JPH04178400A - Bioactive substance - Google Patents

Abstract

NEW MATERIAL:A bioactive substance having an effect for promotion of production of acetylcholinesterase on megakaryocyte-based cells. USE:A preventive and therapeutic agent for diseases accompanied by functional disorders of megakaryocytes and platelet. PREPARATION:For example, human-derived cell strain KHM-5M is cultured in a culture medium at 37 deg.C for 3 days and the resultant cell-floating liquid is then centrifuged using a high-speed refrigerated centrifuge to collect a supernatant. The resultant supernatant is concentrated using an ultrafiltration membrane and the resultant concentrated liquid is dialyzed overnight at 4 deg.C using 50mM sodium acetate buffer solution (pH5.5). The obtained dialysis-treated sample is subjected to cation-exchange chromatography and the obtained active fraction is subjected to electrophoresis using an SDS-polyacrylamide gel. The obtained fraction of 19KD-31KD molecular weight is treated with hydrophobic chromatography, anion-exchange chromatography and reverse phase chromatography in order for purification, thus obtaining the objective bioactive substance.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to a physiologically active substance, a method for producing the physiologically active substance,
The present invention relates to a culture supernatant of a cell line that produces the physiologically active substance and a pharmaceutical composition containing the physiologically active substance as an active ingredient. Since the physiologically active substance of the present invention acts on the megakaryocyte-platelet system, it is particularly useful in the medical field.

<Conventional technology> There are cells such as red blood cells, white blood cells, lymphocytes, and platelets in the blood that circulates in the living body, and each of them has a unique function and is responsible for maintaining the homeostasis of the living body. There is. Elucidating how these blood cells differentiate, mature, and appear in the blood has been a major research theme in the field of hematology for many years. It has become clear that the hematopoietic stem cells differentiate and mature from a single type of multifunctional hematopoietic stem cell, and that humoral factors in the body are involved in the differentiation and maturation process. These humoral factors are expected to be applied as therapeutic agents for diseases accompanied by a decrease in blood cells, but to date, among these humoral factors, erythropoietin, G-C3F, GM- C
Is it a factor such as SF, M-C3F, or various interleukins?
# is separated, and the - part is beginning to be applied as a drug as a differentiation and maturation factor for red blood cells, white blood cells, and lymphocytes.

By the way, research on the megakaryocyte-platelet system has been delayed compared to other blood cell systems, and recently it has been reported that IL-6 promotes the formation of megakaryocytes, which are precursor cells of platelets. (Toshiyuki l5hibash
i etc., Proc, NatlAcad, Sci,
LISA, Vol, 86, p, p, 5953~
5957.1989 and ChoshiyukiIshi
bashi et al., Blood, Vol. 74.
p, p1241-1244.1989). However, IL-6 exhibits a wide variety of actions, one of which is that it is known to be deeply involved in the induction of inflammation as an acute phase reaction protein in vivo.
When used as a medicine, there are concerns that it may be accompanied by strong side effects. Therefore, it is a physiologically active substance other than the above-mentioned IL-6 that promotes differentiation, maturation, and proliferation of megakaryocyte-platelet system! #I want you to leave me.

<Problems to be Solved by the Invention> An object of the present invention is to provide a physiologically active substance that promotes differentiation, maturation, and/or proliferation of the megakaryocyte-platelet system.

Another object of the present invention is to provide a method for producing the physiologically active substance and a culture supernatant of the cell line KHM-5M that produces the physiologically active substance.

Another object of the present invention is to provide a pharmaceutical composition containing the physiologically active substance as an active ingredient for treating diseases accompanied by megakaryocyte and/or thrombocytopenia and for treating megakaryocytes and/or platelets. The aim is to provide them as therapeutic or preventive agents for diseases accompanied by functional abnormalities.

<Means for Solving the Problems> The present inventors have conducted extensive research in order to identify physiologically active substances that promote the differentiation, maturation, or proliferation of the megakaryocyte-platelet system. As a result, the present inventors found that the above-mentioned activity was found in the culture supernatant of the human-derived cell line KHM-5M. The present invention was completed by purifying the target physiologically active substance and clarifying its properties.

That is, the first aspect of the present invention is a physiologically active substance characterized by having an effect of promoting acetylcholinesterase production in megakaryocytic cells.

As the physiologically active substance, mouse megakaryocytic cell line L805
7, those having an effect of promoting acetylcholinesterase production are preferred.

As the physiologically active substance, SDS-PAGE under non-reducing conditions
Preferably, the molecular weight is from about 19 kD to about 31 kD.

Furthermore, the physiologically active substance preferably has one or more of the following properties a) to d).

a) Activity remains after treatment with 1% SDS at 37°C for 60 minutes.

b) Activity remains after treatment with 0.05% trifluoroacetic acid and 150% acetonitrile at 25°C for 60 minutes.

c) Activity remains after treatment with 7M urea at 25°C for 180 minutes.

d) Activity remains after treatment at 25° C. for 240 minutes at pH 2-12.

Furthermore, it is preferable that the physiologically active substance be one whose presence is confirmed in the culture soil of KHM-5M cell line.

The physiologically active substance of the present invention preferably has the activity of promoting any one or more of differentiation, maturation, and proliferation of the megakaryocyte-platelet system.

Furthermore, the physiologically active substance of the present invention is preferably derived from humans, and more preferably can be produced by the human-derived cell line KHM-5M.

The physiologically active substance of the present invention preferably has the amino acid sequence shown below, and more preferably has an amino acid sequence in which X is Cys or Ser.

Glyteu Glu X Asp Gl
y Lys Val Asn Ile X
X tys Lys Gln PhePhe
Val X Phe Lys Asp
Ile Gly (X indicates that the amino acid is not specified. Also, the four X's may be the same or different from each other.) The second aspect of the present invention is one of the following steps. A method for producing a bio-embedded active material according to the first aspect of the invention, characterized in that at least one of the following is carried out.

a) Culture the human-derived cell line KHM-5M.

b) Collect the culture supernatant of the human-derived cell line KHM-5M.

C) Perform at least one purification method selected from the following.

