lysis buffer [0.5.
% NP-40, 10 mM Tris-Cl (pH 8.0), 100
mM NaCl, 3 mM MgCl ₂ , 1000 U / ml RNAsi
n (Promega)] and centrifuged at 12000 rpm for 2 minutes to remove cell nuclei. The supernatant can then be separated and used directly as a source of RNA.

Alternatively, 1 × 10 ⁸ cells are GI
T buffer [4M guanidine thiocyanate, 50 mM Tris-Cl (pH 8.0), 10 mM EDTA (ethylenediaminetetraacetic acid), 0.5% sodium lauryl sarcosine,
After dissolving in 4 ml of 0.1 M mercaptoethanol], 5.
Load 7 ml of 7M CsCl buffer, 80,000 rpm, 2
It was centrifuged at 5 ° C. for about 2 hours. After obtaining the total RNA attached to the lower part of the test tube and the wall in this way, poly (A) ⁺ mRN
In order to separate A purely, using TE buffer as an eluent, an appropriate amount of oligotex-dT was added.
It was added to the obtained RNA. The mixture was heated at 65 ° C. for 5 minutes, then quenched with ice, then 1/10 volume of 5M.
The NaCl solution was added and the mixture was incubated at 37 ° C. for 5 to 10 minutes. Then, centrifuge at 15000 rpm for 5 to 10 minutes,
After removing the supernatant, add sterile distilled water and mix, then again
Heat at 65 ° C. for 5 minutes, then centrifuge at 15000 rpm for 5-10 minutes. The supernatant was directly precipitated with ethanol, then diethylpyrocarbonate (DEPC) -dH
It was possible to obtain the desired mRNA was dissolved in ₂ O.

The mRNA obtained by the above method was added to 65
Denature at ~ 75 ° C for 10 minutes. Denatured mRNA
1-2 μg, oligo (dT) _12-18 (Pharmacia (Pharma
cia)) 0.5 μg, 1 × Taq polymerase buffer (50 mM
Tris-Cl, pH 8.3, 3 mM MgCl ₂ , 250 μg / m
l BSA), dNTP 1.25 mM, MgCl ₂ 2.5 mM,
20 units of RNAsin (BRL) and murine reverse transcriptase (B
RL) 100 units in a test tube containing no RNAse, in which DEPC is brought to a final volume of 20 μl
It was added to the -dH ₂ O. After thoroughly stirring, the mixture was reacted at 37 ° C for 1 hour [Molecular Cloning (Molecular
Cloning), Volume II, 14, pp 20-21].
Then, after heating at 95 ° C. for 5 minutes to terminate the reaction,
Alkali treatment with NaOH gives single-stranded cDNA, which is then used as a template for the polymerase chain reaction (PCR).

In a 0.5 ml Eppendorf tube, 1 × Taq polymerase buffer (50 mM Tris-C) was added.
pH 8.3, 3 mM MgCl ₂ , 250 μg / ml BS
A), dNTP 200 μM, primer 5′-GGGA
AATTGAGGGCTTTCGC-3 'and 5'-C
TGAAGCAATATAGTTGGTGTC-3 '0.2 for each
5 μM (10 pmol), 2 μg of the above-generated cDNA and 0.1 U of Taq polymerase were introduced, and then sterilized dH ₂ O was added and mixed sufficiently so that the total volume of the reaction solution was 10 μl. Then, the test tube was centrifuged for a short time, and the reaction liquid adhering to the wall of the test tube was collected in the lower part of the test tube. The reaction solution was transferred to a glass capillary tube while being careful so that air bubbles did not enter, and both ends of the capillary tube were heated and melted and sealed. Then, the thermal cycler (t
hermal cycler, FTC-2000) for one cycle at 95 ° C for 5 seconds, 55 ° C for 5 seconds, 72 ° C for 15 seconds.
PCR amplification was performed by specifying 35 seconds and repeating 35 to 45 cycles. The amplified DNA was electrophoresed using a 1% Seakem agarose gel, stained with ethidium bromide solution 50 μg / ml and then UV transilluminator.
Confirmed using. As a result, the intensity of ethidium bromide staining in the tissue of the mouse to which the antisense oligodeoxynucleotide against TGF-β of the present invention was administered was much weaker than that in the tissue of the untreated control group. From these results, the antisense oligodeoxynucleotide against TGF-β is
It was found that it binds to the front part of the -β structural gene and specifically suppresses the expression of TGF-β in a concentration-dependent manner. Therefore, based on the results of such concentration-dependent suppression, the amount of antisense oligodeoxynucleotide capable of completely suppressing the expression of TGF-β was determined by the number of cells at the wound site, and then clinically. Used as an ideal dose that can induce effective wound healing.

