JPH0474360B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a novel physiologically active substance. More specifically, it relates to a proteinaceous human-derived physiologically active substance (hereinafter abbreviated as cell differentiation-inducing substance) that is produced by culturing human-derived macrophage-like cells and has a cell differentiation-inducing effect. . The chemical substance of the present invention has the effect of inducing cell differentiation, so it induces the differentiation of tumor cells, especially leukemia cells, into normal cells, and it has the effect of eliminating their proliferation ability, so it is useful for the treatment of cancer. It can be used. [Prior Art] Among the cell groups that control immune reactions, macrophages (hereinafter abbreviated as Mφ) have recently attracted attention. This is because it has become clear that Mφ plays a central role in the body's defense mechanisms, including the processing of antigens through phagocytosis. Furthermore, it has been known that Mφ produces a variety of important physiologically active substances, such as interleukin-1, cancer necrosis factor, and colony formation stimulating factor, in response to extracellular stimuli. Blood cells, rather than hematopoietic stem cells, undergo repeated proliferation and differentiation, mature and become functional cells. During this differentiation and maturation process, leukemia cells have the ability to proliferate and turn into tumors. 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 (J.National Cancer Institute).
Institute) Volume 67, Page 1225 (1981), Cancer
Cancer Research, Vol. 42, p. 3928 (1982)). However, in these reports,
Those obtained from human peripheral blood lymphocytes were Sephadex G-75 (Pharmacia, Sweden).
The molecular weight was 25,000 by gel filtration fractionation using
It was only reported that it was a proteinaceous substance with a molecular weight of 45,000 to
60000 or 100000, is a substance with an isoelectric point of 5 to 7,
It was thought to be sensitive to trypsin and unstable to heat. On the other hand, the differentiation-inducing activity found in the culture supernatant of human T-lymphocytic leukemia cells was determined to be derived from a proteinaceous substance with a molecular weight of 50,000 to 60,000 using acrylamide gel electrophoresis. However, during the isolation process, approximately 60-90% of its activity
% lost (Japanese Society for Tissue Culture Abstracts, p. 43,
(1983)). [Problems to be solved by the invention] In this way, human peripheral blood lymphocytes and human
Regarding the differentiation-inducing activity produced from T-lymphocytic leukemia cells, the properties of the substance that causes this activity are largely unknown, or it is an unstable substance that is highly sensitive to heat and proteolytic enzymes. Furthermore, these activities were weak, and sufficient differentiation of leukemia cells could not be induced unless vitamin A derivatives and the like coexisted. [Means for Solving the Problems] Recognizing the above prior knowledge, the present inventors have discovered that macrophage (Mφ) cells have a proteinaceous physiological structure that is more stable and has a strong cell differentiation-inducing effect. We have continued to conduct intensive studies based on the working hypothesis that active substances will be produced. As a result, we discovered a human-derived protein cell differentiation inducer that acts on human Mφ progenitor cells and has the activity of inducing differentiation into Mφ by itself, leading to the completion of the present invention. [Contents of the Invention] That is, the present invention provides a human-derived proteinaceous physiologically active substance having the following characteristics and having a cell differentiation-inducing effect on leukemia cells. a Molecular weight 50000±5000 (gel filtration method) 50000±5000 (SDS-polyacrylamide electrophoresis method) b Adsorption to lectin column Adsorbs to lentil lectin column, concanavalin A columnc Thermal stability Disqualified after 1 hour at 70℃ Not affected by reducing agent Activity decreases PH stability Stable in the PH range of 2 to 10. f No decrease in differentiation-inducing activity is observed by enzymatic reaction with trypsin and pronase E. In the present invention, a cell differentiation-inducing substance is a substance produced by human-derived Mφ or Mφ-like cells,
It means a substance that has the ability to differentiate at least mouse M-1 cells in vitro and induce phagocytosis. The Mφ-like cells used in the present invention include peripheral blood monocytes, tissue Mφ, and Mφ precursor cells that have been transformed into Mφ-like cells by the action of a differentiation-inducing agent in vitro. Mφ precursor cells are cells that first transform into Mφ-like cells due to the action of a differentiation-inducing agent, or cells that originally have some of the properties of Mφ, but further change to have Mφ properties due to the action of a differentiation-inducing agent. means a cell that Mφ progenitor cells are
Although it can be obtained from primary cells and established cell lines isolated from leukemia patients, established cell lines are preferred because they are easy to obtain in large quantities. Examples of established cell lines used in the present invention include HL-60 cells (Nature, 270
Vol. 347 (1977)) THP-1 cells (Int.J.
