JPS63129993A - Production of human-originated physiologically active substance - Google Patents

Abstract

PURPOSE:To produce a specific human-originated physiologically active substance, by culturing a human leukemia cell differentiable to macrophage-like cell in the presence of a differentiation-inducing substance and a macrophage- activation substance. CONSTITUTION:Human leukemia cell which can be differentiated to macrophage- like cell is cultured in the presence of a differentiation-inducing substance and a macrophage-activation substance to obtain a human-originated physiologically active substance having cell differentiation-inducing activity to leukemia cell. The human-originated physiologically active substance has a molecular weight of 50,000+ or -5,000 by gel-filtration and SDS-polyacrylamide electrophoresis and an isoelectric point of 6.5+ or -1.0 by isoelectric electrophoresis and is thermally stable to endure the inactivation at 70 deg.C. Examples of established cell of leukemia cell are HL-60 cell, THP-1 cell, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel method for producing a physiologically active substance. More specifically, it relates to a method for producing a proteinaceous human-derived physiologically active substance (hereinafter abbreviated as cell differentiation-inducing substance) that has a cell differentiation-inducing effect and is produced by culturing human-derived leukemia*m. .

[Prior Art] Among the cell groups that control immune reactions, macrophages (hereinafter abbreviated as Mφ) have recently attracted attention. Mφ undergoes .I return, matures, and reaches functional cells during the body's defense mechanisms, including the processing of antigens through phagocytosis. Leukemia cells are cells that have the ability to proliferate and become tumors during the differentiation and maturation process. Substances with differentiation-inducing ability induce the differentiation of such tumor cells into cells with normal functions, and by using these substances with differentiation-inducing ability, new cancer treatments are possible. In recent years, it has attracted attention as a method for developing methods.

As substances capable of inducing differentiation, highly safe proteinaceous substances, particularly human-derived proteinaceous substances, are attracting attention and have been actively researched in recent years. To date, it has been reported that stimulation of human peripheral blood lymphocytes with lectins produces differentiation-inducing activity (Journal National Cancer Institute, 1999).
National Cancer Institute)
Volume 67, page 1225 (1981), Cancer Research Volume 42, 3
! 328 pages (1982)). However, in these reports, it was only reported that it was a proteinaceous substance of Hi4o, ooo, but in subsequent research, it was found that the molecular weight was 45.000 to 60 as shown in Japanese Patent Publication No. 1982-28934. , 000 or 100,000
It is a substance with an isoelectric point of 5 to 7, is sensitive to trypsin, and is unstable to heat. On the other hand,
The differentiation-inducing activity found in the culture supernatant of T-lymphocytic leukemia cells was determined by acrylamide gel electrophoresis. It was thought to be derived from a proteinaceous substance of 50,000 to 60,000, but approximately 60 to 90% of its activity was lost during the isolation process (Japan MJ Oribai Hyakukai Abstracts,
p. 43 (1983)).

[Problems to be Solved by the Invention] As described above, the nature of the substance that causes the differentiation-inducing activity produced from human peripheral blood lymphocytes and human lymphocytic leukemia cells is largely unknown. In addition to being unstable substances that are highly sensitive to heat and proteolytic enzymes, the activity of these substances is weak, and vitamin A
Without the coexistence of derivatives, leukemia cells could not be induced to differentiate sufficiently.

Furthermore, since human peripheral blood lymphocytes are prepared by fractionating blood cells collected from human blood, it is difficult to obtain them in large quantities, and industrial production has not been easy. Furthermore, human knee lymphocytic leukemia cells have acquired proliferative properties through maintenance infection with human T-cell leukemia virus, and large-scale cell culture has been problematic in terms of safety.