■Anion exchange chromatography ■Cation exchange chromatography ■Reverse phase chromatography ■Hydrophobic chromatography Each of the above-mentioned chromatographies preferably has the following characteristics.

a) Anion exchange chromatography uses a carrier into which diethylamine ethyl groups are introduced.

b) Cation exchange chromatography uses a carrier into which a sulfopropyl group has been introduced.

c) Reversed phase chromatography uses a butyl group-introduced carrier or polystyrene divinyl Hensen polymer as a carrier.

d) Hydrophobic chromatography uses a carrier into which phenyl groups are introduced.

A third aspect of the present invention is a human-derived cell line KHM-, which is characterized by containing the physiologically active substance of the first aspect of the present invention.
5M culture supernatant.

A fourth aspect of the present invention is a pharmaceutical composition characterized by containing the physiologically active substance of the first aspect of the present invention as an active ingredient.

The contents of the present invention will be explained in detail below.

The physiologically active substance of the first aspect of the present invention has an effect of promoting acetylcholinesterase production in a patented strain characterized by having an effect of promoting acetylcholinesterase production in megakaryocytic cells. is preferred.

Acetylcholinesterase is an enzyme produced when megakaryocytic cells in the dentine neck differentiate and/or mature, so this indicates that the physiologically active substance of the present invention acts on the megakaryocyte-platelet system. It shows.

In addition, as the physiologically active substance of the present invention, SD under non-reduction
Molecular weight measured by S-PAGE is approximately 19 kD to approximately 31
kD is preferred.

As is well known, the molecular weight of physiologically active substances can be determined by gel filtration or S
It varies depending on the measurement method such as DS-PAGE, the type of carrier and molecular weight marker used for measurement, and measurement conditions.
Therefore, the bio-burial active substance of the present invention has a molecular weight of 14.4 kD and 20.1 kD in SDS-PAGE under non-reducing conditions using Pharmacia LMW kit E (manufactured by Pharmacia Co., Ltd.).

The molecular weight is approximately 19k when measured using 30kD, 43kD, 67kD and 94kD molecular weight markers.
D to about 31 kD is preferred.

Furthermore, the physiologically active substance of the present invention preferably has one or more of the following properties a) to d).

a) Activity remains after treatment with 1% SDS at 37°C for 60 minutes.

b) Activity remains after treatment with 0.05% trifluoroacetic acid/50% acetonitrile at 25°C for 60 minutes.

c) Activity remains after treatment with 7M urea at 25°C for 180 minutes.

d) Activity remains after treatment at 25° C. for 240 minutes at pH 2-12.

By the way, the physiologically active substance of the present invention is thought to exist in body fluids and cell culture supernatants where physiologically active substances are expected to exist in general, but when the substances are grouped according to their location, , KHM-sMM! It may be classified into the group that exists in the A cell culture supernatant.

Furthermore, it is preferable that the physiologically active substance of the present invention has the activity of promoting any one or more of differentiation, maturation, and proliferation of the megakaryocyte-platelet system.

Here, the promoting activity for megakaryocyte-platelet system is
It includes not only the above-mentioned activities in the process of platelet production from megakaryocytes, but also any one or more of the activities that promote the differentiation, maturation, and proliferation of megakaryocytes or their progenitor cells. .

By the way, when measuring megakaryocyte-platelet differentiation, thermogenesis, and proliferation, bone marrow cells or megakaryocytic cells are generally used. That is, this is a method in which a test substance is applied to these cells and the appearance of proteins and enzymes specific to megakaryocytes and platelets is measured.

Bone marrow cells and megakaryocytic cells used for such purposes are generally known as human-derived cells or mouse-derived cells, but if mouse-derived cells are used, human rL-3 and human GM-C5F can be used. , the influence of known humoral factors that exhibit species specificity between humans and mice can be excluded. Among them, the established mouse megakaryocytic cell line L8057 is useful (Journal of the Japanese Society of Hematology, Vol. 51, p. 310, Abstract No. 187, 1988). Like mouse megakaryocyte cells, these cells produce acetylcholinesterase (hereinafter abbreviated as AchE) as they mature, so either stain the cells and measure the number of cells that produce AchE. Alternatively, the produced AchE activity is measured with a spectrophotometer (for example, Toshiro Ngasawa et al.
Journal of the Japanese Society of Hematology, Vol. 49, pp. 168B-1695, 19
(see 1986), it is possible to measure the effects of physiologically active substances on the differentiation, maturation, and proliferation of the megakaryocyte-platelet system.

Furthermore, it has been reported that this mouse megakaryocytic cell line L8057 does not react with recombinant human IL-6, recombinant human erythroboietin, mouse IL-3, and recombinant mouse IL-1. Experime
ntal Haematology.

Vol, 18. p, 594. Presentation number 174゜1990
Year). ' The biologically active substance of the present invention is not limited in its origin, but
Preferably, it is of human origin. For example, a culture supernatant of human-derived cells that produce the physiologically active substance of the present invention, a substance obtained from human body fluids such as urine, a substance produced using a human gene for the physiologically active substance of the present invention, etc. be. Among these, those derived from the human-derived cell line KHM-5M are preferable, and particularly those obtained from the culture supernatant of the human-derived cell line KHM-5M are preferable.

Furthermore, the physiologically active substance of the present invention preferably has the amino acid sequence shown below, and particularly preferably has an amino acid sequence that is X, Cys, or Ser in the amino acid sequence.

Gly Leu Glu X Asp Ga
y Lys Val 8s Ile X
X Lys Lys Gin PheP
he Val, X Phe Lys Asp
rle Gly (X indicates that the amino acid is not specified. Also, the four X's may be the same or different.) The method for producing the physiologically active substance of the present invention is not particularly limited, but , preferably manufactured according to the second aspect of the present invention described later.

Next, a manufacturing method according to the second aspect of the present invention will be explained.

A second aspect of the present invention includes at least one of the following steps.
A method for producing a physiologically active substance according to the first aspect of the present invention, which is characterized by carrying out the following steps.

a) Culture the human-derived cell line KHM-5M.

b) Collect the culture supernatant of the human-derived cell line KHM-5M.

C) Perform at least one purification method selected from the following.

■Anion exchange chromatography ■Cation exchange chromatography ■Reverse phase chromatography ■Hydrophobic chromatography In the present invention, it is not necessary to carry out all of the above steps. A method for producing a physiologically active substance of the present invention in which other steps are added to at least one of the above steps as necessary is also included in the method of the present invention.