Cancer) vol. 26, p. 171 (1980)), Mono-1-
207 cells
Pathological Anatomy Histopathy (Virchows Arch.A Path.Anat.and Histol.)
Volume 371, page 15 (1976)). The differentiation-inducing agent used here refers to a substance that induces the transformation of Mφ precursor cells that can transform into Mφ-like cells into Mφ-like cells, such as phorbol esters, diterpene compounds such as meserein, Examples include teleosidin. Among phorbol esters, 4β-hydroxy form is preferred;
Among them, 12-O-tetradecanoylphorbol-
13-acetate (hereinafter abbreviated as TPA) is particularly preferred. The Mφ activator used in the present invention includes vitamin A derivatives (e.g. vitamin A
acid, vitamin A alcohol, vitamin A acetate, vitamin A palmitate), dimethyl sulfoxide, sodium butyrate, hydrocortisone, lipopolysaccharide derived from gram-negative bacteria (hereinafter abbreviated as LPS), lipid A, BCG bacteria, etc. These include wall, muramyl dipeptide, etc., and by using each alone or in appropriate combination, Mφ can be activated and the production of cell differentiation-inducing substances can be promoted. Among these Mφ activators, about 1 to 5000 ng/ml, preferably about
100-3000ng/ml, more preferably about 500-
Particular preference is given to using 2000 ng/ml of vitamin A derivatives, especially vitamin A acid. After culturing human-derived Mφ-like cells for a sufficient time to produce cell differentiation-inducing substances, the culture supernatant was collected,
If cell debris is removed by centrifugation, a solution containing a cell differentiation inducer can be obtained. The solution containing the cell differentiation inducer is purified by an appropriate combination of conventional biochemical separation methods, concentration by ultrafiltration, dialysis desalting, ion exchange chromatography using an anion exchanger, gel filtration, electrophoresis, etc. By doing so, a highly pure cell differentiation inducer can be obtained. The activity of the cell differentiation inducer is measured by measuring the effect of inducing phagocytosis in mouse myeloid leukemia M-1 cells in vitro. The method used by the present inventors is an improvement on Hayashi's method (Toxicology Forum, Vol. 7, p. 50, (1984)). That is, M-1 in the growth phase
Cells 5×10 ⁵ cells/ml (medium: Eagle MEM
+ 2 culture vitamins/amino acids + 10% fetal bovine serum)
Mix the cell differentiation inducer solution (test solution) with the suspension, place 1 ml of it in a 10 ml glass tube, turn it sideways, and incubate the culture solution for 2 days at 37°C in a carbon dioxide incubator.
Centrifuge (1000 rpm, 10 minutes), discard the supernatant, add 1 ml of serum-free culture medium, suspend the cells again, and add polystyrene latex particles (1.004 μm, manufactured by Sekisui Chemical Co., Ltd.) at a concentration of 2 μ/ml. )
After adding and stirring, culture was continued for an additional 4 hours. Wash the cells thoroughly with 1 ml of PBS and centrifuge to remove extracellular latex particles. After repeating this operation twice, use a pipette to suck up the cells that have settled at the bottom of the centrifuge tube, and drop one drop onto a glass slide.
Add one drop of 0.5% eosin solution to this, place a cover glass on it, and observe with a microscope. Excluding dead cells that are stained red, only living cells are counted, including cells that have phagocytosed latex particles and non-phagocytosed cells.
Find the ratio of phagocytic cells. Dilute the test solution appropriately and carry out the above measurements until the ratio of phagocytic cells is 10.