2) In situ hybridization: The target TGF-β gene was plasmid vector pBlue.
Corresponding E from escript (purchased from Clontech)
insert into the coRI cleavage site to generate a recombinant plasmid,
This recombinant plasmid was then transformed into E. coli HB10
1 to obtain a transformant. The transformed E. coli HB101 thus obtained was cultured to obtain a large amount of the desired plasmid, which was cesium chloride (CsCl) gradient centrifuge.
(1 g / ml CsCl, 740 μg / ml ethidium bromide + plasmid DNA, 100,000 rpm, 10 hours). After that, using an RNA labeling kit (manufactured by BM Co., Ltd. (BM Co., Ltd.)),
In v by SP6 (or T3) T7 RNA polymerase
Itro transfer was performed at 37 ° C for 1-2 hours. After the transcription reaction was completed, the template DNA was decomposed by DNase I and ethanol precipitation was performed to recover the RNA probe as a transcription product. Collected RNA probe with 10 mM DTT, 1
0 mM Tris-HCl, 1 mM EDTA (pH 7.6) 50 μ
It was dissolved in 1 and used for hybridization. After completion of the reaction, the same amount of neutralization buffer (300 mM) was added to the reaction mixture.
CH ₃ COONa, pH 6.0, 1% CH ₃ COOH, 10 m
MDTT), followed by ethanol precipitation,
The target probe was collected.

Hybridization reaction (50% deionized formamide, 10 mM Tris-HCl, pH 7.
6, tRNA without RNase 200 μg / ml, 1
× Denhardt's solution, consisting of 10% dextran sulfate, 600 mM NaCl, 0.25% SDS) 1
After adding 0.5 μg of the probe obtained above to 00 μl and mixing, place 50-100 μl of this mixture on each pretreated slide glass to which the mouse wound tissue obtained from 1) above is attached. The slide glass was covered with a cover glass and subjected to hybridization for 16 to 22 hours.

After completion of hybridization, 5 × S
The coverslips were removed from the slides in SC at 50 ° C. and the hybridized products were treated with RNase to remove unwanted signals.

For the hybridized products, DIG
The color reaction was performed using an ELISA DNA labeling and detection kit (BM Co., Ltd.). That is, the hybridized product obtained above was treated with 100 mM Tris-HCl, pH 7.5, 15
After leaving it in 0 mM NaCl (TS) for about 5 minutes, it was taken out and again put in a blocking solution in which dry milk powder containing no fat or sugar in TS was dissolved at a concentration of 1.5% and left at room temperature for 30 minutes. . Then 500-1000 here
0.2 unit of alkaline phosphatase-conjugated anti-digoxigenin antibody diluted with double volume of TS was added, and the mixture was reacted for 30 to 60 minutes. After the reaction was completed, the product was washed twice in TS for 15 minutes each to obtain 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 500 mM Mg.
Washed briefly in Cl ₂ (TSM). Then, nitro blue tetrazolium (NBT) diluted with TSM and 5-
Bromo-4-chloro-3-indolyl phosphate (BCIP)
The solution was stained for 1-2 days. Then, a TE buffer solution (Tris-HCl 10 mmol / liter, EDTA 1 mmol / liter, pH 8.0) was added to stop the color development reaction, and the slide glass was mounted with an aqueous crystal mount. The results were read in the stained state.

As a result, in the control group not treated with the antisense oligodeoxynucleotide, the cytokine mRNA was slightly detected on the first day after the wound occurred, but from the third day, it was mainly large. It was detected in phagocytes. On the other hand, in the antisense oligodeoxynucleotide-treated group, no signal for cytokine mRNA was detected. From this, it can be clearly seen that the expression of the cytokine is suppressed by the antisense oligodeoxynucleotide.

Example 5 With respect to the antisense oligodeoxynucleotide of the present invention, the scar formation inhibitory activity was measured by the following method.

A wound having a diameter of about 6 mm was artificially added to the back site of 40 mice, and then 20 mice were divided into two groups. One group of mice was antisense oligodeoxynucleotide against TGF-β (5′-CCGAGAGCGCGCGA produced by Example 1 after wounding).
ACAGGGC-3 ′) was sprayed onto the wound site in a volume of 50 μM (antisense treated group). The remaining one group was not treated with antisense oligodeoxynucleotides
(Untreated control group). After that, the collagen content and the tensile force at the wound site, which are important parameters of scar formation, were measured over time, and the effect of the antisense oligodeoxynucleotide of the present invention on the inhibition of scar formation was determined.

The results are shown in "Table 1" and "Table 2" below.

