JP2017137268A - Constriction inhibitor, composition, and antiinflammatory agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel constriction inhibitor which can inhibit constriction.SOLUTION: The present invention provides a constriction inhibitor comprising a culture supernatant of mesenchymal cells, and a composition comprising a culture supernatant of CD90 expression positive mesenchymal cells and having a viscosity of 100 cP or more and 1000000 cP or less.SELECTED DRAWING: None

Description

  The present invention relates to a stenosis inhibitor containing a culture supernatant of mesenchymal cells. Furthermore, the present invention relates to a composition having a predetermined viscosity, which contains a culture supernatant of mesenchymal cells. Furthermore, this invention relates to the stenosis inhibitor and anti-inflammatory agent containing the said composition.

  Mesenchymal cells are somatic stem cells that have been pointed out to be present in the bone marrow, and have the ability to differentiate into bone, cartilage and fat as stem cells. Mesenchymal cells are attracting attention as a promising cell source in cell therapy, and are recently known to exist in fetal appendages such as placenta, umbilical cord, and egg membrane.

  Patent Document 1 discloses a method for producing a mesenchymal cell composition comprising a step of enzyme treatment of amniotic membrane with collagenase and thermolysin and / or dispase; and a step of passing the enzyme-treated amniotic membrane through a mesh; And a method for treating graft-versus-host disease, inflammatory bowel disease, systemic lupus erythematosus, cirrhosis, and radiation enteritis using the mesenchymal cell composition as an active ingredient . However, Patent Document 1 does not describe the use of cell culture supernatants for the prevention or treatment of diseases.

It has been reported that the culture supernatant of cells such as mesenchymal cells is effective for the treatment of certain diseases. For example, Patent Document 2 describes a prophylactic / therapeutic agent for enteritis containing a culture supernatant of mesenchymal cells (MSC). US Pat. No. 6,057,038 is associated with or caused by an inappropriate or undesirable immune response, including administering to an individual a therapeutically effective amount of amniotic membrane-derived adherent cells (AMDAC), or conditioned culture medium with AMDAC. Methods for treating individuals having or at risk for developing a disease or disorder are described. Patent Document 4 describes a method for improving pancreatic function comprising the step of administering to a subject STRO-1 ⁺ cells and / or progeny cells thereof and / or soluble factors derived therefrom. U.S. Patent No. 6,057,031 describes a substantially purified population of amnion-derived cells that are negative for expression of protein markers CD90 and CD117. Furthermore, Non-Patent Document 1 describes that the culture supernatant of amnion-derived mesenchymal stem cells exhibits an effect of improving liver fibrosis. As described above, Patent Documents 2 to 4 and Non-Patent Document 1 describe the use of cell culture supernatants for the treatment of diseases. There is no description.

  On the other hand, endoscopic submucosal dissection (ESD) for superficial cancer of the esophagus has rapidly become widespread, and excision techniques that are not limited by the size of the lesion are also progressing. However, if extensive excision is performed, scar stenosis will occur after the operation and cause a decrease in QOL. In clinical practice, endoscopic balloon dilatation is used to prevent stenosis after surgery, steroid ulcers are injected locally, or used internally (Non-patent Documents 2 and 3), and cell sheets obtained from the oral mucosal epithelium are used as clinical trials. However, there are complications, invasiveness of cell collection, and cost.

JP2015-61520A JP 2013-18756 A Special table 2014-501249 gazette JP2015-205894A Special table 2008-538180 gazette

Kubo et al. , Transplantation Direct 2015: 1; e16 Hanaoka et al. Endoscopy 2012: 44; 1007 Yamaguchi et al. Gastrointest Endosc 2011: 73; 1115 Ohki T et al. Gut 2006: 55; 1704-10

  Patent Document 2 described above describes a preventive / therapeutic agent for enteritis containing a culture supernatant of mesenchymal cells (MSC). However, a technique for retaining an active ingredient for a long time at a diseased site is desired. Further, there is no satisfactory means for preventing postoperative stenosis, and provision of new means is desired.

  This invention makes it the subject which should be solved to provide the novel stenosis inhibitor which can suppress stenosis. Furthermore, this invention makes it the problem which should be solved to provide the novel composition which can suppress stenosis and has anti-inflammatory action. Furthermore, this invention makes it a subject to provide the stenosis inhibitor and anti-inflammatory agent containing the said composition.

  The present inventors have intensively studied to solve the above problems. First, human amnion-derived mesenchymal cells are cultured in a serum-containing medium (αMEM), and when the cells become confluent, they are replaced with a serum-free medium. After further culturing, the medium was collected and centrifuged to obtain a culture supernatant. This culture supernatant was suspended in carboxymethylcellulose to prepare a gel composition. After applying the above gel composition to the esophagus after porcine esophageal ESD (endoscopic submucosal dissection), the esophageal stenosis was significantly suppressed, and the effect was comparable to that of steroid local injection. Demonstrated. Moreover, when the gel composition was enemamed into a rat TNBS (2,4,6-trinitrobenzenesulfonic acid) enteritis model, it was found that the inflammation findings of the large intestine were significantly improved. The present invention has been completed based on these findings.

That is, according to this specification, the following invention is provided.
[1] A stenosis inhibitor comprising a culture supernatant of mesenchymal cells.
[2] The stenosis inhibitor according to [1], wherein the mesenchymal cells are human amnion-derived mesenchymal cells.
[3] The stenosis inhibitor according to [1] or [2], which is a digestive tract stenosis inhibitor.
[4] The stenosis inhibitor according to any one of [1] to [3], which is an esophageal stenosis inhibitor.
[5] A composition comprising a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP or more and 1000000 cP or less.
[6] The composition according to [5], wherein the mesenchymal cells are human amnion-derived mesenchymal cells.
[7] The composition according to [5] or [6], wherein the culture supernatant is a serum-free conditioned medium.
[8] The composition according to any one of [5] to [7], wherein the composition is a gel composition containing a thickener.
[9] The composition according to [8], wherein the thickener is a polysaccharide.
[10] The composition according to [8] or [9], wherein the thickener is carboxymethylcellulose.
[11] The composition according to any one of [5] to [10], which is used as a pharmaceutical, a food or drink, or a cosmetic including a quasi drug.
[12] Any of [8] to [10], comprising a step of suspending a thickener in the culture supernatant of mesenchymal cells positive for CD90 expression and gelling the resulting suspension. A method for producing the composition according to 1.
[13] A culture supernatant of mesenchymal cells positive for CD90 expression was obtained by culturing mesenchymal cells positive for CD90 expression in a serum-containing medium and then in a serum-free medium. The method according to [12], which is a culture supernatant.
[14] A stenosis inhibitor comprising the composition according to any one of [5] to [11].
[15] The stenosis inhibitor according to [14], which is a gastrointestinal stenosis inhibitor.
[16] The stenosis inhibitor according to [14] or [15], which is an esophageal stenosis inhibitor.
[17] An anti-inflammatory agent comprising the composition according to any one of [5] to [11].