[Means for Solving the Problems] The inventors of the present invention recognized the prior knowledge described in Section 2 and concluded that macrophage (N・1φ) cells have superior stability and a strong cell differentiation-inducing effect. Based on the working hypothesis that human N
We have discovered a human-derived proteinaceous cell differentiation inducer that acts on 1φ progenitor cells and has the activity of inducing differentiation into Mφ by itself, completed the invention, and filed a patent application. However, even when using leukemia cells, which are Mφ progenitor cells, the previous application proposed that after changing the Mφ progenitor cells into Mφ-like cells with a substance capable of inducing differentiation (hereinafter abbreviated as a differentiation inducing agent), differentiation is induced. It was very time-consuming to wash and remove the agent, and it was difficult to process a large amount of cells. Therefore, we have repeatedly investigated ways to easily obtain cell differentiation-inducing substances. By culturing,
The present invention was completed by discovering that it is possible to safely handle large amounts of cells and to obtain large amounts of cell differentiation-inducing substances.

[Contents of the invention] That is, the present invention has the following characteristics by culturing human leukemia cells that differentiate into macrophage-like cells in the presence of a substance capable of inducing differentiation and a macrophage activating substance, and a) Molecule j! l 50.000±5
,000 (gel filtration) 50.000±5.000 (
S D S-polyacrylamide electrophoresis method) b) Isoelectric point 6.5 ± 1.0 (isoelectric focusing method) C)
The present invention relates to a method for producing a human-derived physiologically active substance that does not become inactivated at 70'C and has cell differentiation-inducing activity against leukemic cells.

In the present invention, the cell differentiation inducer is Hi!・Origin M
A substance produced by φ-like cells in vitro.

The human leukemia cells capable of differentiating into Mφ-like cells used in the present invention correspond to Mφ precursor cells, and only change into Mφ-like cells by the action of a differentiation-inducing agent. Cells or cells that originally have some of the properties of Mφ, but which change to have the properties of Mφ by the action of a differentiation-inducing agent. Mφ progenitor cells can be obtained from primary cells isolated from leukemia patients, established cell lines, etc., but established cell lines are preferred because they are easy to obtain in large quantities. Leukemia cell lines (an example of human cells used in the present invention include HL-60 cells (Nature (N
ature), vol. 270, p. 347 (1977)),
T HP-1 cells (International Journal Cancer (Int, 1. Cancer) vol. 26, 1
7! (1980), Mono-1-207*
Virchows Virchows Bathological Anatomy Hisdopathology
Arch, A Path, Anat.

anrl Ili pieces, OL, vol. 371, p. 15 (1
976)). The differentiation-inducing agent used here refers to a substance that induces the transformation of leukemic cells that can transform into N1φ-like cells into Mφ-like cells, such as:
Among these, 12-0-tetradecanoylphorbol-13-acetate (hereinafter abbreviated as TPA) is preferable.
is particularly preferred.

As the Mφ activated substance 71 used in the present invention,
Vitamin A derivatives (e.g., vitamin A acid, vitamin A
alcohol, vitamin A acetate, vitamin A palmitate), dimethyl sulfoxide, butyric acid sodium salt,
Hydrocortiscin, Durum-negative bacterium-derived Noriboborisatsuryite (hereinafter abbreviated as LPS), Livid A, BCGW
Examples include the bacterial cell wall of Nato, muramyl dipeptide, etc., and by using each alone or in an appropriate combination, Mφ can be activated and production of cell differentiation-inducing substances can be promoted. Among these Mφ activators, about 1 to
5000ng/ml, preferably about 100-3000
ng/ml, more preferably about 500-2000 ng
Particular preference is given to using vitamin A derivatives of /m I, especially vitamin A acids.

After culturing human-derived white-faced disease cells for a sufficient period of time to produce a cell differentiation inducer, the culture supernatant is collected and the cell layer is removed by centrifugation, followed by purification by appropriately combining the activities of the cell differentiation inducer. By doing so, it is possible to obtain a highly pure cell differentiation inducer.