%, the activity amount of the cell differentiation inducer in the present invention is defined as 1 unit (U)/ml, which is the reciprocal of the dilution rate of the sample of the cell differentiation inducer. 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 inducer of the present invention produced by culturing the above cells will be described in detail. A Molecular weight: 50000±5000 (gel filtration method) Dulbets cholic acid buffer (potassium chloride 0.2
g/, Sodium chloride 8g/, Potassium phosphate 0.2g/, Disodium phosphate 1.15
Superose6+ equilibrated with a solution containing 0.01% polyethylene glycol (g/, PH7.4)
Superose12 (manufactured by Pharmacia (Sweden))
The fraction is fractionated by a gel filtration method using M-1 cells, and the cell differentiation-inducing activity by inducing phagocytosis in M-1 cells is measured. B Molecular weight: 50000±5000 (SDS-polyacrylamide electrophoresis method) Segrest et al.'s method [Method in Enzymology 28-B
Vol., p. 54 (1972)], electrophoresis was performed in Tris/glycine/SDS (PH8.3). A molecular weight calibration curve is created using a standard 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) Using an isoelectric focusing device (SJ1071EC type) manufactured by Atto Co., Ltd., 5 containing Pharmalite (manufactured by Pharmacia, PH4-8) and glycerol was used.
% polyacrylamide plate gel. 0.04M DL-glutamic acid on the anode side, 0.2M on the cathode side
Perform a 50 minute pre-run at 700V using L-histidine. Next, apply the sample and apply 1 at 700V.
Perform electrophoresis at 500V for 16 hours. After the electrophoresis is complete, cut out the gel into 2.5 mm width pieces, and then separate each gel piece.
Extraction is performed with 0.2 ml of 0.02 M Tris-HCl buffer (PH8.2) containing 0.15 M sodium chloride, and each extract is evaluated for cell differentiation-inducing activity using M-1 cells. D Thermostability The cell differentiation inducer of the present invention was diluted 3 times with PH7.4 phosphate buffer containing 0.01% polyethylene glycol, heated at a predetermined temperature for a predetermined time, and then M-1 The cell differentiation inducing activity will be evaluated using cells. The cell differentiation inducing substance of the present invention is 70
It did not lose its activity even after being treated at ℃ for 1 hour.
It is a thermally stable physiologically active substance. E Adsorption to Lectin Column Pack various commercially available lectin-immobilized resins into a commercially available Sepacol Mini Column (manufactured by Bio-Rad).
After thorough washing with 50mM phosphate buffer (PH7.5) containing 150mM sodium chloride, the cell differentiation inducer sample of the present invention is added and washed with the same buffer. Next, elution is performed with an eluent containing various sugars. When concanavalin-A and lentil lectin columns were used, adsorption to the lectin column was observed;
Cell differentiation inducing activity is eluted with a 0.2M α-methyl-d-mannoside solution. F Effect of reducing agent on disulfide bonds Dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) as a reducing agent on disulfide bonds was added to the solution of the cell differentiation inducer of the present invention, and the mixture was heated at 37°C. Let it react for 4 hours.
After the reaction, phagocytosis-inducing activity against M-1 cells is measured. The activity decreases when 50mM dithiothreitol is added. G PH stability In the solution containing the cell differentiation inducer of the present invention,
Add 4 times the amount of buffer solution of various PH and incubate at 37℃ for 24 hours.
After heating, the pH is returned to neutral and the cell differentiation inducing activity for M-1 cells is measured. No decrease in activity was observed in the pH range of 2 to 10. H Protease stability Solution containing the cell differentiation inducer of the present invention (PH
Add proteolytic enzyme trypsin or pronase-E (200 units) to 7.4) and react at 37°C for 3 hours. After the reaction, no decrease in cell differentiation inducing activity for M-1 cells was observed. When examining the effect of protease under the same conditions as in the above experiment except for adding 0.1% SDS,
It is observed that the cell differentiation inducing activity of pronase-E when 0.1% SDS is added disappears. I Physiological effects on human leukemia cells Human monocytic leukemia cells (THP-1) and human promyelocytic leukemia cells (HL-60) were incubated in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a carbon dioxide incubator. The cells in the growth phase are thoroughly washed with phosphate buffer. The cells were placed in RPMI-1640 supplemented with 10% fetal bovine serum and 10 nM vitamin A acid.