Further, according to the present invention, the following inventions are also provided.
(A) A culture supernatant of mesenchymal cells used for stenosis suppression.
(B) A composition used for suppressing stenosis, comprising a culture supernatant of mesenchymal cells positive for CD90 expression, and having a viscosity of 100 cP or more and 1000000 cP or less.
(C) A composition used for an anti-inflammatory agent, comprising a culture supernatant of mesenchymal cells that are positive for CD90 expression, and having a viscosity of 100 cP to 1000000 cP.
(D) A method for suppressing stenosis, comprising a step of administering to a subject in need of stenosis suppression an effective suppression amount of a culture supernatant of mesenchymal cells.
(E) a step of administering to a subject in need of stenosis suppression an effective amount of a composition containing a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP to 1000000 cP A method for suppressing stenosis.
(F) A step of administering an effective amount of a composition containing a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP to 1000000 cP to a subject who needs suppression of inflammation A method for suppressing inflammation, comprising:
(G) Use of a culture supernatant of mesenchymal cells positive for CD90 expression for the production of a stenosis inhibitor.
(H) Use of a composition containing a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP or more and 1000000 cP or less for producing a stenosis inhibitor.
(I) Use of a composition containing a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP or more and 1000000 cP or less for the production of an anti-inflammatory agent.

  ADVANTAGE OF THE INVENTION According to this invention, the novel stenosis inhibitor which can suppress stenosis is provided. Moreover, according to the composition having a predetermined viscosity of the present invention, stenosis can be suppressed and further inflammation can be suppressed.

FIG. 1 shows the results of confirming surface antigen expression in amniotic membrane MSC with a flow cytometer. FIG. 2 shows the positional relationship of the lengths a, b1, and b2 in the measurement of the stenosis rate. FIG. 3 shows the measurement result of the stenosis rate. STD indicates a control group, CM indicates a gel group, and Steroid indicates a steroid group. FIG. 4 shows an example of measurement of the fiber thickness of the stenosis by masson-trichrome staining. FIG. 5 shows the results of macroscopic findings (left side) and masson-trichrome staining. FIG. 6 shows the measurement results of the fiber thickness of the stenosis. STD indicates a control group, CM indicates a gel group, and Steroid indicates a steroid group. FIG. 7 shows the measurement results of the number of activated fibroblasts. STD indicates a control group, CM indicates a gel group, and Steroid indicates a steroid group. FIG. 8 shows the measurement results of the pathological score. Control indicates a control, αMEMGel indicates an uncultured αMEM gel, and CMmel indicates a gel containing the culture supernatant of mesenchymal cells. FIG. 9 shows the staining result of CD68 positive macrophages. Control indicates a control, αMEMGel indicates an uncultured αMEM gel, and CMmel indicates a gel containing the culture supernatant of mesenchymal cells. FIG. 10 shows the staining result of myeloperoxidase positive neutrophils. Control indicates a control, αMEMGel indicates an uncultured αMEM gel, and CMmel indicates a gel containing the culture supernatant of mesenchymal cells. FIG. 11 shows the measurement results of the TNFα mRNA expression level. Control indicates a control, αMEMGel indicates a non-cultured α-MEM gel, and CMmel indicates a gel containing a mesenchymal cell culture supernatant.

[1] Explanation of terms “Fetal appendage” in the present specification refers to egg membrane, placenta, umbilical cord and amniotic fluid. Furthermore, the “egg membrane” is a fetal sac containing fetal amniotic fluid and consists of an amnion, chorion and decidua from the inside. Of these, the amnion and chorion originate from the fetus. “Amnion” refers to a transparent thin film with poor blood vessels in the innermost layer of the egg membrane. The inner layer of the amniotic membrane (also called the epithelial cell layer) is covered with a layer of secretory epithelial cells to secrete amniotic fluid, and the outer layer of the amniotic membrane (also called the extracellular matrix layer, which corresponds to the stroma) contains mesenchymal stem cells. Including.

As used herein, “mesenchymal cell (MSC)” refers to mesenchymal and progenitor cells (meaning cells having the ability to differentiate into cells that constitute one or more various organs or tissues). Refers to a cell that satisfies the definition of a pluripotent mesenchymal cell (MSC) proposed by The International Society for Cellular Therapy (see below).
In addition, “mesenchymal stem cells (MSC)” is used without distinction from “mesenchymal stromal cells (Mesenchymal stromal cells)”.

Definition of pluripotent mesenchymal cells i) Adhesive to plastic under culture conditions in standard medium.
ii) The surface antigens CD105, CD73, and CD90 are positive, and CD45, CD34, CD14, CD11b, CD79alpha, CD19, and HLA-DR are negative.
iii) Differentiation into bone cells, adipocytes, and chondrocytes under culture conditions.

[2] Preparation of Mesenchymal Cells The origin of the mesenchymal cells used in the present invention is not particularly limited, but is preferably a mammal (eg, human, cow, mouse, rat, pig, monkey, dog, cat, etc.) Yes, more preferably a human. The tissue from which the mesenchymal cells are collected is not particularly limited, and the mesenchymal cells can be obtained from tissues such as bone marrow, adipose tissue, egg membrane (including amniotic membrane), placenta, umbilical cord, and dental pulp. Preferably, the mesenchymal cells used in the present invention are mesenchymal cells collected from amniotic membrane.
The mesenchymal cells used in the present invention are positive for CD90 expression, preferably positive for CD105, CD73, and CD90, and negative for CD45, CD34, CD11b, CD19, and HLA-DR.

  A mesenchymal cell used in the present invention comprises a step of enzymatically treating the above tissue (eg, amniotic membrane) with collagenase and / or metal proteinase; and a step of passing the enzyme-treated tissue (eg, amniotic membrane) through a mesh Can be prepared.

  In one example, human amniotic membrane can be treated only once with an enzyme solution containing collagenase and metalloproteinase adjusted to an optimal concentration, and the digested solution after the enzyme treatment can be passed through a mesh. The epithelial cell layer containing the basement membrane that is not digested by collagenase and metal proteinase adjusted to the optimal concentration remains in the mesh, and the MSC contained in the extracellular matrix layer that is digested by collagenase and metal proteinase adjusted to the optimal concentration Since it passes, the MSC can be recovered by recovering the cells that have passed through the mesh.

  Although the amniotic membrane-derived MSC separation method is not particularly limited, for example, the following method can be used. Physically harvest amniotic membrane from human fetal appendages. The amniotic membrane is washed with an isotonic solution such as physiological saline, and the amniotic membrane is immersed in a solution containing an enzyme containing an optimal concentration of collagenase and metal proteinase, followed by shaking and stirring at an appropriate temperature. Since the epithelial cell layer is not separated into individual cells by the enzyme treatment that targets only the extracellular matrix, it remains in a layer structure. Therefore, when the obtained amniotic fluid is passed through the mesh, the epithelial cell layer remains on the mesh. The MSC contained in the extracellular matrix layer can pass through the mesh and be collected.