The activity of the cell differentiation inducer was measured in vitro by measuring the effect of inducing phagocytosis in mouse myeloid leukemia M-1 cells. The method used by the present inventors is an improvement on Hayashi's method (Toxicology Forum, Vol. 7, p. 50, (1984)). That is, N1-1 cells 5XIO5ce in the phylum
lls/ml (Medium: Eagle MEM + 2 medium vitamins and amino acids + 10% tallow serum) Mix the cell differentiation inducer solution (test solution) with the suspension, put 1 ml of it into a 10 ml glass tube, turn it sideways, and add carbon dioxide gas. In the incubator, 37
After culturing at ℃ for 2 days, centrifugation treatment (+000 rpm, 1
0 minutes), discard the supernatant, add 1 ml of serum-free culture medium, suspend the cells again, and add polystyrene latex particles at a concentration of 2 μm/ml (1,004 μm:
(manufactured by Sekisui Chemical Co., Ltd.) and stirred for a further 4 hours. Place this thin strip on top and observe it under a microscope. Dyed red, death! Count the cells that have phagocytosed one latex particle and the non-phagocytic cells for only living cells, excluding the bag, and find the ratio of phagocytic cells. Dilute the test solution appropriately,
Perform the above measurements, and calculate the amount of activity of the cell differentiation inducer in the present invention using the reciprocal of the dilution of the sample of the cell differentiation inducer necessary for the ratio of phagocytic cells to be 10%.
Defined as 1 single samurai (U)/it.

Hereinafter, the activity amount of the cell differentiation-inducing substance in the present invention is shown in units measured by this phagocytosis measurement method.

The properties of the cell differentiation-inducing substance of the present invention, which can be produced by culturing the above-mentioned arcuate, will be described in detail.

A) Molecular weight: 50.000±5.000 (gel filtration illegal) Dalhera cholic acid buffer solution (potassium chloride 0.2g/
l, sodium chloride 8g/1. Potassium phosphate 0.
2y, /1. Sodium phosphate 1.15g/1.
5uperose 6 + equilibrated with a solution containing 0.01χ polyethylene glycol at pH 7.4)
5uperose 12 (Method of Fal5e2rest et al. [Method in Enzymology (Meth
od in Enzvmology) Volume 28-8, 5
Tris/glycine/5D according to page 4 (1972)
Electrophoresis was performed at pH 8.3. A molecular weight calibration curve is created using a standard Lt molecular weight kit (manufactured by Pharmacia), and the molecular weight is determined by evaluating the phagocytosis-inducing activity of M-1 cells of the extract from the fractionated gel.

From the above results, it can be seen that the cell differentiation-inducing substance of the present invention does not have a subunit structure.

C) Isoelectric point: 6.5 ± 1.0 (isoelectric focusing method) Isoelectric focusing device (SJ107IEc manufactured by At-Co., Ltd.)
Pharmalite (manufactured by Pharmacia, pH
A 5% polyacrylamide plate gel containing 4 to 8) and glycerol is prepared. 0.04 M DL- on the anode side
Perform pre-phoresis at 700v for 50 minutes using glutamic acid and 0.2M L-histidine on the cathode side. Subsequently, a sample is applied and electrophoresis is performed at 700V for 1 hour and at 500V for 16 hours. After the electrophoresis was completed, the gel was cut into a 2.5 mm width, and each gel piece was soaked in 0.02 M Lithium salt 11 (pH 8) containing 0.15 M sodium chloride.
, 2) Extract with 0.2 ml, and for each extract, M
The cell differentiation inducing activity will be evaluated using -1 cells.

D) Thermostability The cell differentiation inducer of the present invention is diluted 3 times with a pH 7.4 phosphate buffer containing 0.01x polyethylene glycol, heated at a predetermined temperature for a predetermined time, and then −
Evaluation 111i of cell differentiation inducing activity using 1 cell is performed. The cell differentiation-inducing substance of the present invention is a thermally stable physiologically active substance that does not lose its activity even after heat treatment at 70°C for 1 hour.

E) Adsorption to lectin columns Various commercially available lectin immobilization resins were packed into a commercially available Sepacol mini column (manufactured by Bio-Rat), and 50 mM phosphate buffer (pH 7.5) containing 150 mM sodium chloride was added.
After thorough washing with water, the sample of the cell differentiation inducer of the present invention is added, washed with the same buffer, and then eluted with elution lα containing various saccharides.

When concanavalin-A and lentil lectin columns were used, adsorption to the lectin column was observed, and in both columns 0.2M α-methyl-d-
Cell differentiation inducing activity is eluted by the mannoside solution.