Here, each of the above steps will be described in sequence.

The step of culturing the human-derived cell line KHM-5M in a) above includes:
This may be carried out under culture conditions that allow the cells to proliferate.

That is, the human-derived cell line KHM-5M may be cultured at 37° C. in a medium containing serum and growth factors such as fetal bovine serum and insulin at concentrations suitable for propagating the cell line. The medium is DME, which is currently commonly used.
M (Dulbecco's Modified Ea
gle's Medium), I M D M
(Iscove's Modified Dulbec
The medium may be appropriately selected from media such as Co's Medium) and RPMI 1640.

To give an example of a preferred method, the cells are cultured under conditions suitable for proliferation, and then the cells are cultured under conditions suitable for recovering the physiologically active substance of the present invention, such as complete synthesis without serum etc. It is best to transfer it to a medium and culture it.

It is also possible to culture the cells using individual animals.

That is, the cells can be transplanted into an animal, or cultured using the animal's body fluids in a diffusion chamber installed inside or outside the animal.

Furthermore, the cells cultured using an individual animal can also be taken out from the individual animal, dispersed, and cultured in an appropriate growth medium.

When culturing the cells using an individual animal, any animal that can proliferate the cells can be used, but mice, rats, and hamsters that have had their thymus removed or have been treated with anti-thymus antibodies can be used. Alternatively, it is preferable to use nude mice or nude rats.

The step of collecting the culture supernatant of the human-derived cell line KHM-5M in b) above involves, for example, collecting the culture supernatant from the medium or body fluid in which the human-derived cell line KHM-5M was cultured as described in a). This is the process of collecting.

That is, after culturing the human-derived cell line KHM-5M, the medium is removed from the culture container, and if necessary, the cells in the medium and the culture supernatant are separated by means such as centrifugation or filtration, The culture supernatant may be collected.

The method for purifying the physiologically active substance of the present invention from the culture supernatant thus obtained is not particularly limited, and includes salting-out method, ultrafiltration method, isoelectric precipitation method, Kell filtration method, ion exchange chromatography. , hydrophobic chromatography, various types of affinity chromatography such as antibody affinity chromatography, chromatofocusing methods, adsorption chromatography, reversed-phase chromatography, etc., and many documents and books, such as "Biochemistry Experiment Course 1 Chemistry of Protein Purchasing" ”
(Nippon Seikagaku Kinen, 1976, Tokyo Kagaku Dojin), an appropriate method may be selected from among the methods described, and the selected method may be performed in an appropriate order. However, the C
), the physiologically active substance of the present invention can be easily purified.

In step C), a liquid containing the physiologically active substance of the present invention is
This is a step of subjecting the product to at least one purification method selected from anion exchange chromatography, cation exchange chromatography, reversed phase chromatography, and hydrophobic chromatography.

Here, the liquid containing the physiologically active substance of the present invention includes, for example, the culture supernatant of tissues and cells that produce the physiologically active substance of the present invention, including the human-derived cell line K) fM-5M, and the present invention. These include a culture medium in which vine cells or microorganisms expressing the physiologically active substance of the present invention are cultured, a body fluid containing the physiologically active substance of the present invention, and the like.

In step C), the anion exchange chromatography is preferably an anion exchange chromatography using a carrier into which a diethylaminoethyl group is introduced, and the cation exchange chromatography is preferably an anion exchange chromatography using a carrier into which a sulfopropyl group is introduced. Cation exchange chromatography is preferred, reversed phase chromatography is preferably reversed phase chromatography using a carrier into which a butyl group is introduced or polystyrene divinylbenzene polymer is used as a carrier, and hydrophobic chromatography is preferably , hydrophobic chromatography using a carrier into which a phenyl group is introduced is preferred.

Next, the culture supernatant of the third aspect of the present invention will be explained.

The culture supernatant of the third aspect of the present invention is a culture supernatant of the human-derived cell line KHM-5M, which is characterized by containing the physiologically active substance of the first aspect of the present invention.

The culture supernatant of the present invention may be any culture supernatant obtained by culturing KHM-5M, and may be a completely synthetic medium as exemplified in the explanation section regarding the second aspect of the present invention, or a medium containing serum or the like. or animal body fluids.

In addition, the present inventors used the human-derived cell line KHM-5M as
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on August 9, 1990 (Accession number: Tetsukoken Bacteria Collection No. 11).
[No. i'54 (FERMP-11654)).

Next, the pharmaceutical composition of the fourth aspect of the present invention will be explained.

The pharmaceutical composition of the present invention is characterized by containing the physiologically active substance of the first aspect of the present invention as an active ingredient.

Even if the pharmaceutical composition of the present invention is prepared by simply treating the physiologically active substance shown in the first aspect of the present invention with a pharmaceutically necessary process such as freeze-drying or sterile filtration, Although it is capable of providing effects in the medical field, it may also contain pharmaceutically acceptable auxiliary ingredients in addition to the physiologically active substance of the present invention.

These auxiliary ingredients include bases, stabilizers, preservatives, preservatives, emulsifiers, suspending agents, solubilizers, solubilizers, lubricants, flavoring agents, colorants, fragrances, soothing agents, and excipients. It refers to excipients, binders, thickeners, buffering agents, etc. Specifically, they include calcium carbonate, lactose, sucrose, sorbitol, mannitol, starch,
Examples include amylopectin, cellulose conductor, gelatin, cocoa butter, distilled water for injection, aqueous sodium chloride solution, Ringer's solution, glucose solution, human serum albumin (H5A), and the like.

When preparing the pharmaceutical composition of the present invention, for example, refer to the Pharmaceutical Excipient List (published by the Pharmaceutical Affairs and Regulations Committee of the Tokyo Pharmaceutical Manufacturers Association and the Pharmaceutical Affairs and Regulations Research Committee of the Osaka Pharmaceutical Association), and select the appropriate auxiliary ingredients. Just choose. Further, the amount of the auxiliary component to be used may be appropriately selected from a pharmaceutically acceptable range depending on the drug form of the pharmaceutical composition.