An example of an amnion-derived MSC separation method will be described in detail.
1. Aseptically collect human fetal appendages from elective cesarean section in the operating room.
2. Aseptically and manually remove the amniotic membrane from the fetal appendage.
3. Transfer the amniotic membrane to a sterile container (disposable cup) and wash it several times with an isotonic solution such as physiological saline to remove the adhering blood.
4). Roughly cut the amniotic membrane with a scalpel or scissors (this step may be omitted). The amniotic membrane may be used after being stored in a medium at 2 ° C. or higher and 8 ° C. or lower for 24 hours or longer and 48 hours or shorter.
5. The amniotic membrane is immersed in a solution containing collagenase adjusted to an optimal concentration and thermolysin and / or dispase, and shaken and stirred at 37 ° C. and 60 rpm for 90 minutes using a constant temperature shaker.
6). As a result, the epithelial cell layer remains one layer, and the MSC contained in the extracellular matrix layer floats in the enzyme-containing solution.
7). Falcon cell strainer (100 μm mesh) is set in a sterilized tube (Falcon 50 mL tube), and the cell-containing solution after digestion of amniotic membrane is filtered by free fall. The epithelial cell layer remains on the mesh, and only the MSC passes through the mesh. To do.
8). The MSC-containing solution that has passed through the mesh is diluted with Hank's balanced salt solution, and then mesenchymal cells are pelleted by centrifugation at 400 × g for 5 minutes.
9. Dilute with αMEM supplemented with 10% fetal bovine serum (FBS), inoculate in plastic flask and culture.

The combination of enzymes used for the separation of amnion-derived MSCs is preferably a collagenase that digests only collagen and a metalloproteinase (eg, thermolysin and / or dispase) that cleaves the N-terminal side of nonpolar amino acids.
As described above, a combination of collagenase and metalloproteinase (eg, thermolysin and / or dispase) is preferably used, but an enzyme (or combination thereof) that releases MSC and does not degrade the epithelial cell layer can also be used. The preferred combination of collagenase and metalloproteinase (eg thermolysin and / or dispase) can be determined by microscopic observation or flow cytometry after enzyme treatment. The concentration is preferably such that the epithelial cell layer is not decomposed and MSC contained in the extracellular matrix layer is released.

  The concentration of collagenase is preferably 50 CDU / mL or more, more preferably 75 CDU / mL or more, still more preferably 100 CDU / mL or more, still more preferably 125 CDU / mL or more, and further preferably 150 CDU / mL or more. Further, the concentration of collagenase is not particularly limited, but for example, 1000 CDU / mL or less, 900 CDU / mL or less, 800 CDU / mL or less, 700 CDU / mL or less, 600 CDU / mL or less, 500 CDU / mL or less, 400 CDU / mL or less, 300 CDU / mL or less. / ML or less. Here, CDU (collagen digestion unit) is defined as the amount of enzyme that produces amino acid and peptide corresponding to 1 μmol of leucine in 5 hours at 37 ° C. and pH 7.5 using collagen as a substrate. The concentration of metalloproteinase (eg, thermolysin and / or dispase) is preferably 100 PU / mL or more, more preferably 125 PU / mL or more, further preferably 150 PU / mL or more, more preferably 175 PU / mL or more, and further preferably 200 PU. / ML or more. Further, the concentration of the metal proteinase is preferably 800 PU / mL or less, more preferably 700 PU / mL or less, further preferably 600 PU / mL or less, further preferably 500 PU / mL or less, and further preferably 400 PU / mL or less. Here, PU (protease unit) is defined as the amount of an enzyme that produces an amino acid and a peptide corresponding to 1 μg of tyrosine per minute at 35 ° C. and pH 7.2 using lactate casein as a substrate. Within the above enzyme concentration range, amniotic membrane MSC contained in the extracellular matrix layer can be efficiently released while preventing contamination of amniotic epithelial cells contained in the epithelial cell layer of amniotic membrane. If the collagenase concentration is less than 50 CDU / mL, or the metalloproteinase concentration is less than 100 CDU / mL, digestion of the extracellular matrix layer of the amniotic membrane may be insufficient, and the recovery rate of the amniotic MSC may be significantly reduced. When the concentration of the metal proteinase is greater than 800 CDU / mL, epithelial cells are mixed into the amniotic digestive fluid as the epithelial cell layer of the amniotic membrane is decomposed, so that the recovery rate of the amniotic MSC may be relatively lowered. A combination of preferable concentrations of collagenase and / or metalloproteinase can be determined by microscopic observation of the amniotic membrane after the enzyme treatment or flow cytometry of the obtained cells.

  The enzyme treatment is preferably performed by immersing the amniotic membrane washed with physiological saline or the like into the enzyme solution and stirring the mixture with a stirrer or a shaker, but other stirring means may be used. In short, the MSC may be efficiently released. . As a result, MSC contained in the extracellular matrix layer is released. The enzyme treatment can be preferably performed by stirring at 10 rpm / min to 100 rpm / min for 30 minutes or more using a stirrer or shaker. The upper limit of the enzyme treatment time is not particularly limited, but it can be generally within 6 hours, preferably within 3 hours, for example, within 90 minutes. The enzyme treatment temperature is not particularly limited as long as the object of the present invention can be achieved, but is preferably 30 ° C. or higher and 40 ° C. or lower, more preferably 30 ° C. or higher and 37 ° C. or lower.

  At this time, it is important to combine the concentrations so that the epithelial cell layer does not decompose. This is because when the epithelial cells are mixed, the MSC content is relatively lowered.

  By filtering the enzyme solution containing the released MSC through the mesh, only the released cells pass through the mesh, and the undegraded epithelial cell layer cannot pass through the mesh and remains in the mesh form. Can be easily recovered. At this time, the pore size (size) of the mesh is not particularly limited as long as it does not contradict the object of the present invention, but is preferably 40 μm or more and 200 μm or less, more preferably 40 μm or more and 150 μm or less, and further preferably 70 μm or more and 150 μm or less. It is particularly preferably 100 μm or more and 150 μm or less. By making the pore size (size) of the mesh within the above range, it is possible to prevent the cell viability from being lowered by dropping by natural dropping without applying pressure.

  Nylon mesh is preferably used as the mesh material. A tube containing nylon mesh of 40 μm, 70 μm, or 100 μm, such as a Falcon cell strainer that is widely used for research, can be used. Moreover, the medical mesh cloth (nylon and polyester) currently used by hemodialysis etc. can be utilized. Furthermore, an arterial filter (polyester mesh, 40 μm or more and 120 μm or less) used during extracorporeal circulation can also be used. Other materials, such as stainless mesh (wire mesh), can also be used.

  When MSC is passed through a mesh, natural fall (free fall) is preferred. Although forced mesh passage such as suction using a pump or the like is possible, in order to avoid damaging the cells, the pressure as low as possible is desirable.

MSCs passed through the mesh can be collected by centrifugation after diluting the filtrate with double or more medium or balanced salt buffer. As the balanced salt buffer, buffer solutions such as Dulbecco's phosphate buffer (DPBS), Earl balanced salt (EBSS), Hanks balanced salt (HBSS), and phosphate buffer (PBS) can be used. The collected cells can be grown by culture if desired. The medium is α MEM (Alpha Modification of Medium Medium Eagle), M199, or a medium based on these, containing DMB, F12, RPMI1640, or these. Medium is less proliferative. Desirably, αMEM medium containing 10% or more of bovine serum or human serum is cultured in a plastic dish flask in a 5% CO ₂ and 37 ° C environment.