F) Effect of reducing agents on disulfide bonds Dithiothreitol (DTT) as a reducing agent on disulfide bonds,
Or 2-mercaptoethanol (2-ME),
Added to the solution of the cell differentiation inducing substance of the present invention, at 37°C.
Let it react for 4 hours.

After the reaction, the phagocytosis-inducing activity on M-1 cells is measured. A decrease in the activity is observed when 50 mM dithiothreitol is added.

G) pH stability To the solution containing the cell differentiation inducing substance of the present invention, four times the amount of buffer solution of various pH was added, and after heating at 37°C for 24 hours, the pH was returned to neutral, and the pH was adjusted to neutral. Measure the cell differentiation inducing activity for -1 cells. In the pH range of 2 to 10,
No decrease in activity was observed.

■) No decrease in cell differentiation inducing activity due to protease stability was observed. The effect of protease was investigated under the same conditions as in the above experiment except that 0.1% SDS was added.
It was observed that the cell differentiation inducing activity of pronase-E when S was added disappeared.

1) Physiological effect on human leukemia cells Human monocytic leukemia cells (THP-1) and human promyelocytic leukemia cells (HL-60) were incubated at 37°C with carbon dioxide in RP M l-1640 medium containing 10% fetal bovine serum. After each cell was cultured in a gas incubator and the cells between the growth stages were thoroughly washed with phosphate buffer, the cells were transferred to RPM I-11340 medium supplemented with 5% live fetal serum and 10 nM hitamine (enriched with hitamine and amino acids). Then, suspend each cell at 2 x 105 cells/ml medium. Cell suspension? αlO
Oμm and cell differentiation inducer solution (test solution) 100111
The mixture with the above was placed in a 96-well plate and cultured for 3 days at 37°C under 5% charcoal and carbon dioxide atmosphere.

A solution of 0.2% nitroblue tetrazolium (NET, Sigma) (0.2 μg/ml me dissolved in TPA-containing medium) IO07zl was added for an additional 45 min.
NBT was applied to T HP-1 cells and HL-60 cells by
Not only is the reducing ability induced, but the latex particles also have the ability to adhere to food oil, culture vessel walls, yeast bactericidal ability, i-phosphatase activity, β-glucuronidase activity, etc.
As a characteristic, it is commonly used for cell identification (“Manual
of macrophage methodology (Manual
of Macropha, ge Me↑, hodolo
gy), Marcel Detzker, USA, 1981.
"Illustrated White Blood Cells, Kinpodo, 1982") enhancement of the activity of various indicators was observed.

As is clear from the above explanation, the novel physiologically active substance of the present invention acts on human and mouse myeloid leukemia cells, which are Mφ precursor cells, and induces differentiation into Mφ-like cells. Bringing the equator of various functions unique to Mφ,
The molecular weight in solution, that is, the molecular weight at the time of gel filtration is 50
．． 000±5,000, and the molecular weight by 5DS-polyacrylamide gel electrophoresis is 50.000±5,00.
0 and has binding properties to concanavalin and lectin such as lentilretin. Therefore, the cells of the present invention can be cultured in various synthetic media suitable for culturing higher animal cells. is used.

As a typical medium, for example, RP M l-1640
Medium, Eagle's MEM medium, Dulbecco's modified MEM
Medium, a-MEM medium,) Jam's medium, 199
A medium such as McCoy 5A medium, I5cove medium, etc. may be used alone or in an appropriate mixture. The compositions of these media are described in "Cell, Iori Culture Manual," Kodansha, 1982. Serum-derived proteins such as albumin, insulin, and transferrin, and animal serum such as human serum, fetal bovine serum, bovine serum, and horse serum may be added to these media alone or in appropriate combinations. In addition, if necessary, in order to prevent contamination by microorganisms, for example, 10-100 units of penicillin/m11g1t! ! , streptomycin sulfate 10~
100μg/ml medium, kanamycin sulfate 40-60I
Antibiotics such as 1 g/ml medium may be added. Control of the pH of the culture solution, TII, and carbonate ion concentration:
For the A node, e.g. 10-60 mM Hepes[4-(
2-Hadroxyethyl)-1-Vivera is preferred.