The dosage of the pharmaceutical composition of the present invention is appropriately selected depending on the condition, age, sex, weight, etc. of the patient receiving treatment, and
The administration method depends on the patient's condition: oral administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intravenous administration,
Although various administration methods are selected, such as intraarterial administration and rectal administration, intravenous administration is particularly preferred.

The pharmaceutical composition can be used to treat or prevent diseases accompanied by a decrease in megakaryocytes and/or platelets, diseases accompanied by abnormal functions of megakaryocytes and/or platelets, or to reduce or prevent side effects of anticancer drugs. It will provide an effective means.

<Examples> The present invention will be explained in more detail below with reference to Examples, but these are shown as examples of implementation, and the present invention is not limited to these examples. Furthermore, the abbreviations used in the following description are based on the abbreviations commonly used in the field.

(Example 1) Culture of human-derived cell line KHM-5M (1) Subculture of human-derived cell line K) iM-5M The human-derived cell line KHM-5M was subcultured by the following method.

First, 5% inactivated fetal bovine serum (hereinafter abbreviated as FBS),
Human-derived cell line K) IM-5M was added to 100 ml of RPM11640 medium (manufactured by Gibco).
The cells were implanted at a concentration of X10' cells/mj2 and cultured at 37° C. for 3 days in a culture flask (manufactured by Becton Dickinson) with a bottom area of 175 square centimeters.

After culturing, the medium was removed, washed twice with RPM 11640 medium, and the cells were detached with about 10 μL of trypsin solution containing EDTA, and then washed with RPMI 1640 medium.
Add 40 ml of culture medium and use a low-speed refrigerated centrifuge (Tommy R
L-100, manufactured by Tomy Seiko Co., Ltd.).

The medium was removed by centrifugation at 1000 rpm for 10 minutes. After repeating this operation further, 5%F
Add BS/RPMI 1640 medium and bring the cell concentration to 4.
A cell suspension was prepared at 11 x 10' cells/m and cultured in a culture flask at 37°C.

Thereafter, subculture was performed in the same manner.

(2) Culture of the human-derived cell line KHM-5M In order to recover the physiologically active substance of the present invention from the culture supernatant of the human-derived cell line KHM-5M, the human-derived cell line KHM-5M was cultured using the following method.
M-5M was cultured.

First, 10% BS (bovine serum, Arpine Scientific)/RPMI1640 medium! Human-derived a-cell line KHM-5M was incubated at approximately 2X10' cells/m using
j2 concentration was prepared, and it was planted in a 5ft roller bottle (manufactured by Tanehashi Kikaiten Co., Ltd.) and cultured for 3 days at 37°C for 30 rotations.

After culturing, remove the medium and use RPM11640 medium 2.
The 50 mj medium was replaced with 1 fL of IMDM medium (manufactured by Gibco) at 25 ml, and cultured at 37°C for an additional 3 days with 30 rotations.

(Example 2) Purification of the physiologically active substance of the present invention (Part 1) Example 1 (2)
The cell suspension obtained according to
The cells were centrifuged at xg and 4°C for 20 minutes to obtain a culture supernatant.
This culture supernatant was concentrated and purified by the following method.
In addition, among the following operations, concentration and dialysis were performed at 10°C or lower unless otherwise specified, and chromatography was performed at approximately 25°C unless otherwise specified.

(1) Concentration The above culture supernatant 21j2 was concentrated using an ultrafiltration membrane (Spiral Cartridge 5IY3, manufactured by Amicon), and further filtered for 180 m using an ultrafiltration membrane (YM5, manufactured by Amicon).
It was concentrated to j2.

Using a dialysis membrane with a molecular weight cutoff of 3500, 36 ml of the obtained concentrate was diluted with 50 mM sodium acetate buffer (pH 5).
．． 5) overnight at 4°C.

(2) Cation exchange chromatography The sample after dialysis was centrifuged at approximately 90,000 xg and 4°C for 20 minutes, and the supernatant was collected. The collected supernatant was subjected to cation exchange chromatography as follows.

That is, this supernatant was mixed with sp-Toyopearl 65 equilibrated in advance with 50 mM sodium acetate buffer (pH 5.5).
08 column (2.2 cmφ x 20 cm, manufactured by Tosoh Corporation), and the column was washed with the same buffer. After washing,
0 to 0.8 M while monitoring the protein amount in the elution fraction by measuring absorbance at a wavelength of 280 mm.
NaCj2750mM sodium acetate buffer (pH 5,
5) using the linear concentration gradient method at a flow rate of 3 mfl.
Elution was performed at /min.

Collect a portion from each fraction and adjust B to a final concentration of 01%.
After adding SA (bovine serum albumin, manufactured by Sigma), P
Dialyzed overnight against BS-. After dialysis, use a sterile Mirex GV filter (manufactured by Nippon Millibore Industries Co., Ltd.)
Perform sterilization filtration using 0.1% BSA/rMD.
It was diluted with M and used as a sample for activity measurement.

The activity of the obtained sample was measured by the method described in Example 5. As a result, the NaCj2 concentration ranged from about 0.005M to about 0.
．． It was active or thickened on both sides around 25M.

This active fraction was collected and used in the experiment described later.

(3) SDS ~ polyacrylamide Kel dynamic method Using the active fraction obtained in Example 2 (2) as a sample, La
emmli method (Nature, vol. 227, p. 680,
SDS-polyacrylamide gel run (hereinafter abbreviated as SDS-PAGE) was carried out as follows according to the method published in 1970).

First, using a dialysis membrane with fractionated molecules i5000, the sample was Dialysis was performed at 4°C for 1 hour against IMI-Lis-HCl M solution (pH 6.8), and the sample after dialysis was passed through an ultrafiltration membrane (Ultrafree CL, manufactured by Nippon Millibore Industries Co., Ltd.) with a molecular weight cutoff of 5000. and concentrated.

60 μk was collected from the concentrated sample and 10% SD
S20Atu, glycerin 15μ℃, 0 1% BPB
(Bromphenol blue) was added at 5 μC and reacted at 37C for 1 hour. 16% T gel (length 6cm x width 8cm, thickness 1.5m
Electrophoresis was carried out for 2.2 hours at 15 mA using a 100-m (Tefco, Inc.).