2 for each medium (vitamin and amino acid enriched)
Suspend at ×10 ⁵ cells/ml medium. Cell suspension 100μ and cell differentiation inducer solution (test solution)
Pour the mixture with 100μ into a 96-well plate and incubate at 37℃.
Cultivate for 3 days under 5% carbon dioxide mixed air.
100μ of 0.2% nitro blue tetrazolium (NBT, Sigma) solution (medium containing 0.2μg/ml TPA)
After incubating for an additional 45 minutes, observe under a microscope. Cells with blue pigment deposited are observed as NBT reducing ability positive cells. By culturing with the cell differentiation inducer of the present invention for 3 days, NBT was induced in THP-1 cells and HL-60 cells.
It not only induces reducing ability, but also has the characteristics of Mφ, such as the ability to eat latex particles, the ability to adhere to the culture vessel wall, the ability to kill yeast, acid phosphatase activity, and β-glucuronidase activity, and is commonly used for cell identification. (Manual of Macrophage Methodology)
Methodology), 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. It brings about the enhancement of various functions unique to Mφ, and the molecular weight in solution,
In other words, the molecular weight of gel filtration is 50,000 ± 5,000, the molecular weight of SDS-polyacrylamide gel electrophoresis is 50,000 ± 5,000, and it has binding properties to lectins such as concanavalin-A and lentilretin. The cell differentiation-inducing substance of the invention is considered to be a glycoprotein that does not have a subunit structure. Next, the method for producing the cell differentiation-inducing substance of the present invention will be described in detail. Various synthetic media suitable for culturing higher animal cells are used for culturing the cells used in the present invention.
Typical media include RPMI-1640 medium, Eagle's MEM medium, Dulbecco's modified method, etc.
MEM medium, α-MEM medium, Ham's medium, 199
A medium such as McCoy 54 medium, Iscove's medium, etc. may be used alone or in an appropriate mixture. The compositions of these media are described in "Cell Tissue Culture Manual, Kodansha, 1982". Serum-derived proteins such as albumin, insulin, and transferrin, and animal sera such as human serum, fetal bovine serum, bovine serum, and horse serum may be added to these media alone or in appropriate combinations. Also,
If necessary, to prevent contamination by microorganisms, antibiotics such as penicillin 10-100 units/ml medium, streptomycin sulfate 10-100 μg/ml medium, kanamycin sulfate 40-60 μg/ml medium, etc. can be added. can. In order to control the pH of the culture solution and adjust the carbonate ion concentration, e.g.
60mM Hepes[4-(2-hadroxyethyl)-
1-piperazineethanesulfonic acid] may also be used. The material of the culture container is not particularly limited, but it is preferably made of plastic, glass, or metal, which allows cell proliferation and has excellent cell adhesion. In order to produce a cell differentiation inducer, human-derived Mφ-like cells are added to about 5 x 10 ⁴ to 5 x 10 ⁵ cells, preferably about 8 cells per cm ² of the culture vessel surface, using an appropriate medium. Plant them so that there are ⁴ x 10 to ⁵ x 10. Then, the Mφ activator is added. Approximately 35-38 mL of culture vessel containing cells and Mφ activator
℃, preferably about 37℃, in air containing about 5 to 10% carbon dioxide, and under conditions of about 90 to 100% humidity.