αMEM is generally glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, L-histidine, L-isoleucine, Amino acids such as L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-ascorbic acid, biotin , Choline chloride, calcium D-pantothenate, folic acid, niacinamide (nicotinamide), pyridoxal hydrochloride, thiamine hydrochloride (vitamin B1), riboflavin (vitamin B2), vitamin B12, myo-inositol and other vitamins, calcium chloride, sulfuric acid Magnesium, potassium chloride, sodium bicarbonate Um, sodium chloride, containing inorganic salts such as sodium dihydrogen phosphate. αMEM includes, for example, adenosine, cytidine, guanosine, uridine and other ribonucleosides, 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, thymidine and other deoxyribonucleosides, D-glucose, You may contain lipoic acid, phenol red, sodium pyruvate, etc. An example of a preferred αMEM composition is 0.67 mM glycine, 0.28 mM L-alanine, 0.50 mM L-arginine, 0.33 mM L-asparagine, 0.23 mM L-aspartic acid, 0.59 mM L-cysteine, 0 10 mM L-cystine, 2.0 mM L-glutamine, 0.51 mM L-glutamic acid, 0.20 mM L-histidine, 0.40 mM L-isoleucine, 0.40 mM L-leucine, 0.40 mM L-lysine, 10 mM L-methionine, 0.19 mM L-phenylalanine, 0.35 mM L-proline, 0.24 mM L-serine, 0.40 mM L-threonine, 0.049 mM L-tryptophan, 0.20 mM L-tyrosine, 0.39 mM L-valine, 0.28 mM L-ascorbic acid, 4.1 × 0 ^-4 mM biotin, 7.1 × 10 ^-3 mM choline chloride, 2.1 × 10 ^-3 mM D- calcium pantothenate, 2.3 × 10 ^-3 mM folic acid, 8.2 × 10 ^-3 mM niacin Amide, 5.0 × 10 ⁻³ mM pyridoxal hydrochloride, 3.0 × 10 ⁻³ mM thiamine hydrochloride, 2.7 × 10 ⁻⁴ mM riboflavin, 1.0 × 10 ⁻³ mM vitamin B12, 0.011 mM myo Inositol, 1.8 mM calcium chloride, 0.81 mM magnesium sulfate, 5.3 mM potassium chloride, 26.2 mM sodium bicarbonate, 117.2 mM sodium chloride, 1.0 mM sodium dihydrogen phosphate, 5.6 mM D-glucose, 9 7 × 10 ⁻⁴ mM lipoic acid, 0.027 mM phenol red, 1.0 mM sodium pyruvate.

  MSCs obtained by culturing may be peeled off from a plastic dish flask and then seeded and cultured in a new plastic dish flask, or may be stored frozen by any method known in the art. Also good. As a preferred cryopreservation method, for example, the method described in Patent Document 1 (Japanese Patent Laid-Open No. 2015-61520) can be exemplified, but the method is not limited thereto. The cryopreserved MSC may be thawed, seeded in a plastic dish flask, and cultured.

  The expression marker of MSC can be confirmed by any detection method known in the art. Examples of expression markers include, but are not limited to, surface antigens or intracellular expression proteins. Examples of methods for detecting surface antigens and / or intracellularly expressed proteins include, but are not limited to, flow cytometry. In flow cytometry using a fluorescence-labeled antibody, when 5% or more of the measured number of cells that emit fluorescence more strongly than a negative control (isotype control) is detected, the cell is “positive” for the marker. It is determined. As the fluorescently labeled antibody, any antibody known in the art can be used. For example, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), peridinin chlorophyll protein-cyanine 5 And the like, but not limited to antibodies labeled with .5 (PerCP-Cy5.5) or the like.

  In another example, mesenchymal cells used in the present invention can be prepared from the above tissues (eg, bone marrow) using a stem cell separation device.

Although the bone marrow origin MSC separation method is not specifically limited, For example, it can carry out with the following method. Bone marrow fluid is collected from the human iliac crest using a puncture needle, and heparin is added and thoroughly mixed by inversion. A physiological saline solution is passed through a column (filter) attached to a bone marrow MSC separation device (manufactured by Kaneka) to remove air in the column. 2 mL of bone marrow fluid is passed through the column with a syringe pump, and the mesenchymal cells are captured on the column. 2 mL of physiological saline is passed from the same direction, and bone marrow fluid, red blood cells, white blood cells, and platelets remaining on the column are washed and removed. 2 mL of cell culture medium (αMEM) containing 15% of fetal bovine serum (FBS) is vigorously passed in the direction opposite to the direction in which the bone marrow fluid has flowed to recover the mesenchymal cells from the column. 5 mL of a cell culture solution (αMEM) containing 15% of fetal bovine serum is added to 2 mL of the obtained cell suspension, transferred to a plastic dish, and cultured in an environment of 5% CO ₂ and 37 ° C.

[3] Culture supernatant of mesenchymal cells In the present invention, a culture supernatant of mesenchymal cells is used. The culture supernatant of mesenchymal cells is not particularly limited as long as it is a culture solution in which mesenchymal cells are cultured. Examples of the medium for culturing mesenchymal cells when obtaining the above culture supernatant include the above-mentioned medium, which may be a serum-containing medium or a serum-free medium, preferably a serum-free medium. It is. That is, the culture supernatant of mesenchymal cells is preferably a serum-free conditioned medium.

  As used herein, “serum-free medium” means a medium to which no serum is added. The serum-free medium may contain any additive useful for cell growth and / or maintenance. Examples of such additives include proteins, peptides, fatty acids, lipids, growth factors, serum substitutes (for example, Knockout ™ Serum Replacement (KSR) (manufactured by Thermo Fisher Scientific), Chemically Sensitive Lipid Concentrate And GlutaMAX (trademark) (manufactured by Thermo Fisher Scientific) and the like, but are not limited thereto.

  As a procedure for preparing a culture supernatant of mesenchymal cells, first, the mesenchymal cells are cultured using a medium containing serum (for example, αMEM medium containing 10% or more bovine human serum). When the cells become macroscopically confluent, the cells are preferably washed with a washing solution (for example, phosphate buffered saline (PBS)) and then cultured in a serum-free medium (for example, αMEM). The lower limit of the confluence rate is not particularly limited, but is usually 70% or more, preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, and further preferably 90% or more. The upper limit of the confluence rate is not particularly limited, but is usually 100% or less, preferably 99% or less, more preferably 98% or less, further preferably 97% or less, more preferably 96% or less, and still more preferably 95%. % Or less. The lower limit of the culture time in the serum-free medium is not particularly limited, but is usually 12 hours or longer, preferably 18 hours or longer, more preferably 24 hours or longer, more preferably 30 hours or longer, more preferably 36 hours or longer, Preferably it is 42 hours or more. Further, the lower limit of the culture time in the serum-free medium is not particularly limited, but is usually 96 hours or less, preferably 84 hours or less, more preferably 72 hours or less, further preferably 66 hours or less, more preferably 60 hours or less. More preferably, it is 54 hours or less. After culturing in a serum-free medium, the culture medium is collected, and if desired, centrifuged (for example, at 3,000 rpm for 5 minutes), and the supernatant is collected to obtain a culture supernatant of mesenchymal cells. . In the present invention, the culture supernatant of mesenchymal cells can be used as a stenosis inhibitor. The stenosis inhibitor will be described later.

[4] Composition of the present invention One embodiment of the present invention relates to a composition comprising a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP to 1000000 cP.