[0049]

[Table 1]

[0050]

[Table 2]

As can be seen from the results of "Table 1" and "Table 2" above, in the rat treated with the antisense oligodeoxynucleotide against TGF-β of the present invention at the wound site, the collagen content was higher than that in the non-treated control group. Although significantly lower, the tensile force was rather high despite such low collagen content. In addition, the administration of the antisense oligodeoxynucleotide of the present invention does not delay the wound healing and heals the morphology closer to the normal tissue as compared with the control group, and is so perfect that it cannot be visually distinguished. Exhibited tissue regeneration power.

These results indicate that when a wound site is treated with an antisense oligodeoxynucleotide against TGF-β, TGF- is a phenomenon that generally occurs at the wound site.
It is presumed that it was obtained not only by suppressing the expression induction of various cytokines such as β, but also by suppressing fibrocyte division and connective tissue hyperplasia by growth factors. It is also desirable to administer such antisense oligodeoxynucleotides as soon as possible, ie immediately after the wound has developed, for increased efficacy and less angiogenesis.

Example 6 In vitro experiment using antisense oligodeoxynucleotide against TNF-α In vitro of mouse macrophage cell line RAW264.7
The antisense oligodeoxynucleotide against TNF-α was injected into the culture medium to observe the inhibitory effect on the formation of nitric oxide (NO), which is known to be involved in the anticancer and antitumor effects. This test shows that the formation of NO is carried out by the expression of a gene for nitric oxide synthetase (NOS), which is closely related to the expression of TNF-α [Reference: Journal of Immunology]. (Journal of Immunology) 1
46, 114-120, 1991 and Journal of Immunology 149,
3290-3296, 1992].

Mouse macrophage cell line RAW26
200 μl of 4.7 in 96-well plate (Nunc)
DMEM medium containing 10 U / ml of γIFN and 10 ng / ml of LPS (lipopolysaccharide) (Dulbecco's Modified Eagle's).
s Medium) + 5% fetal bovine serum + 0.5% penicillin /
200 μl of streptomycin) was added. Also, TNF
Antisense oligodeoxynucleotides against α were added at concentrations of 0.1 μM, 1 μM and 10 μM, respectively, and for control, all test tubes were treated with no antisense oligodeoxynucleotide against TNF-α.
It was cultured at 7 ° C for 48 hours. Then, the method of Ding et al. [Reference: Journal of Immunology (J
ournal of Immunology) 141, 2407-241
2, 1988], the color reaction between 100 μl of the culture solution and the same amount of Griess reagent was used to perform TNF-
The amount of nitrite secretion mediated by γIFN and LPS (calculated as NO _{2 and} expressed in μM / liter) in the presence or absence of antisense oligodeoxynucleotides against α was measured to determine antisense oligodeoxy for nitrite production. The effect of nucleotides was evaluated. The results are shown in "Fig. 2".

As can be seen from the results shown in FIG.
NO production is suppressed by antisense oligodeoxynucleotides in a concentration-dependent manner. From this result, TNF
Antisense oligodeoxynucleotides against fibrosis-inducing cytokines including -α are known to be expressed during wound formation TNF-α, TGF-β
It can be inferred that the expression of fibrogenesis-inducing cytokines such as can be directly suppressed.

As described above, the antisense oligodeoxynucleotide against the fibrogenesis-inducing cytokine of the present invention is secreted in a large amount from the wound site and promotes scar formation, such as TGF-β, TNF-α and PDGF. Since it suppresses the gene expression of cytokines, it can be used as a useful drug for inhibiting the formation of scars at the wound site and allowing the wound site to be healed without any difference in appearance from normal tissues.

The more relevant and important features of the present invention have been outlined above so that the detailed description of the invention can be better understood and its contribution to the art is fully appreciated. Those skilled in the art will appreciate that the concepts and particular embodiments described herein are readily available on the basis of modification or design of other structures to serve the same purpose of the invention. Furthermore, one of ordinary skill in the art will appreciate that such equivalent structures do not depart from the scope of the invention as claimed.

[0058]

INDUSTRIAL APPLICABILITY The antisense oligodeoxynucleotide of the present invention binds to sequences complementary to mRNAs of fibrogenesis-inducing cytokines such as TGF-β, TNF-α and PDGF, and suppresses gene expression of these substances. By doing so, it is possible to prevent the formation of fibrosis-inducing cytokines in the wound and scar formation due to the fibrosis of the tissue, and to heal the wound site so that it has an appearance and tensile force almost similar to those of normal tissue.

[Brief description of drawings]

FIG. 1 TGF-β 1 and 2 (lanes 2 and 3), TNF-α (lane 4), PDGF 1 and 2
(Lanes 5 and 6) and φX174 / Hae III
The drawing substitute photograph which shows the result of the electrophoresis of the antisense oligodeoxynucleotide with respect to (molecular size marker; lane 1).