By culturing for hours, a cell differentiation-inducing substance is produced and released into the culture supernatant. The pH of the medium is
It is preferably maintained at about 6.0-7.5 during the culture period. In addition, when using Mφ progenitor cells, Mφ
The progenitor cells are transformed into MΦ-like cells by treatment with a differentiation inducer such as TPA at about 5 to 5000 ng/ml medium, preferably about 50 to 1000 ng/ml medium, and then differentiated. The inducing agent is removed by washing, and the resulting Mφ-like cells are used and cultured as described above to obtain a cell differentiation inducer. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but it goes without saying that the present invention is not limited thereto. In the following description, "%" represents capacity percentage (V/V%) unless otherwise specified. Also, unless otherwise specified,
Cultivation was carried out at 37°C, humidity of 90-100%, and air containing 5% carbon dioxide. Example 1 Human acute monocytic leukemia THP-1 cells were
10% fetal bovine serum, 50 units/ml penicillin and
Contains 50μg/ml streptomycin
Cell density 6 x ¹⁰ cells/ml in RPMI-1640 medium
Prepare a cell suspension and add 10ml of it to
It was implanted into 100 tissue culture plastic Petri dishes (diameter 8 cm) and treated with 12-O-tetradecanoylphorbol-13- as a differentiation inducer.
Acetate (TPA) was added at a concentration of 1 μg/ml, and cultured at 37° C. in air containing 5% carbon dioxide for 24 hours. After that, the culture medium was removed and vitamin A acid (RA) was added as an Mφ activator without TPA.
10 ml of fresh serum-free RPMI-1640 medium containing 1 μg/ml (contains penicillin and streptomycin)
and continued culturing. After 72 hours, the culture supernatant of each date was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris, and the activity of the cell differentiation inducer in the supernatant was measured. That is, 5×10 ⁵ cells/ml of M-1 cells in the proliferation phase (medium: Eagle
Mix the obtained culture solution with the MEM + 2 culture vitamins/amino acids + 10% fetal bovine serum) suspension and 1 ml of it.
Place it in a 10 ml glass tube, turn it sideways, and culture it at 37℃ for 2 days in a carbon dioxide incubator. Centrifuge it (1000 rpm, 10 minutes), discard the supernatant, add 1 ml of serum-free culture solution, and repeat. The cells are suspended, polystyrene latex particles (1.004 μm, manufactured by Sekisui Chemical Co., Ltd.) at a concentration of 2 μ/ml are added, stirred, and then cultured for an additional 4 hours. this cell
Wash thoroughly with 1 ml of PBS and centrifuge to remove extracellular latex particles. After repeating this operation twice, use a pipette to suck up the cells that have settled at the bottom of the centrifuge tube, and drop one drop onto a glass slide. to this
Add one drop of 0.5% eosin solution, place a cover glass, and observe with a microscope. Excluding dead cells that are stained red, only living cells are counted, including cells that have phagocytosed the latex particles and non-phagocytic cells, to determine the ratio of phagocytic cells. Dilute the culture solution appropriately and perform the above measurements, and use the reciprocal of the dilution rate of the culture solution required to make the ratio of phagocytic cells 10%.
The active amount of the cell differentiation inducer of the present invention, defined as 1 unit (U)/ml, is as follows:
It was 77 units/ml. The adsorption of the obtained culture solution to lectin was investigated. Various commercially available lectin-immobilized resins were packed into a commercially available Sepacol minicolumn (manufactured by Bio-Rad), thoroughly washed with 50mM phosphate buffer (PH7.5) containing 150mM sodium chloride, and culture solution sample was added. After washing with a buffer solution, elution was performed with an eluent containing various saccharides. When concanavalin-A (Con-A) and lentil lectin columns were used, adsorption to the lectin column was observed, and in both columns 0.2Mα-
Cell differentiation-inducing activity was eluted by the methyl-d-mannoside solution. 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 mixed with Dulbecco's phosphate buffer (0.2 g of potassium chloride, 8 g of sodium chloride, 0.2 g of potassium phosphate, 1.15 g of dibasic sodium phosphate). /, PH7.4) equilibrated with a solution containing 0.01% polyethylene glycol.
It was fractionated by gel filtration using +Superose 12 (manufactured by Pharmacia), and the cell differentiation-inducing activity by inducing phagocytosis in M-1 cells was measured.