  The viscosity of the composition of the present invention is 100 cP or more at 25 ± 1 ° C., preferably 200 cP or more, more preferably 300 cP or more, further preferably 400 cP or more, and further preferably 500 cP or more. More preferably, it is 600 cP or more, More preferably, it is 700 cP or more, More preferably, it is 800 cP or more, More preferably, it is 900 cP or more, More preferably, it is 1000 cP or more, More preferably, it is 2000 cP or more, Preferably it is 3000 cP or more, More preferably, it is 4000 cP or more, More preferably, it is 5000 cP or more, More preferably, it is 10000 cP or more, More preferably, it is 20000 cP or more, More preferably, it is 50 And at 00cP or more, still more preferably at least 100000CP, more preferably at least 200000CP, particularly preferably at least 500 000 cP. Further, the upper limit of the viscosity of the composition of the present invention is 1000000 cP or less at 25 ± 1 ° C., more preferably 900000 cP or less, further preferably 800000 cP or less, further preferably 700000 cP or less, and further preferably Is 600,000 cP or less. If the viscosity is less than 100 cP, dripping tends to occur when the composition is applied to the digestive tract, and it becomes difficult to stay in the affected area for a long time when the composition is injected. Moreover, when a viscosity is larger than 1000000 cP, injection | pouring to a digestive tract will become difficult. The viscosity of the gel composition can be measured using, for example, a B-type rotational viscometer, a cone / plate-type rotational viscometer, a capillary viscometer, a falling ball viscometer, or the like.

  The form of the composition of the present invention is not particularly limited as long as it has the above viscosity, and may be any of gel, cream, ointment and the like.

The composition of the present invention is preferably a gel composition comprising a mesenchymal cell culture supernatant and a thickener.
The thickener used in the gel composition is not particularly limited as long as the gel composition can be prepared by gelling the culture supernatant. Specific examples of the thickener include carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxymethylethylcellulose, methylcellulose, carboxymethylhydroxyethylcellulose sodium, and cationized cellulose. Polysaccharides), gum arabic, xanthan gum, carrageenan, juran gum, guar gum, tragacanth gum, caraya gum, tara gum, psyllium seed gum, locust bean gum, pullulan, curdlan, fur cerelan, alginic acid, sodium alginate, polyglutamic acid, agar, pectin, gelatin , Collagen, elastin, glucomannan, chitosan, sorbi Lumpur, sodium chondroitin sulfate, sodium polyacrylate, polyvinyl alcohol, carboxyvinyl polymers, polyvinyl pyrrolidone, dextrin, dextran, alpha-cyclodextrin, but glycerin and the like, are not particularly limited. Only one thickener may be used, or two or more thickeners may be used in combination.

  When the composition of the present invention includes a thickener, the composition of the present invention may further contain any factor for inducing and / or promoting gelation by the thickener. Examples of such factors include, but are not limited to, metal ions. The metal ion is particularly preferably a divalent metal ion, and examples thereof include, but are not limited to, calcium ion and magnesium ion.

  When the composition of the present invention contains a thickener, the lower limit of the content of the thickener is not particularly limited, but is usually 0.3% by mass or more, preferably 0.5% by mass or more, more preferably 1 It is at least mass%, more preferably at least 1.5 mass%, even more preferably at least 2 mass%. Moreover, when the composition of this invention contains a thickener, the upper limit of content of a thickener is although it does not specifically limit, Usually, 10 mass% or less, Preferably it is 9 mass% or less, More preferably, it is 8 mass%. Hereinafter, it is more preferably 7% by mass or less, further preferably 6% by mass or less, and further preferably 5% by mass or less.

  The composition of the present invention can be used as a pharmaceutical (including quasi-drugs), a food or drink, or a cosmetic, but the form of use is not particularly limited.

[5] Method for producing composition When the composition of the present invention is a gel composition, the gel composition is prepared by adding a thickener to the culture supernatant of the mesenchymal cells obtained in [3] above. And suspended. The addition amount of the thickener can be appropriately determined in consideration of the type of thickener to be used, the viscosity intended for the gel composition, and the like. The gel composition means a composition in a gelled state. Gelation means that the whole or part of a composition containing a mesenchymal cell culture supernatant and a thickener is in a state where the viscosity is increased and the fluidity is lost or decreased from the state of the solution. For example, it means solidifying in a jelly form, becoming a solid exhibiting thixotropy, becoming a viscous fluid, and the like.

  The gel composition of the present invention contains a culture supernatant of a mesenchymal cell and a thickener. The gel composition of the present invention may be composed of only a mesenchymal cell culture supernatant and a thickener, or may contain other components other than a mesenchymal cell culture supernatant and a thickener. May be. Examples of other components include, but are not particularly limited to, preservatives, colorants, humectants, ultraviolet absorbers, and antioxidants. Examples of preservatives include, but are not limited to, paraoxybenzoates, benzalkonium chloride, chlorobutanol, cresol, and the like. Examples of the colorant include Red No. 2, Red No. 3, Red No. 102, Red No. 104, Red No. 105, Red No. 106, Yellow No. 4, Yellow No. 5, Blue No. 1, Blue No. 2, and Blue No. 3 However, it is not limited to these. Examples of the humectant include petrolatum, but are not limited thereto. Examples of the ultraviolet absorber include, but are not limited to, octyl salicylate, sinoxate, tetrahydroxybenzophenone, and the like. Examples of the antioxidant include, but are not limited to, ascorbic acid and α-tocopherol.

  When the composition of this invention is a cream, a cream composition can be manufactured by a normal formulation. Examples of cream bases include higher fatty acid esters, lower alcohols, hydrocarbons, polyhydric alcohols (eg, propylene glycol, 1,3-butylene glycol, etc.), higher alcohols (eg, 2-hexyldecanol, cetanol, etc.), Those selected from emulsifiers (for example, polyoxyethylene alkyl ethers, fatty acid esters, etc.), water, absorption accelerators and anti-rash agents can be used alone or in admixture of two or more.

  When the composition of the present invention is an ointment, the ointment composition can be produced by a normal formulation. Examples of the ointment base include higher fatty acids or higher fatty acid esters (for example, adipic acid, myristic acid, palmitic acid, stearic acid, oleic acid, adipic acid ester, myristic acid ester, palmitic acid ester, stearic acid ester, oleic acid). Esters), waxes (eg, beeswax, whale wax, ceresin, etc.), surfactants (eg, polyoxyethylene alkyl ether phosphates, etc.), higher alcohols (eg, cetanol, stearyl alcohol, cetostearyl alcohol, etc.) , Silicone oil (eg, dimethylpolysiloxane), hydrocarbons (eg, hydrophilic petrolatum, white petrolatum, purified lanolin, liquid paraffin), glycols (eg, ethylene glycol, diethylene glycol, propylene glycol) , Polyethylene glycol, macrogol, etc.), vegetable oil (eg, castor oil, olive oil, sesame oil, turpentine oil, etc.), animal oil (eg, mink oil, egg yolk oil, squalane, squalene, etc.), water, absorption enhancer or rash prevention What is chosen from an agent can be used individually or in mixture of 2 or more types.

[6] Stenosis inhibitor and anti-inflammatory agent The present invention further includes
-A stenosis inhibitor containing a culture supernatant of mesenchymal cells;
A stenosis inhibitor comprising a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP to 1000000 cP; and a mesenchymal cell positive for CD90 expression An anti-inflammatory agent comprising a composition having a viscosity of 100 cP to 1000000 cP:
About.

  According to the present invention, there are provided the culture supernatant of the above-described mesenchymal cells used for suppressing stenosis and the above-described composition of the present invention used for suppressing stenosis. According to this invention, the composition of this invention mentioned above used for inflammation suppression is provided.