FIG. 2 is a graph showing the effect of antisense TNF-α on the secretion of nitrite mediated by γIFN and LPS.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12N 15/11 15/16 15/24 15/28 (71) Applicant 593193701 Hontech John HUN-Taeg CHUNG Korea Cholula Bukto, Iri, Nam Jung Dong 1ga, No. 224-5 Namsung Apartment 1-301 (72) Inventor Hong Tech John Cholla Bukto, Iris, Nam Jung Dong 1Ga, No. 224.5 Namsung Apartment 1-301

Claims (14)

[Claims] 1. A fibrogenesis-inducing cytokine mRNA
Antisense oligodeoxynucleotides and derivatives thereof, which are capable of suppressing the expression of genes for fibrogenesis-inducing cytokines by binding to a complementary sequence to. 2. The antisense oligodeoxynucleotide according to claim 1, wherein the sequence of the antisense oligodeoxynucleotide is modified with phosphorothioate. 3. The fibrogenesis-inducing cytokines are transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TN.
F-α), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), interleukin-1 (IL-1) and interleukin-6 (I).
The antisense oligodeoxynucleotide according to claim 1, which is selected from the group consisting of L-6). 4. The fibrogenesis-inducing cytokine is TGF.
The antisense oligodeoxynucleotide according to claim 3, which is selected from the group consisting of -β, TNF-α and PDGF. 5. The fibrogenesis-inducing cytokine is TGF-
β and the antisense oligodeoxynucleotide has the following sequence with respect to mRNA of TGF-β: 5′-CCGAGAGCGCGAACAGGGC-3 ′; 5′-GGTGTGTGGTGGGGAGGG-3 ′; 5′-GGCTGGGGGTCACCCC-3 ′; 3 ';5'-CCCGGAGGGGCGGGCAT-3';5'-GGCAAAAGGTTAGGAG-3';5'-GAAAGCTGAGGCTCC-3';5'-GAGAAGGGCGCAGGTG-3';5'-GTGGAGGGGAGGCTT-3T; The antisense oligodeoxynucleotide according to claim 4, wherein the antisense oligodeoxynucleotide comprises: 6. The fibrogenic cytokine is TNF-
α and the antisense oligodeoxynucleotide is the following sequence for the mRNA of TNF-α: 5′-AGCTTTCAGTGCTCAT-3 ′; 5′-GGTGTCTCTTTCCAGG-3 ′; 5′-TAGCTGGTCCTCTGC-3 ′; 5′-CCTGCCTGGCAGCTT- The antisense oligodeoxynucleotide according to claim 4, which has 3 ′; or 5′-GGTGGGCGCTGCCACGAT-3 ′. 7. The fibrogenesis-inducing cytokine is PDGF.
And the antisense oligodeoxynucleotide is P
The following sequence for DGF mRNA: 5'-CCAGCAGGCGATTCAT-3 ';5'-AACCGCGTAGATCGAG-3';5'-ATCAGGCGCTCAGGC-3';5'-GACCAGACGCAGGGTA-3'; or 5'-ACAGGTGGACGCGC-3 '. The antisense oligodeoxynucleotide according to claim 4, which comprises. 8. A wound at a wound site, which comprises an effective amount of at least one antisense oligodeoxynucleotide with respect to the mRNA of the fibrogenesis-inducing cytokine according to any one of claims 1 to 7. A composition for inhibiting scar formation during healing. 9. An antisense oligodeoxynucleotide 5′-CAACACG for TGF-β mRNA.
The scar formation inhibitor composition according to claim 8, which contains GGTTCAGGTAC-3 ′. 10. An antisense oligodeoxynucleotide 5′-CATGCT against TNF-α mRNA.
The scar formation inhibitor composition according to claim 8, which contains TTCAGTGCTCAT-3 ′. 11. An antisense oligodeoxynucleotide 5'-CCAGCAG for PDGF mRNA.
The scar formation inhibitor composition according to claim 8, which comprises CGATTCAT-3 ′. 12. The scar formation inhibitor composition according to claim 8, which further comprises a pharmaceutically acceptable carrier, adjuvant or excipient. 13. The scar formation inhibitor composition according to claim 8, which is formulated in the form of an injection, a spray, an ointment or a cream. 14. An antisense oligodeoxynucleotide for mRNA of a fibrogenesis-inducing cytokine is synthesized by an automatic DNA synthesizer; linker sequences are linked to both ends of the synthesized sequence, and the linked sequence is then converted into an appropriate sequence. Insertion into a plasmid to obtain a recombinant plasmid;
A transformed host bacterium is transformed with the obtained recombinant plasmid; the obtained transformant is cultured to express a large amount of an antisense oligodeoxynucleotide of interest. 9. A method for purely mass-producing an antisense oligodeoxynucleotide against mRNA of a fibrogenesis-inducing cytokine according to any one of 7 above.