Cell differentiation-inducing substance activity was observed in the fraction with a molecular weight of 50,000±5,000. Tris/glycine/SDS (PH
8.3), electrophoresis was performed. A molecular weight calibration curve was prepared using a standard molecular weight kit (manufactured by Pharmacia), and the molecular weight was determined by evaluating the phagocytosis-inducing activity of M-1 cells of the extract from the fractionated gel. The activity of a cell differentiation inducer was observed in the fraction. For isoelectric point measurement, isoelectric focusing was performed using the following method. That is, using an isoelectric focusing device (Model SJ1071EC) manufactured by Atto Corporation,
Pharmalite (manufactured by Pharmacia, PH4-8)
A 5% polyacrylamide plate gel containing glycerol was prepared. Pre-phoresis was performed at 700V for 50 minutes using 0.04M DL-glutamic acid on the anode side and 0.2M L-histidine on the cathode side. Subsequently, a sample was applied and electrophoresis was performed at 700V for 1 hour and at 500V for 16 hours. After the electrophoresis, the gel was cut into a 2.5 mm width, and each gel piece was extracted with 0.2 ml of 0.02 M Tris-HCl buffer (PH8.2) containing 0.15 M sodium chloride. When the cell differentiation inducing activity was evaluated, the isoelectric point was PH: 6.5±1.0. The thermostability of the cell differentiation inducer was investigated using the following method. The culture solution was diluted 3 times with PH7.4 phosphate buffer containing 0.01% polyethylene glycol, heated at a given temperature for a given time, and then tested for cell differentiation inducing activity using M-1 cells. We conducted an evaluation.

[Table] In order to examine the influence of reducing agents on disulfide bonds, dithiothreitol (DTT) or
2-mercaptoethanol (2-ME) was added to the culture solution and reacted at 37°C for 4 hours. After the reaction, phagocytosis-inducing activity against M-1 cells was measured.

[Table] PH stability is shown below. After adding 4 times the volume of various buffers of PH2, 4, 7.3, 9, and 10 to the culture solution containing the cell differentiation inducer and warming it to 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 the pH range of 2 to 10. Next, stability against proteolytic enzymes was investigated. A proteolytic enzyme trypsin or Bronase-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
The cell differentiation-inducing activity for -1 cells was measured, but no decrease in activity was observed under any conditions. When the effect of protease-E was examined under the same conditions as in the above experiment except for the addition of 0.1% SDS, the cell differentiation-inducing activity was completely abolished by pronase-E when 0.1% SDS was added. . Furthermore, in order to confirm the physiological effects on human leukemia cells, we added human monocytic leukemia cells (THP-1) and human promyelocytic leukemia cells (HL-60) to RPMI-1640 medium containing 10% fetal bovine serum. ℃, in a carbon dioxide gas incubator, and after thoroughly washing the cells in the growth phase with phosphate buffer, RPMI- to which 10% fetal bovine serum and 10 nM vitamin A acid was added.
They were each suspended in 1640 medium (vitamin and amino acid enriched) at 2×10 ⁵ cells/ml medium. Add a mixture of 100μ of cell suspension and 100μ of culture medium to 96%
The cells were placed in a hole plate and cultured for 3 days at 37°C under an air mixture of 5% carbon dioxide gas. A solution of 0.2% nitro blue tetrazolium (NBT, Sigma) (0.2μ
Add 100 μg/ml TPA-containing medium) and add 45 μg/ml TPA-containing medium)
After incubation for a minute, the cells were observed under a microscope. NBT-reducing ability-positive cells with blue pigment deposited are THP-
29% in 1 cell, 21% in HL-60 cell
was observed. Example 2 Human acute monocytic leukemia THP-1 cells were
10% fetal bovine serum, 50 units/ml penicillin and
Contains 50μg/ml streptomycin
Cell density 6 x ¹⁰ cells/ml in RPMI-1640 medium
Prepare a cell suspension, inoculate 10 ml of it into a tissue culture plastic Petri dish, add TPA to 100 ng/ml, and incubate at 37°C in air containing 5% carbon dioxide. The cells were cultured for 24 hours. After that, the culture medium was removed, TPA-free, and the Mφ activating substances (vitamin A) shown in the table below were added.