  According to the present invention, there is further provided a method for suppressing stenosis, comprising the step of administering to the subject in need of stenosis suppression an effective amount of the above-described mesenchymal cell culture supernatant. The present invention further provides a method for suppressing inflammation, comprising the step of administering to the subject in need of inflammation suppression an effective amount of the above-described composition of the present invention.

  The present invention further provides the use of the culture supernatant of mesenchymal cells for the production of a stenosis inhibitor, and the use of the composition of the present invention described above for the production of a stenosis inhibitor. . The present invention further provides the use of the composition of the present invention described above for the manufacture of an anti-inflammatory agent.

  It is presumed that administration of the mesenchymal cell culture supernatant suppresses fibrosis by suppressing fibroblast activation at the administration site, thereby suppressing stenosis. The action mechanism of suppression is not limited to the above. In addition, it is speculated that administration of the composition of the present invention suppresses infiltration of inflammatory cells, thereby suppressing inflammation and preventing and / or treating enteritis. And / or the mechanism of action of treatment is not limited to the above.

  The stenosis inhibitor and anti-inflammatory agent of the present invention can be used as pharmaceuticals (including quasi-drugs), foods and drinks, or cosmetics, preferably in the form of a composition, but the usage forms are not particularly limited. .

The site of stenosis referred to in the present invention is not particularly limited as long as it may cause stenosis. For example, the digestive tract, upper respiratory tract (nasal cavity, pharynx, larynx), lower respiratory tract (trachea, main bronchus), etc. Can be mentioned. The digestive tract is a collective term for digestive and absorption tubes, and means, for example, the oral cavity, esophagus, stomach, and intestinal tract (duodenum, small intestine, large intestine, rectum, colon).
The stenosis may be a transient stenosis or a chronic stenosis.
The stenosis inhibitor of the present invention can be used as a prophylactic and / or therapeutic agent for stenosis.

  The anti-inflammatory referred to in the present invention means prevention, treatment or symptom improvement of inflammation. Inflammation is a general term for various reactions that the immune system responds to invasion such as infection, physical stimulation, chemical stimulation, and allergic reactions. In general, inflammation is caused by the interaction of exogenous factors such as external stimuli and endogenous factors such as abnormalities and constitutional factors on the living body side.

  The anti-inflammatory agent of the present invention can be used as a prophylactic and / or therapeutic agent for inflammatory diseases. Inflammatory diseases include enteritis (colitis, enterocolitis, enterocolitis, proctitis), asthma, inflammatory bowel disease, appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, Cholangitis, cholecystitis, conjunctivitis, cystitis, lacrimal inflammation, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, epicondylitis, epididymis, fasciitis, connective inflammation, gastritis , Gastroenteritis, hepatitis, purulent spondylitis, laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, nephritis, ovitis, testitis, osteomyelitis, otitis, pancreatitis, parotitis, Pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, interstitial pneumonia, pneumonia, prostatitis, pyelonephritis, rhinitis, fallopianitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, grape Examples include but are not limited to meningitis, vaginitis, vasculitis, and vulvitis.

The method for administering the stenosis inhibitor of the present invention is not particularly limited, and examples thereof include direct administration (preferably application) or oral administration to the local area where stenosis should be suppressed. As a method for oral administration, for example, a stenosis inhibitor can be directly taken, or it can be taken by discharging a stenosis inhibitor filled in a syringe, a tube or the like, but is not limited thereto.
Although the administration method of the anti-inflammatory agent of this invention is not specifically limited, For example, administering directly to a disease site is mentioned. When used as an anti-inflammatory agent for enteritis, direct administration to the intestine (preferably the rectum) can be mentioned, and enema administration (intrarectal injection) is preferred. However, the administration method is not limited to the above.

  The doses of the stenosis inhibitor and anti-inflammatory agent of the present invention are such that when administered to a subject, an inhibitory, preventive or therapeutic effect can be obtained as compared to a subject not administered. The specific dose can be appropriately determined depending on the administration form, administration method, purpose of use, age, weight and symptoms of the subject. As an example, the upper limit of the dose when suppressing or preventing stricture of the esophagus is 0.1 mL / kg body weight or more, preferably 0.2 mL in terms of the volume of gel per kg body weight of the administration subject per administration. / Kg body weight or more, more preferably 0.5 mL / kg body weight or more, further preferably 0.8 mL / kg body weight or more, and the lower limit of the dose is 10 mL / kg body weight or less, preferably 5 mL / kg body weight or less, more Preferably it is 3 mL / kg body weight or less. As another example, the upper limit of the dose when treating enteritis is 0.2 mL / kg body weight or more, preferably 0.5 mL / kg in terms of the volume of gel per kg body weight of the administration subject per administration. Body weight or more, more preferably 1 mL / kg body weight or more, further preferably 1.2 mL / kg body weight or more, and the lower limit of the dose is 10 mL / kg body weight or less, preferably 5 mL / kg body weight or less, more preferably 3 mL / kg body weight or less. In the prevention of esophageal stricture and the treatment of enteritis, the above dose can be administered once or more and not more than 3 times a day, preferably once a day. The administration period in the prevention of esophageal stricture and the treatment of enteritis is not particularly limited. For example, the administration period can be 1 day to 1 month, and can be administered at an appropriate interval such as once a day to once a week. it can.

  The dosage form of the stenosis inhibitor and anti-inflammatory agent of the present invention is not particularly limited as long as it can be safely administered to mammals including humans. For example, injections, solutions, extracts, Aerosols, patches, cataplasms, gels, creams, ointments, transdermal preparations, suspensions, emulsions and the like can be mentioned. These preparations can be produced, for example, by the method described in the Japanese Pharmacopoeia, but are not limited thereto.

  The stenosis inhibitor and anti-inflammatory agent of the present invention can be used for food and drink. In addition, the stenosis inhibitor and anti-inflammatory agent of the present invention can be contained in foods, nutritional functional foods, foods for specified health use, nutritional supplements, nutrients, beverages, feeds, and added to general foods. You can also. For example, it can be used by appropriately adding to bread, pasta, miscellaneous cooking, cooked rice, biscuits, crackers, cakes, confectionery, gum, candy and the like.

  When using the stenosis inhibitor and anti-inflammatory agent of the present invention for food and drink, the lower limit of the content of the stenosis inhibitor and anti-inflammatory agent in the food and drink is not particularly limited, but usually 0.1% by mass or more , Preferably 0.3% by mass or more, more preferably 0.5% by mass or more, and further preferably 1% by mass or more. Moreover, although the upper limit of content of the constriction inhibitor and anti-inflammatory agent in food / beverage products is not specifically limited, Usually, it is 80 mass% or less. Although the said food / beverage products can be ingested from a mouth, for example, it is not limited to this.

   The stenosis inhibitor and anti-inflammatory agent of the present invention can also be used for cosmetics. Examples of final product forms as cosmetics include, but are not limited to, ointments, lotions, lotions, creams, non-woven cloth impregnated cosmetics (masks), packs, and the like.