In addition, as a molecular weight marker, Pharmacia LMW
Kit E (manufactured by Pharmacia), 14,4kD,
20.1kD, 30kD, 43kD, 67kD and 9
A 4kD marker was used.

After the electrophoresis is completed, the gel is cut into 2 mm wide pieces perpendicular to the electrophoresis direction, and each gel piece is divided into 0.2 mm and 4 mjl.
%BSA/PBS- overnight for extraction.

After extraction, remove the gel pieces and add IM KCIL 10 μJ2.
was added and centrifuged at 10,000×g and 4° C. for 10 minutes. The supernatants were collected, and using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000, I MDM
The culture medium was dialyzed overnight at 4°C.

After dialysis, perform sterilization filtration using a sterile Mirex GV filter (previously listed), and remove 0.1% BSA/IMDM.
It was diluted with water and used as a sample for activity measurement.

The activity of each sample was measured by the method described in Example 5. However, the L8057 cells were cultured at 37° C. for 4 days after adding the sample. As a result, it was confirmed that the active substance was extracted from the cut out pieces of Kel.

From the position of this gel piece, the activity is estimated to have a molecular weight of approximately 19
It was thought to originate from a substance of kD to about 31 kD.

(4) Hydrophobic chromatography The active fraction obtained in Example 2 (2) was subjected to hydrophobic chromatography in the following manner.

First, the active fraction was concentrated using YM5 (previously mentioned), and then added to a 1.5M ammonium sulfate/10mM sodium phosphate buffer (
(pH 7.0) for 3 hours. After dialysis, about 9
Centrifugation was performed at 0,000 xg and 4°C for 20 minutes, and the supernatant was collected.

This supernatant was preliminarily mixed with 1.5M ammonium sulfate/10
Phenyl-5PW column (7.5 mmφX'7.5
cm, manufactured by Tosoh Corporation). After washing the column with the same buffer, while monitoring the protein amount in the elution fraction,
5M-0M Ammonium Sulfate 710 m M Sodium Phosphate &! Elution was performed using a buffer solution (pH 7.0) using a linear concentration gradient method at a flow rate of o and 5 mi/min.

A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed overnight against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 01% BSA/IMDM to prepare a sample for activity measurement.

The activity of the obtained sample was measured by the method described in Example 5, and it was found that ammonium sulfate? R degree about 0.6 to about 0
．． Activity was observed around 9M.

(5) Anion exchange chromatography Example 2 (2)
The active fraction obtained was subjected to anion exchange chromatography in the following manner.

First, the above active fraction was concentrated using YM5 (already mentioned), and the obtained concentrated M solution was added to 25 mM Tris-HCl M solution (pH 8,0) using a dialysis membrane with a molecular weight cutoff of 5000.
I3! Dialysis was performed for a period of time. After dialysis, at about 9000oXg, 4
Centrifugation was performed for 20 minutes at ℃, and the upper groove was collected.

This supernatant was preliminarily mixed with 25mM Tris-HCl M solution (pH 8).
DEAE-5pw column (7,5m) equilibrated with
mφX75 mm, manufactured by Tosoh Corporation), and the column was washed with the same buffer. After washing, use OM to IM NaCf1/25mM Tris-HCl buffer (pH 8,0) at a flow rate of 0.5m while monitoring the protein amount in the elution fraction.
Elution was performed using a linear concentration gradient method at ρ/min.

A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed overnight against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 0.1% BSA/IMDM to prepare a sample for activity measurement.

When the activity of the obtained sample was measured by the method described in Example 5, the NaCu concentration was about 0.05M to about 0.35M.
Two peaks of activity were observed in nearby fractions.

(6) Reverse phase chromatography Of the active fractions obtained in Example 2 (5), both fractions corresponding to the latter peaks were subjected to reverse phase chromatography in the following manner.

First, the active fraction was concentrated using YM5 (previously described), and the resulting f14 condensate was dialyzed against 0.1% trifluoroacetic acid for 3 hours using a dialysis membrane with a molecular weight cutoff of 5000.

After dialysis, centrifugation was performed at approximately 90,000×g and 4° C. for 20 minutes, and the supernatant was collected. This supernatant was mixed with YMCAP8 that had been equilibrated with 0.1% trifluoroacetic acid in advance.
03 C4 column (4.6 mm φ x 250 mm, manufactured by Yamamura Kagaku Kenkyusho), and the column was washed with 0.1% trifluoroacetic acid. After washing, use 0% to 90% acetonitrile 10.1% trifluoroacetic acid at a flow rate of 0.5 ml using a linear concentration gradient method while monitoring the protein amount.
Elution was performed at 1.7 minutes.

Collect a portion from each fraction and adjust B to a final concentration of 01%.
After adding SA, the mixture was dialyzed overnight against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 01% BSA/IMDM to prepare a sample for activity measurement.

The activity of the obtained sample was measured by the method described in Example 5, and the activity was found to be at an acetonitrile concentration of about 60% to
It was recognized in about 80% of cases.

(Example 3) Purification of the physiologically active substance of the present invention (Part 2) 108 mj2 of a concentrated solution of the culture supernatant of the human-derived cell line KHM-5M obtained according to the method described in Example 2 (1) was purified. The sample obtained by dialysis using the method described in Example 2 (1) was purified using the following method.

In addition, among the following operations, concentration and dialysis were performed at 10°C or lower unless otherwise specified, and chromatography was performed at approximately 25°C unless otherwise specified.

(1) Cation exchange chromatography The sample after dialysis was centrifuged at approximately 90,000×g and 4° C. for 20 minutes, and the supernatant was collected. The collected supernatant was subjected to cation exchange chromatography as follows.

That is, this supernatant was applied to an sp-Toyovar 650S column (2.2 cm φ x 20 cm, manufactured by Tosoh Corporation) that had been equilibrated in advance with 50 mM sodium acetate buffer (pH 5,0), and the column was washed with the same II buffer. . After washing, while monitoring the protein amount, OM~1. OMNa
Elution was performed using a Cn150mM sodium acetate buffer (PH5,0) by a linear concentration gradient method at a flow rate of 3ff+1/min.

A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed overnight against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 0.1% BSA/IMDM to prepare a sample for activity measurement.

The activity of the obtained sample was measured by the method described in Example 5, and the activity was found to be at NaCJ2 concentrations of about 0.15M to
It was observed in the fraction around 0.35M.