In order to produce cell differentiation-inducing substances, human leukemia cells are grown in an appropriate medium at a concentration of about 1 liter per ml of medium.
XIO3-4 X l 06 pieces, preferably about 4 x 1
Suspend to make 05-2X06 cells and transfer to culture container 1
+W Equipped. Next, a substance capable of inducing differentiation (differentiation inducing agent) and a substance with Mφ activity are added. A culture vessel containing cells, a differentiation inducer, and Mφ activated vIJ substance was prepared for approximately 35 minutes.
~38° (:, preferably at about 37°C, in air containing about 5-10% carbon dioxide, at a temperature of about 90-100%,
By culturing for 40 to 100 hours, a cell differentiation inducer is produced and released into the culture supernatant.

The pH of the medium is preferably maintained at about 6.0 to 7.5 throughout the culture.

Next, the present invention will be explained in more detail with reference to Examples, and it goes without saying that the present invention is not limited thereto. In the following description, "%" represents capacity percentage (V/V%) unless otherwise specified. Unless otherwise specified, cultivation was carried out at 37°C and humidity 90-10°C.
1 in air containing 0% and 5% carbon dioxide.

Example 1 Human acute monocytic leukemia T HP-1 cells at 1.50
Serum-free RP M 1 containing units/ml of penicillin and 5071 g/ml of streptomycin
Prepare a cell suspension in 1640 medium at a cell density of 6xlo5 cells/ml, and add 10 ml of the suspension to 100
12-0-tetradecanoylphorbol-13-acyl (TPA) was used as a differentiation inducer, and vitamin A acid (RA) was used as an Mφ activator. Each! 7zg/
ml and cultured for 72 hours at 37°C in air containing 5% carbon dioxide. After 72 hours, the tqi supernatant of each dish was collected and centrifuged at 300 rpm for 10 minutes to remove the cell layer, and then the activity of the cell differentiation inducer in the supernatant was measured. That is, 5 x 10 M-1 cells in the iv propagation stage.
ce!Is/ml (Medium: Eagle MEM + 2 volumes of vitamins and amino acids + 10% tallow baby serum) Mix the obtained culture solution with the suspension, take 1 ml of it into a 10 ml glass tube, turn it sideways, and add carbon dioxide gas. After culturing at 37°C for 2 days in an incubator, centrifuge the cells (1000 rpm, 10 minutes), discard the supernatant, add 1 ml of serum-free culture medium, resuspend the cells, and bring the cells to a concentration of 2 μI/ml. After adding polystyrene latex particles (1,o04zzm, manufactured by Block Chemical Co., Ltd.) and stirring, the mixture was further cultured for 4 hours.

The cells are thoroughly washed with 1ml of PBS and centrifuged to remove extracellular latex particles. After repeating this procedure twice, pipette the cells that have settled at the bottom of the centrifuge tube.
Drop one portion onto a glass slide. 10.
Add 5% eosin solution 1'Ilt, place a cover glass, and observe with a microscope. Dead cells stained red 1
For living cells only, count the cells that have phagocytosed latex particles and non-phagocytic cells, and determine the ratio of phagocytic cells. Dilute the culture medium Rm appropriately and perform the above measurements.The reciprocal of the dilution rate of the culture medium required to make the ratio of phagocytic cells 0% is defined as 1 unit (U)/ml. The active form of the cell differentiation inducer of the present invention was 138 units/ml in the obtained culture solution.

The adsorption of the obtained culture solution to lectin was investigated. Various commercially available lectin-immobilized resins were packed into a commercially available Sepacol mini column (manufactured by Biorat) and 150 TOM
When a column containing 50 mM phosphorus λ containing sodium chloride of

The molecular weight and isoelectric point of the cell differentiation-inducing activity adsorbed and eluted on the Con-A column were measured.