  When the stenosis inhibitor and anti-inflammatory agent of the present invention are used for cosmetics, the lower limit of the content of the stenosis inhibitor and anti-inflammatory agent in cosmetics is not particularly limited, but is usually 0.1% by mass or more, preferably Is 0.3% by mass or more, more preferably 0.5% by mass or more, and further preferably 1% by mass or more. Moreover, although the upper limit of content of the stenosis inhibitor and anti-inflammatory agent in cosmetics is not particularly limited, it is usually 80% by mass or less. For example, the cosmetic can be applied to or spread on the skin, but is not limited thereto.

  The present invention will be specifically described in the following examples, but the present invention is not limited to the examples.

Example 1: Preparation of gel composition Human amniotic MSC was prepared according to the method described in Example 1 of Patent Document 1 (Japanese Patent Laid-Open No. 2015-61520) (provided that collagenase 300 CDU / mL and thermolysin 250 PU / mL). The conditions were separated and cultured. Specifically, it is as follows.

  The amniotic membrane was manually separated from the pregnant human fetal appendages of a waiting cesarean section with informed consent. After washing twice with Hank's balanced salt solution (without Ca · Mg), 1 g of the obtained amniotic membrane was placed in a container and purified collagenase (CLSPA, Worthington, Standards> 500 CDU / mg) 300 CDU / mL and thermolysin ( A total of 5 mL of Hank's balanced salt solution (containing Ca · Mg) containing 250 PU / mL (Wako Pure Chemical, Standard> 7000 PU / mg) was added, and the mixture was shaken and stirred with a shaker at 37 ° C. for 90 minutes and 60 rpm. The obtained mixture was diluted with 10% fetal bovine serum (FBS) -added αMEM (Alpha Modification of Minimum Essential Medium) and then filtered using a nylon mesh filter (pore size: 100 μm), and the filtrate containing amniotic membrane MSC was collected. did. The filtrate containing amniotic membrane MSC was centrifuged at 400 × g for 5 minutes, and the supernatant was removed. The obtained cell pellet was suspended in αMEM containing 10% FBS, seeded on a plastic dish, and amnion MSCs were allowed to adhere and culture.

  The result of confirming the expression of the surface antigen in the amniotic membrane MSC with a flow cytometer is shown in FIG. In the amniotic membrane MSC obtained above, the surface antigens CD105, CD73, and CD90 were positive, and CD45, CD34, CD11b, CD19, and HLA-DR were negative. CD14 and CD79alpha are also negative. Furthermore, this amniotic membrane MSC can be differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.

  Amniotic membrane MSCs that had been subjected to adhesion culture until the confluence rate was 90% or more and 95% or less were treated with ethylenediaminetetraacetic acid (EDTA) and trypsin and detached from the plastic dish. The resulting cell suspension was centrifuged and the supernatant was removed. The obtained cell pellet was suspended in αMEM containing 10% FBS, seeded on a plastic dish, and amnion MSCs were allowed to adhere and culture.

  When the cells became macroscopically confluent, the adherent cells were washed three times with phosphate buffered saline (PBS), and then cultured in serum-free medium (αMEM) for 48 hours. Thereafter, the medium was collected and centrifuged at 3,000 rpm × 5 minutes, and the supernatant was collected to obtain a culture supernatant. To the culture supernatant, carboxymethylcellulose (CMC) (sodium carboxymethylcellulose manufactured by Wako Pure Chemical Industries, Ltd., or Marumishi Pharmaceutical's carmellose sodium powder “Maruishi”) is added so that the final concentration is 2% by mass or 5% by mass. The gel composition was prepared by suspending and gelling. As a comparative example, a liquid composition was prepared by adding CMC (carboxymethylcellulose sodium manufactured by Wako Pure Chemical Industries, Ltd.) to the culture supernatant so that the final concentration was 1% by mass.

  The viscosity of a liquid composition having a CMC concentration of 1% by mass and a gel composition (with a CMC concentration of 2% by mass and 5% by mass) were measured by the following method. As a result, the viscosity of the liquid composition having a CMC concentration of 1% by mass is 88 cP, the viscosity of the gel composition having a CMC concentration of 2% by mass is 704 cP, and the viscosity of the gel composition having a CMC concentration of 5% by mass is 135600 cP. The viscosity of the gel composition having a carmellose sodium concentration of 2% by mass was 3544 cP, and the viscosity of the gel composition having a carmellose sodium concentration of 5% by mass was 476000 cP.

(Measuring method of viscosity of gel composition)
The viscosity of the gel composition was measured at 25 ± 1 ° C. using a B-type rotational viscometer (BROOKFIELD VISCOMETER DV2T, manufactured by Eiko Seiki Co., Ltd.).

Example 2: Prevention of esophageal stricture <Test method>
The esophageal ESD model was performed on a 20 kg female domestic pig with the submucosal layer peeled off at the same position in each pig at a major axis of 5 cm and 3/4 circumference. Specifically, ESD was performed using an ESG100 electrical generator and an IT nanoknife (manufactured by Olympus), and the endoscope used was GIF-Q240 manufactured by Olympus. The day when ESD is performed and Day1.

Immediately after ESD, a culture supernatant gel (20 mL) containing 5% by mass of carboxymethylcellulose prepared in Example 1 was injected and applied to the ulcer surface. The culture supernatant gel was applied to Day 1, 8, and 15 at the time of endoscopic observation (gel group: n = 3). A non-cultured αMEM gel was used as a control (control group: n = 3). The non-cultured αMEM gel was applied to Day 1, 8, and 15. In addition, a method of locally injecting Trimcinolone 80 mg in 4 mL physiological saline currently used in daily clinical practice into Day 1 at 0.5 mL each on the ulcer surface was also performed (steroid group: n = 3).
The pig was slaughtered on Day 22, the esophagus was removed, and the histopathological evaluation was performed by measuring the stenosis rate.

<Evaluation and results>
(Measurement of esophageal stricture rate)
The esophageal stenosis rate was determined by the following formula.
Stenosis rate (%) = {1-a / (b1 + b2) × 1/2} × 100
a indicates the length of the short axis of the maximum stenosis site, and b1 and b2 indicate the length of the short axis of the normal mucosa. The positional relationship of the lengths a, b1, and b2 is shown in FIG.

The results of measuring the esophageal stenosis rate for the control group, gel group, and steroid group are shown in FIG. In FIG. 3, * and ** indicate the following.
* P <0.001 vs STD ANOVA and Tukey's multiple comparison test ** P <0.0001 vs STD ANOVA and Tukey's multiple comparison test

  For the porcine esophageal ESD model, the esophageal stenosis rate was 86.3 ± 2.0% in the control group, 56.3 ± 7.1% in the gel group (P <0.01 vs control), and 49.3 ± in the steroid group. 4.2% (P <0.01 vs control).

(Thickness of stenotic fiber)
The fiber thickness of the stenosis was measured by masson-trichrome staining. Masson-trichrome staining is a method of staining collagen fibers with aniline blue (http://www.mutokagaku.com/products/reagent/pathology/masson_formalin/).
An example of the state of masson-trichrome staining is shown in FIG. The distance indicated by the arrow in the right diagram of FIG. 4, that is, the distance from the line connecting both ends of the mucosal muscle plate to the deepest part of the fibrous tissue was defined as the fiber thickness of the stenosis.