The active fraction was collected and further purified by anion exchange chromatography using the following method.

(2) Anion exchange chromatography Example 3 (1)
The active fraction obtained was concentrated with YM5 (previously described),
The obtained concentrate was dialyzed for 3 hours against 25 mM Tris-HCl buffer (PH8.0) using a dialysis membrane with a molecular weight cutoff of 5000. For post-dialysis samples, approximately 90
Centrifuge for 20 minutes at 000xg and 4°C.
The supernatant was collected.

This supernatant was added in advance to 25mM Tris-HCl buffer (PH8,
DEAE-5pw column (7.6m) equilibrated with
mφ×75 mm, manufactured by Tosoh Corporation), and the column was washed with the same buffer. After washing, while monitoring the amount of protein, the flow rate was adjusted to 0.0. Elution was performed at 5 ml in 11 minutes.

A portion of each fraction was collected, and after adding BSA to a final concentration of 0.1%, it was dialyzed overnight against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 0.1% BSA/IMDM to prepare a sample for activity measurement.

When the activity of the obtained sample was measured by the method described in Example 5, the NaCJZ concentration was about 0.05M to about 0.3M.
Two peaks of activity were observed in the fraction around 5M.

The elution pattern is shown in Figure 1.

(3) Reverse phase chromatography Of the active fractions obtained in Example 3 (2), both fractions corresponding to peak B in FIG. 1 were subjected to reverse phase chromatography in the following manner.

First, the above active fraction was concentrated using YM5 (already mentioned), and the resulting concentrated solution was mixed against 0.1% trifluoroacetic acid/10% acetonitrile for 3 hours using a dialysis membrane with a molecular weight cutoff of 8000. Dialyzed.

After dialysis, centrifugation was performed at approximately 90,000×g and 4° C. for 20 minutes, and the supernatant was collected. This supernatant was applied to a PRLP-S column (4.6 mm Φ x 50 m
The column was then washed with 0.1% trifluoroacetic acid/10% acetonitrile. After washing, monitor the amount of protein in the eluted fraction and add 0.1% trifluoroacetic acid/10% to 90%.
Elution was performed using acetonitrile using a direct M a gradient method at a flow rate of 0.5 mρ/min.

Collect a portion from each fraction and adjust B to a final concentration of 01%.
After adding SA, the mixture was dialyzed overnight against PBS- using a dialysis membrane with a molecular weight cutoff of 6,000 to 8,000. After dialysis, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the mixture was diluted with 01% BSA/IMDM to prepare a sample for activity measurement.

When the activity of the obtained sample was measured according to the method described in Example 5, activity was observed in the fraction with an acetonitrile concentration of about 30%.

(4) SDS-PAGE A part of the active fraction finally obtained in Example 3 (3) was
The mixture was dried under reduced pressure using a centrifugal concentrator (Speedback Concentrator, manufactured by Sahant), and subjected to SDS-PAGE in the following manner.

First, a sample dried under reduced pressure was mixed with 1% SDS10.
005% B P B / 2
It was dissolved in 0mM Tris-HCl buffer (pH 8,0) and reacted at 100°C for 2 minutes. The sample after the reaction was subjected to electrophoresis using Fast System (manufactured by Pharmacia). After electrophoresis,
When stained with Coomassie blue, the molecular weight was approximately 2.
Dense bands were observed only at positions from 3 to about 27 kD.

In addition, as a molecular weight marker, Pharmacia LMW
Kit E (already published) was used.

(Example 4) Determination of amino acid sequence (1) Amino acid sequence of protein derived from Beak B Example 3 (
The active fraction obtained in 3) was dissolved in 50% acetic acid to prepare a sample, and its N-terminal amino acid sequence was determined using Model 477A Protein Sequencing System-120A PTH Analyzer (manufactured by Abride Biosystems). did. That is, PTH amino acid was detected by ultraviolet absorption at 270 nm, and the amino acid was identified based on the retention time of standard PTH amino acid (manufactured by Applied Biosystems), which had been previously separated using the same method.

As a result, it was confirmed that the physiologically active substance of the present invention had the following amino acid sequence.

Gly Leu Glu X Asp Gly L
ys Val^sn Ila X X Lys
Lys Gin PhePheνal X Phe
Lys Asp IIeGly (X indicates that the amino acid is not specified. Also, the four X's may be the same or different.) shows a location where an amino acid could not be identified, but if an amino acid cannot be identified using the method shown here, the amino acid at that location is likely to be Cys or Set.

(2) Amino acid sequence example 3 of protein derived from peak 8 (
Among the active fractions obtained in 2), the fraction corresponding to peak A in FIG. 1 was subjected to reversed phase chromatography by the method described in Example 3 (3).

As a result, activity was observed in the fraction with an acetonitrile concentration of about 30%.

This active fraction was transferred to a SpeedVac concentrator (
(already mentioned) to dryness under reduced pressure, and then the SDS-
It was subjected to PAGE.

That is, the sample dried under reduced pressure was mixed with 20% glycerol 10.01% BPB/2% SDS 10.0625M
It was dissolved in Tris-HCl buffer (pH 6, 8), and the entire amount was subjected to SDS-PAGE. In addition, SDS-PAG
E is 15% according to Laemmli's method (already published)
The test was carried out using T gel (14 cm long x 14 cm wide, 1.0 mm thick).

The protein molecules separated in the polyacrylamide gel were then separated using the method of Matsudaira et al.
, Biol, Chem.

Vol, 262, pJ, 10035-10038
．． Electroplotting was carried out on a polyvinylidene-difluoride (hereinafter abbreviated as PVDF) membrane according to (1987).

That is, after washing the polyacrylamide gel after electrophoresis with distilled water, transfer
amino ] -1-p "apanesulfo
tnicacid (manufactured by Dojindo Chemical Research Institute Co., Ltd.)
(pH 11,0)) for 5 minutes to equilibrate.

The PVDF membrane was rinsed with 100% methanol and then immersed in the transfer buffer described above. moreover,
A filter paper is immersed in a transfer buffer, and the above-mentioned polyacrylamide gel is sandwiched between the filter paper and the above-mentioned PVDF membrane, and a plotting device (manufactured by Biometra) is used.
blotting was carried out for 3 hours at a constant current of 1 milliampere/cm2.