For molecular weight measurement, the active fraction of the cell differentiation inducer was
Dulbecco's phosphate buffer (potassium chloride 0.2g/I
, sodium chloride 8 g/l, potassium monophosphate 0
．． 2g/1. Sodium phosphate 1.15g/1.
5uperose 6 + equilibrated with a solution containing o, otx polyethylene glycol at pH 7.4)
Fractionation was performed by a gel filtration method using 5uperose 12 (manufactured by Pharmacia), and the cell differentiation-inducing activity induced by Don edible oil in M-1 cells was measured, and the following molecules were found:
The activity of the cell differentiation inducer was observed in both cells of 150,000±5,000.

Tris/glycine/5DS(p)I 8.3 for molecular type determination by 5DS-polyacrylamide electrophoresis
), electrophoresis was performed. Standard molecular weight - activity of differentiation inducer was observed.

For isoelectric point measurement, isoelectric focusing was performed by the following method. That is, a 5% polyacrylamide plate gel containing Pharmalite (manufactured by Pharmacia, pH 4 to 8) and glycerol was prepared using an isoelectric focusing device (S, J I0? IEC type) manufactured by At-Co., Ltd. . Using 0.04M DL-glutamic acid on the anode side and 0.2M L-histidine on the cathode side, 700μ = 50
A pre-run for 1 minute was performed. Next, try it! 4 and 70
Electrophoresis was performed at 0V for 1 hour and at 500V for 116 hours. After the electrophoresis was completed, the gel was cut out into 2.5 mm width, and each gel piece was soaked in 0.02 mm solution containing 0.15 M sodium chloride.
M+-Lis-HCl buffer overnight (pH 8,2') 0.2
When the cell differentiation inducing activity of each extract was evaluated using M-1 cells, the isoelectric point was found to be pH: 6.5±1.0.

The thermostability of cell differentiation-inducing substances was investigated using the following method. Dilute the culture solution 3 times with pH 7.4 phosphate buffer containing 0.01χ polyethylene glycol,
Heating at a predetermined temperature for a predetermined time Table 1 Experiment number Conditions M-1 cell content % (relative value %) 1 (No treatment) 58% (100%) 256°C 91
5 minutes 57% (98%) 3 60 minutes
59% (102%) 4 70℃ 15 minutes
54% (93%) 5 60 minutes
53% (91%) 61006C15 minutes 22%
(38%) To examine the influence of reducing agents on disulfide bonds, dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) was cultured) α
and reacted at 37°C for 4 hours. After reaction, M-
The dietary oil-induced activity against 1 cell was measured.

Table 2 Experiment number Conditions M-1 cell content % (relative value %) 1 (No treatment) 54% < 100%) 2 2MEO
,1% 49% (91%)3 1%
46% (85%)4 DT75mM 43%
(80%) 5 50 mM 13% (2
4%) The pH stability is shown below. Culture containing cell differentiation inducer; at night, 4 times the amount of pH 2, 4, 7, 3, 9, 10
Various buffer solutions were added thereto, and the mixture was heated at 37°C for 24 hours, the pH was returned to neutral, and the cell differentiation-inducing activity for M-1 cells was measured. No decrease in activity was observed in any pH range from 2 to 10.

Therefore, the stability against proteolytic enzymes was investigated.

Proteinase trypsin or pronase-E (200 units) was added to the culture medium containing the cell differentiation inducer, and the mixture was reacted at 37°C for 3 hours. After reaction, M-1
Cell differentiation inducing activity for cells was measured, but no decrease in activity was observed under any conditions. - The effect of protease was examined under the same conditions as in the above experiment except that 0.1% SDS was added.
1. of 1% SDS. Cell differentiation inducing activity was completely abolished by pronase-E during post-infusion.

Furthermore, to confirm the physiological effect on human leukemia's@, RPMI-1640 containing 10% fetal bovine serum
Human monocytic leukemia cells (THP-1) were cultured in a human gas incubator, and after thoroughly washing the cells in the expanded Mltll with phosphate buffer, 10% tallow serum and 10 nM vitamin R P M with AM added,
R-1640 medium (fortified with vitamins and amino acids),
2XIO5 cells/ml medium for each! ? 3 It became cloudy.