acid (RA), LPS, succinylated ConA (S-
10 ml of fresh serum-free RPMI-1640 medium (containing penicillin and streptomycin)
and continued culturing. The culture supernatant of each date was collected at 72 hours, centrifuged at 3000 rpm for 10 minutes, and then the activity of the cell differentiation inducer in the supernatant was measured. Activity of cell differentiation inducer is 20 units/ml
The active fraction obtained from the above culture supernatant by adsorption treatment on a Con-A column and desorption treatment with an α-methyl-d-mannoside solution is as follows:
It has a molecular weight of 50,000 (SDS-polyacrylamide electrophoresis method), and does not inactivate when treated at pH 2 to 10.
It maintained its activity even after heat treatment at ℃.

[Table] Example 3 Using human acute promyelocytic leukemia HL-60 cells, 12-O-tetradecanoylphorbol-13 shown in Table 4 was used as an agent for inducing differentiation into Mφ cells.
-acetate (TPA), phorbol-12,13-didecanoate (PDD), meserein (MEZ),
Culture was carried out in the same manner as in Example 2, except that 1 μg/ml of vitamin A acid was used as the Mφ activator, and a culture supernatant was obtained. The activity of the cell differentiation-inducing substance in the culture supernatant was measured, and the following studies were conducted on the culture supernatant in which the activity was observed. The active fraction obtained by adsorption onto a Con-A column and desorption treatment with an α-methyl-d-mannoid solution has a molecular weight of approximately
50,000 (SDS-polyacrylamide electrophoresis method), and did not lose its activity even after heat treatment at 70°C.

[Table] Example 4 20ml of peripheral blood from a healthy person supplemented with 1000 units of heparin
was diluted 2 times, layered on top of Ficoll Hipaque solution (Pharmacia), subjected to specific gravity centrifugation at 800 rpm for 30 minutes, and mononuclear cells were collected from the intermediate layer.
The mononuclear cells were cooled to 4°C and contained 10% fetal bovine serum.
The cells were planted in 10 petri dishes (8 cm in diameter) for tissue culture that had been pretreated with RPMI-1640 medium overnight. The dates were cultured for 3 hours at 37°C in air containing 5% carbon dioxide. Non-adherent cells were removed by washing with serum-free RPMI-1640 medium. Average of 5 x 10 ⁶ pieces per dateshile (1 x 10 ⁵ pieces/cm ² )
of cells were obtained. More than 90% of the cells obtained were monocytes. The date is added to the final concentration of vitamin A.
Serum-free supplemented with acid 1μg/ml and LPS 1μg/ml
RPMI-1640 medium (fortified with vitamins and amino acids) 15
ml and cultured for 3 days at 37°C in air containing 5% carbon dioxide. After 3 days, the culture solution is collected and centrifuged at 3000 rpm to remove cell debris. The phagocytosis-inducing activity of mouse M-1 cells in the resulting culture solution was measured and found to be 44 units/ml. By formulating the cell differentiation-inducing substance of the present invention, which has been explained in detail above, by appropriately subjecting it to conventional pharmaceutical manufacturing methods such as heat treatment, filter sterilization, freeze-drying, dispensing, etc. can be obtained.

Claims (1)

[Scope of Claims] 1. A human-derived proteinaceous physiologically active substance having cell differentiation-inducing action on leukemia cells, having the following properties. a Molecular weight 50000±5000 (gel filtration method) 50000±5000 (SDS-polyacrylamide electrophoresis method) b Adsorption to lectin column Adsorbs to lentil lectin column and concanavalin A column. c Thermal stability: Does not deactivate after 1 hour at 70°C. d Effects of reducing agents Activity decreases. e PH stability Stable within the PH range of 2 to 10. f No decrease in differentiation-inducing activity was observed due to the enzymatic reaction with trypsin and pronase E.