After plotting, the PVDF membrane was taken out and washed in distilled water for 5 minutes, and then the PVDF membrane was dissolved in 50% methanol.
% Coomassie Blue R250 solution for 5 minutes.
After staining, it was thoroughly destained in a 50% methanol and 10% acetic acid solution. Furthermore, this membrane was washed in distilled water for 5 minutes,
Air dried. As a result, a band around 25 kD was observed on the membrane, so that part was cut out and placed on a cartridge block of a sequencer, and the cartridge block was assembled.

Amino acid sequence analysis was performed using the Model 477A Protein Sequencing System-120 APTH Analyzer (
The test was carried out by the method described in Example 4 (1) using the above-described method.

As a result, it was confirmed that the physiologically active substance of the present invention had the following amino acid sequence.

Gly Leu Glu Y Asp Gly L
ys Val Asn l1eY Y Y Y
Gln Phe Phe Val (Y indicates that the amino acid is not specified. In addition, the five Y's may be the same or different.) (Example 5) Activity of the physiologically active substance of the present invention Measurement of the differentiation, maturation and/or growth promoting activity of the novel physiologically active substance of the present invention against the megakaryocyte-platelet system was confirmed by the following method.

(1) Preparation of cell suspension for activity measurement First, mouse megakaryocytic cell line L8057 was mixed with 1% Nut.
Ridoma-5P (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.)/IMDM medium was used to prepare cells at 3x104 cells/mfl, and cultured at 37°C for 3 days.

After the culture is complete, use a low-speed refrigerated centrifuge (already mentioned) for 10
Cells were collected by centrifugation at 00 rpm for 5 minutes. The collected cells were treated with 2% Nutridoma-5P.
/0, 3X10 in 1% BSA/I MDM medium
The cell suspension was dispersed at a ratio of 'cells/m It, to obtain a cell suspension for activity measurement.

(2) Using the activity measurement sample obtained in Activity Measurement Example 2 or 3,
Activity was measured by the following method.

First, a 96-well multiplate (manufactured by Becton Dickinson) was prepared, and the experimental group received the above sample, and the control group received 01% BSA/I MDM.
Added μIt/well.

The above cell suspension for activity measurement was added at 100 μC to each well of this multiplate, and cultured at 37°C for 3 days.

After the culture is complete, place the plate in a centrifuge (H-161,
Centrifugation was performed at 1500 rpm for 5 minutes using a Kokusan Centrifuge Co., Ltd., to remove 150 μk of supernatant from each well, and instead, 150 μk of supernatant was added to PBS- prepared at pH 7.2.
50μ2 each was added. Next, 0.315% DTNB
/1% sodium citrate to 1% Triton X-1
00 was diluted 10 times and added to each well in an amount of 50 μl.
Absorbance at a wavelength of 405 nm was measured at MCC).

Next, 20 μl of 7.5 mM acetyl thiocorile iodide solution was added to each well, and after reacting at 25°C for 30 minutes, the absorbance at a wavelength of 405 nm was measured, and the difference between the two absorbances, i.e. ΔOD 408 (30
min −0+n1n) was calculated.

In addition, DTN B is 5.5'-dithiobis-(
2-nitrobenzoic acid). In addition, PBS-
was used.

When a significant difference from the control was observed, it was determined that there was an effect on the megakaryocyte-platelet system.

(Example 6) Stability of the physiologically active substance of the present invention Example 1 (
The cell suspension TL solution obtained according to 2) was incubated at 8000×g for 4
The mixture was centrifuged at ℃ for 20 minutes to obtain a culture supernatant. The following experiment was conducted using this culture supernatant as a sample.

(1) Stability against 1% SDS SDS (manufactured by Nacalai Tesque Co., Ltd.) was prepared at 2%, and 50 μρ of the SDS solution was added to the Imfl sample and incubated at 37° C. for 1 hour. Then,
1M potassium phosphate buffer (PH7,5) at 150 μC was added, and the mixture was centrifuged at 10,000×g and 4°C for 5 minutes to obtain 1 mj2 of supernatant. The obtained supernatant was dialyzed as follows using a dialysis membrane with a molecular weight cutoff of 5000.

That is, dialysis was repeated twice for 3 hours against 500 mj2 of PBS- at 4°C, then 50 mj2 at 4°C.
A 3 hour dialysis was performed against IMDM. After dialysis, 10 μm of 10% B5A was added, and sterile filtration was performed using a sterile Millex GV filter (previously listed).
．． The sample was diluted with 1% BSA/IMDM to prepare a sample for activity measurement, and the activity was measured by the method described in Example 5. However, the culture of Shi8057 cells after adding the sample is at 37°C.
The reaction time was 60 minutes after the addition of 7.5 mM acetylthiocholine iodide for 4 days. The results are shown in Table 1.

Table 1 (2) Stability against trifluoroacetic acid/acetonitrile A 0.1% trifluoroacetic acid (manufactured by Nacalai Tesque Co., Ltd.)/acetonitrile (manufactured by Nacalai Tesque Co., Ltd.) solution was prepared, and 1 ml of the solution was added to the same amount of sample. ,
Incubate for 1 hour at 25°C. Next, centrifugal concentrator (Speedvac Concentrator-5 already mentioned)
The mixture was concentrated for 30 minutes, diluted with PBS-LTlmfL, and then subjected to the following dialysis treatment.

That is, dialysis was repeated twice for 3 hours against 500 mj2 of PBS- at 4°C, then 50 mj2 at 4°C.
Dialysis for 3 hours was performed against l1 IMDM. After dialysis, add 10% B5A at 10μ℃ and perform sterilization filtration using a sterile Millex GV filter (previously listed).
．． The sample was diluted with 1% BSA/IMDM to prepare a sample for activity measurement, and the activity was measured by the method described in Example 5. However, culture of L8057 cells after sample addition is at 37°C.
The reaction time was 60 minutes after the addition of 7.5 mM acetylthiocholine iodide for 4 days. The results are shown in Table 2.