A mixture of 100 al of cell suspension and 100 μm of culture #ff was placed in a 96-well plate and cultured for 3 days at 37° C. under air mixed with 5% carbon dioxide gas. A solution of 0.2% nitro blue tetrazolium (NBT, Sigma) (0.2 μg
locμl of TPA-containing medium) was added thereto, and after culturing for an additional 45 minutes, observation was made under a microscope. NBT-reducing ability-positive cells with blue pigment deposited are T HP
-33% in iL-60 cells, 2 in iL-60 cells
It was observed in 7%.

Example 2 Using human acute monocytic leukemia T HP-1 cells,
A cell suspension containing 6XIO5 cells/ml was prepared in the same manner as in Example 1, 10 ml of the suspension was implanted into 10 plastic vetri dishes for silk culture, and 12-0- was used as a differentiation inducer. Tetradecanoylphorbol
13-acetate (TPA), culture of horuboyu 1. The supernatant was collected, centrifuged at 3000 rpm for 10 minutes, the cell layer was removed, and the activity of the cell differentiation inducer was measured.

For each culture supernatant, the active fraction obtained by adsorption treatment on a Con-A column and desorption treatment with α-methyl-d-mannoside solution α has a molecular weight of approximately 50.
000 (S D S-polyacrylamide electrophoresis method), it was not deactivated by treatment at pH 2 to 10, and 706
In the heat treatment of C, C also retained its activity.

(Margin below) Table 3 Experiment number Differentiation inducer Cell differentiation inducer Mφ activator I TPA long/ml 97 units/m1RA 17B/m1 2 TPA 100 ng/ml 121R
A1. u g/ml 3 T P A 1000 ng/ml 1
09RA O, Iμg/ml L P S lOμg/ml 4 PDD 1000 ng/ml 85
RA Iμg/ml 6 M EZ 1000 ng/ml
91RA 1 μg/ml (comparative example) TPA 1000 ng/ml
13S-ConA 20 tt g/ml Example 3 Using human acute monocytic leukemia THP-1 cells,
A cell suspension was prepared in the same manner as in Example 1, except that the cell concentration was changed, and 10 ml of the suspension was implanted into 10 M1 tissue culture fusuma plastic Petri dishes.
Add PA to 100 ng/ml and RA to 1 ug/ml, and incubate at 37°C in air containing 5% carbon dioxide.
Cultured for hours. After 72 hours, the culture supernatant of each dish was collected, centrifuged at 3000 rpm for 10 minutes, the cell layer was removed, and the activity of cell differentiation derivative 0 in the supernatant was measured. By acrylamide electrophoresis using each culture supernatant, it was found that the culture supernatant was not inactivated by treatment at pH 2 to 1O at 70°C.
It did not lose its activity even after heat treatment.

(See margin below) Table 4 Experiment number Cell concentration Cell differentiation inducer 1
1 X 105/ml 8G units/m12
4 X 105/ml +043
8 x 105/ml 1294
13 x 105/ml 735
Culture was carried out in the same manner as in Example 2, except that 25 × 10 5 /ml 41 cells were used, and a culture supernatant was obtained. When we measured the activity of cell differentiation inducers in the culture supernatant, we found that
An activity of 119 units/ml was observed. The active fraction obtained by adsorption treatment on a ConA column and desorption treatment with an α-methyl-d-mannoside solution has a molecular weight of approximately 5.
0,000 (5DS-polyacrylamide electrophoresis method), and retained its activity even after pretreatment at 70°C.

By formulating the cell differentiation-inducing substance of the present invention, which has been explained in detail above, by appropriately subjecting it to conventional methods for manufacturing pharmaceuticals, such as heat treatment, sterilization by filtration, freeze-drying, and dispensing,
It is possible to obtain new medicines that have never existed before.

Claims (1)

[Claims] (1) Human leukemia cells capable of differentiating into macrophage-like cells are cultured in the presence of a substance capable of inducing differentiation and a macrophage activating substance, and have the following characteristics: a) Molecular weight 50,000 ±5,000 (gel filtration method) 5
0,000±5,000 (SDS-polyacrylamide electrophoresis) b) Isoelectric point 6.5±1.0 (isoelectric focusing) c) Thermostable leukemia cells that do not inactivate at 70°C Method for producing a human-derived physiologically active substance having cell differentiation-inducing activity against