The results of macroscopic findings (left side) and masson-trichrome staining for the control group, the gel group, and the steroid group are shown in FIG.
Moreover, the measurement result of the fiber thickness of a constriction part is shown in FIG. In FIG. 6, * and ** indicate the following.
* P <0.05 vs STD ANOVA and Newman-Keuls multiple comparison test ** P <0.05 vs STD ANOVA and Newman-Keuls multiple comparison test

  The fiber thickness of the stenosis is 1,609 ± 418 μm in the control group, whereas it is 833 ± 26 μm (P <0.01 vs control) in the gel group, and 944 ± 250 μm (P <0) in the steroid group. .05 vs control).

(Number of activated fibroblasts)
The number of activated fibroblasts was measured by α-SMA staining. The primary antibody was an anti-α-SMA antibody manufactured by Sigma-Aldrich, and reacted at room temperature for 60 minutes at 1000-fold dilution. Observation was performed in a 40-fold field, and the number of stained positive cells was counted.

The measurement result of the number of activated fibroblasts is shown in FIG. In FIG. 7, * and ** indicate the following.
* P <0.001 vs STD ANOVA and Newman-Keuls multiple comparison test ** P <0.001 vs STD ANOVA and Newman-Keuls multiple comparison test

  The number of activated fibroblasts in the control group is 68.3 ± 5.7 / 40-fold field, whereas the gel group is 26.8 ± 8.6 / 40-fold field (P <0.00. 05 vs control) and the steroid group was 20.6 ± 2.3 / 40-fold field (P <0.01 vs control).

(Summary)
From the above results, it was shown that the application of the gel composition of the present invention containing the culture supernatant suppresses fibrosis and suppresses esophageal stricture by suppressing the activation of fibroblasts.

Example 3 Treatment of Enteritis <Test Method>
The enteritis model was prepared by enemarating a 30% ethanol solution of 15 mg / mL TNBS (2,4,6-trinitrobenzenesulfonic acid) to 10-week-old SD rats (enteritis group). The culture supernatant gel (400 μL) containing 2% by mass of carboxymethylcellulose prepared in Example 1 was enema-administered 3 hours after TNBS administration and the next day and the next day (enteritis + gel group) (n = 9). One week after administration of TNBS, the rat rectum was sacrificed after observation with an endoscope, and the histopathological evaluation of the rectum was performed.
In the control group, 30% ethanol was enema administered instead of TNBS (n = 7), and in the enteritis group, an αMEM gel that had not been cultured was enema administered instead of the culture supernatant gel (n = 9). .

<Evaluation and results>
(Pathology score)
The pathological score was evaluated by the method described in the column “Colonoscopic Assessment” of PLoS One 2012: 7; e33360. That is, 5 of “Change of the vascular pattern”, “Mucosal granularity”, “Strictures”, “Bleeding”, and “Ulcecrs”. For each parameter, the score was “0” for “Yes” and 1 for “No”, and the total score was obtained (the total score was between 0 and 5).

  The measurement result of the pathological score is shown in FIG. In FIG. 8, * is p <0.01 vs control, † is p <0.05 vs. TNBS + αMEM gel is shown. For the rat enteritis model, the pathological score was significantly improved by administration of the culture supernatant gel.

(Immunostaining)
CD68 staining was performed as follows. As the primary antibody, an anti-CD68 antibody manufactured by AbD Serotec was used, and the mixture was reacted at a dilution of 100 times for 60 minutes at room temperature.
The staining of myeloperoxidase was performed as follows. The primary antibody was an anti-MPO antibody manufactured by Thermo, and reacted at 4 ° C. overnight at a 300-fold dilution.
Cells were observed in a 40 × field and the number of stained positive cells was counted.

The staining results of CD68 positive macrophages are shown in FIG. 9, and the staining results of myeloperoxidase positive neutrophils are shown in FIG. The unit of the vertical axis in the graphs of FIG. 9 and FIG. 9 and 10, * indicates p <0.05 vs. control, † indicates p <0.05 vs. TNBS + αMEM gel is shown (Newman-Keuls method).
As a result of the above immunohistochemical staining, it was found that administration of the culture supernatant gel significantly suppressed infiltration of CD68 positive macrophages and myeloperoxidase positive neutrophils.

(TNFα mRNA expression level)
The mRNA expression level of TNFα in the colon was measured as follows.
RNA was extracted from rectal tissue using RNeasy mini kit (Qiagen), and reverse transcription was performed using Quantec Reverse Transfer Kit (Qiagen). PCR was performed from the obtained cDNA using primers for TNFα and βactin using Platinum SYBR Green qPCR SuperMix-UDG with ROX. The gene expression level was expressed as a relative value with respect to βactin.

  FIG. 11 shows the measurement results of the TNFα mRNA expression level. The unit of the vertical axis in FIG. 11 indicates “relative expression”. In FIG. 11, * is p <0.05 vs. control, † is p <0.05 vs. TNBS + αMEM gel is shown (Newman-Keuls method). It was found that administration of the culture supernatant gel suppresses the mRNA expression level of TNFα.

(Summary)
From the above results, it was found that enema administration of the culture supernatant gel suppressed inflammatory cell infiltration and improved the pathology of the enteritis model.

Example 4: Culture-supernatant-containing beverage 1.0 parts by weight of culture supernatant gel containing 5% by mass of carboxymethylcellulose prepared in Example 1, 10.7 parts by weight of fructose-glucose liquid sugar, 0.18 parts by weight of citric acid A culture supernatant-containing beverage consisting of 1 part, 0.04 part by weight of trisodium citrate, and 88.08 parts by weight of water was prepared. The obtained culture supernatant-containing beverage was not agglomerated, separated, solidified, etc., and maintained a transparent state.

Claims (17)

A stenosis inhibitor comprising a culture supernatant of mesenchymal cells. The stenosis inhibitor according to claim 1, wherein the mesenchymal cells are human amnion-derived mesenchymal cells. The stenosis inhibitor according to claim 1 or 2, which is a digestive tract stenosis inhibitor. The stenosis inhibitor according to any one of claims 1 to 3, which is an esophageal stenosis inhibitor. A composition comprising a culture supernatant of mesenchymal cells positive for CD90 expression and having a viscosity of 100 cP or more and 1000000 cP or less. The composition according to claim 5, wherein the mesenchymal cells are human amnion-derived mesenchymal cells. The composition according to claim 5 or 6, wherein the culture supernatant is a serum-free conditioned medium. The composition according to any one of claims 5 to 7, wherein the composition is a gel composition containing a thickener. The composition according to claim 8, wherein the thickener is a polysaccharide. The composition according to claim 8 or 9, wherein the thickener is carboxymethylcellulose. The composition according to any one of claims 5 to 10, which is used as a pharmaceutical, a food or drink, or a cosmetic including a quasi-drug. The method according to any one of claims 8 to 10, comprising a step of suspending a thickener in a culture supernatant of mesenchymal cells positive for CD90 expression and gelling the resulting suspension. A method for producing the composition. The culture supernatant of mesenchymal cells positive for CD90 expression was obtained by culturing mesenchymal cells positive for CD90 expression in a serum-containing medium and then culturing in a serum-free medium. The method of claim 12, wherein A stenosis inhibitor comprising the composition according to any one of claims 5 to 11. The stenosis inhibitor according to claim 14, which is a digestive tract stenosis inhibitor. The stenosis inhibitor according to claim 14 or 15, which is an esophageal stenosis inhibitor. An anti-inflammatory agent comprising the composition according to any one of claims 5 to 11.