Table 2 (3) Stability against 7M urea Urea (manufactured by Nacalai Tesque Co., Ltd.) was dissolved in PBS- to prepare a 7M solution. Using a dialysis membrane with a molecular weight cutoff of 5000, sample 1 m at 25°C.
jlL was dialyzed against 100 mu of the above 7M urea solution for 3 hours. Furthermore, dialysis was repeated twice against 500 mu of PBS- at 4°C for 3 hours, and then at 4°C.
Dialysis was performed against 50 ml of IMDM for 3 hours. After dialysis, 0 μρ of 10% B5Al was added, sterilization filtration was performed using a sterile Mirex GV filter (previously described), and the sample was diluted with 0.1% BSA/I MDM to prepare a sample for activity measurement, as described in Example 5. Activity was measured using the method described below. However, the L8057 cells were cultured at 37°C for 4 days after addition of the sample, and the reaction time after addition of the 7.5mM acetylthiocholine iodide solution was 60 minutes.

The results are shown in Table 3.

Table 3 (4) Effect of pH First, the following solutions were prepared.

0.1M potassium chloride/hydrochloric acid buffer (pH 2,0) 0.1M citric acid/sodium hydroxide buffer (pH 4,
0 and pH 6,0) 0.1M Tris-HCl buffer (PH 8,0) 0.1M Glycine-sodium hydroxide buffer (p Hi O) 0.1M Potassium chloride-HCl buffer (pH 8,0) Using a dialysis membrane with a molecular weight of 5000, 1 ml of each sample was dialyzed against 100 mJ2 of each of the above solutions for 4 hours at 25°C.Furthermore, dialysis was performed for 2 hours against 200 mJl of PBS- at 4°C. Repeat three times, then at 4°C.
Dialysis was performed for 3 hours against IMDM at 200 mA. After dialysis, add 10 μm of 10% BSA, perform sterilization filtration using a sterile Mirex GV filter (previously listed), dilute with 0.1% BSA/IMDM to prepare a sample for activity measurement, and prepare as described in Example 5. Activity was measured using the method described below. However, the reaction with the 7.5M acetylthiocholine iodide solution was carried out for 60 minutes. The results are shown in Table 4.

Table 4 (Reference example) Known human and mouse physiologically active substances and LPS
(lipopolysaccharide) was prepared at various dA degrees and used as samples to determine the differentiation, maturation, and/or
Alternatively, the activity against proliferation was measured by a method similar to that described in Example 5. The results are shown in Table 5.

In addition, the result is ΔOD 405 (30min -0m
1nl was significantly higher than the control group, it was shown as +, and when no significant difference was observed, it was shown as -.

Table 5 <Effects of the Invention> The present invention improves the differentiation, maturation, and maturation of megakaryocyte-platelet cells.
Alternatively, a physiologically active substance that promotes proliferation, a method for producing the physiologically active substance, and a cell line KH that produces the physiologically active substance.
A culture supernatant of M-5M is provided.

The present invention also provides a pharmaceutical composition containing the physiologically active substance as an active ingredient.

The physiologically active substance and pharmaceutical composition of the present invention are useful for treating diseases accompanied by megakaryocyte and/or thrombocytopenia, and for treating diseases accompanied by megakaryocyte and/or thrombocytopenia.
Or used for the treatment or prevention of diseases accompanied by platelet function abnormalities.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the elution pattern of activity in Example 3 (2). D280? Active Cfu1! ! ft6 ratio)

Claims (14)

[Claims] (1) A physiologically active substance characterized by having an effect of promoting acetylcholinesterase production in megakaryocyte cells. (2) The megakaryocyte cell line is mouse megakaryocyte cell line L80.
57. The physiologically active substance according to claim 1. (3) The physiologically active substance according to claim 1 or 2, which has a molecular weight of about 19 kD to about 31 kD as measured by SDS-PAGE under non-reducing conditions. (4) The physiologically active substance according to any one of claims 1 to 3, having one or more of the following properties a) to d). a) Activity remains after treatment with 1% SDS at 37°C for 60 minutes. b) Activity remains after treatment with 0.05% trifluoroacetic acid/50% acetonitrile at 25°C for 60 minutes. c) Activity remains after treatment with 7M urea at 25°C for 180 minutes. d) Activity remains after treatment at 25° C. for 240 minutes at pH 2-12. (5) The physiologically active substance according to any one of claims 1 to 4, which is a substance whose presence is confirmed in the KHM-5M cell line culture supernatant. (6) The physiologically active substance according to any one of claims 1 to 5 has an activity of promoting any one or more of differentiation, maturation, and proliferation of the megakaryocyte-platelet system. Physiologically active substances. (7) Claims 1 to 6, wherein the physiologically active substance is of human origin.
The physiologically active substance according to any one of. (8) The physiologically active substance according to any one of claims 1 to 7, wherein the physiologically active substance is produced by the human-derived cell line KHM-5M. (9) The physiologically active substance according to any one of claims 1 to 8, wherein the physiologically active substance has the amino acid sequence shown below. [There is a gene sequence] (X indicates that the amino acid is not specified. Also, 4
The X's at the locations may be the same or different. ) (10) X in the amino acid sequence is Cys or Ser
The physiologically active substance according to claim 9. (11) The method for producing a physiologically active substance according to any one of claims 1 to 10, characterized in that at least one of the following steps is carried out. a) Culture the human-derived cell line KHM-5M. b) Collect the culture supernatant of the human-derived cell line KHM-5M. c) Perform at least one purification method selected from the following. [1] Anion exchange chromatography [2] Cation exchange chromatography [3] Reversed phase chromatography [4] Hydrophobic chromatography (12) The method for producing a physiologically active substance according to claim 11, wherein each of the purification methods is performed as follows. a) Anion exchange chromatography is performed using a carrier into which a diethylaminoethyl group has been introduced. b) Perform cation exchange chromatography using a carrier into which a sulfopropyl group has been introduced. c) Perform reverse phase chromatography using a carrier into which butyl groups have been introduced or using a polystyrene divinylbenzene polymer as a carrier. d) Hydrophobic chromatography is performed using a carrier into which phenyl groups have been introduced. (13) Human-derived cell line KHM-, which contains the physiologically active substance according to any one of claims 1 to 10.
5M culture supernatant. (14) A pharmaceutical composition comprising the physiologically active substance according to any one of claims 1 to 10 as an active ingredient.