JPH02100697A - Production of differentiation-inducing substance derived from human - Google Patents

Abstract

PURPOSE:To produce bioactive substance having cell differentiating and inducing (differentiate tumor cell to cell having normal function) activity by culturing meacrophage-like cell derived from human infected with mycoplasma. CONSTITUTION:A cell derived from human contaminated with mycoplasma is cultured, or cell and crushed material of mycoplasma performed of inactivation treatment is added to a culturing system of cell derived from human to produce cell differentiating and inducing substance in said culturing system. In said process, differentiating and inducing agent such as pholbol esters, diterpene-based compound represented by mezerein or teleocidin is preferably added to the culturing system. Resultant differentiating and inducing substance has an amino acid sequence expressed by the formula (X is amino acid impossible or difficult to identify by Edman decomposing method) in amino chain end and has about 51000 molecular weight (be gel filtration technique).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a novel human-derived differentiation-inducing substance having cell differentiation-inducing activity. More specifically, the present invention relates to a method for producing a macrophage substance derived from a human infected with mycoplasma.

In the present specification, amino acids are represented by 1 according to the PeIUB Committee on Biochemical Nomenclature (CBN), and the following abbreviations are used, for example. In addition, when optical isomers are possible for amino acids, etc., the L-isomer is indicated unless otherwise specified.

Pro: proline residue Leu: leucine residue lie: isoloinone residue Val nivaline residue Ala: alanine residue So, macrophages (hereinafter,
(abbreviated as Mφ) is attracting attention.

Mφ plays a central role in the body's defense mechanism, including the in-vivo processing of antigenic substances through A-phagocytosis. Furthermore, it has been known that Mφ produces various important physiologically active substances, such as interleukin-1, tumor necrosis factor, and colony stimulation factor, in response to extracellular stimuli.

Blood cells mature from hematopoietic stem cells to functional cells through proliferation and differentiation. During this differentiation and maturation process, leukemia cells have the ability to proliferate and become tumors. Differentiation-inducing substances promote the differentiation of tumor cells into cells with normal functions, and are expected to be applied to new cancer treatment methods.

Among these, human-derived protein differentiation inducers, which are expected to be highly safe, have been actively researched.

Early studies showed that differentiation-inducing substances produced by lectin-stimulated human peripheral blood lymphocytes (Journal
Of Su7! I-Naru Cancer Institute
J, 11a (iH31 Cancer In5ti and a patent application has been filed, but this substance has not been isolated and its properties determined.

Although it is possible to use human-derived normal cells as cells that produce such differentiation-inducing substances, it is difficult to prepare a sufficient amount of cells. On the other hand, cell lines are easy to use because they can proliferate cells indefinitely.

There are no particular restrictions on the cell line, but it is preferable to use a leukemic cell line that corresponds to Mφ progenitor cells and has the ability to produce large amounts of differentiation-inducing substances.
(Cell lines such as 1, -60 and U937 are applicable.

Mycoplasma has a size of 125 to 150n+*, and is an organism that can contaminate cultured cells and reproduce itself. Mycoplasma characteristically lacks a cell wall, so the morphology of the fungus exhibits a certain degree of pleomorphism.

When mycoplasma contaminates cultured cells, mutations occur in the properties of the cell membrane system of the cells, resulting in changes in cell morphology. In addition, mutations in the metabolism of nucleic acids, lipids, proteins, etc., changes in the amino acid requirements of cells, shortening of the lifespan of normal cells, and furthermore, when normal cells with tumorigenic cells and mouse tile cells are mixedly cultured, tumor cells -It is expected to produce feron (Tissue Culture Vol. 8, p. 91 (
+982)). As a result, it has been reported that when combined with mycoplasma-contaminated tumor cells, the natural killer cells in the tile cells increased their interferon production.

[Problems to be Solved by the Invention] As described above, it is possible to use glycoprotein physiologically active substances that exhibit differentiation-inducing activity produced from human-derived normal cells, human T-cell leukemia cell lines, human myeloid leukemia cell lines, etc. The aim is to produce larger quantities.

[Means for Solving the Problems] The present inventors have developed a human-derived glycoprotein cell differentiation inducer that acts on human Mφ progenitor cells and has the activity of inducing differentiation into Mφ by itself. After discovering and isolating and purifying the target substance present in trace amounts in the culture solution, the natural killer cells in the tile cells respond to the substance by ink so ooo±soo
o (Use a culture system containing Mycoplasma cells and crushed material treated with non-reducing SDS Boria.
After acting on the cells to transform them into Mφ-like cells, the target substance was produced in the culture solution.

[Contents of the invention] That is, the present invention refers to proline residues as Pro, loino residues as Leu, isoloinone residues as Ile,
Valino residue is Val, alanine residue is A la,
When an amino acid that cannot be identified or is difficult to identify using the Edman degradation method is expressed as x, the amino terminal amino acid sequence is Pro-Le, u-Pro-11e-X -Pro-4.
al-X -Ala-X -X -Alalle-X
-x -Pro- is a glycoprotein physiologically active substance having the following properties, and has the following effects: When producing human-derived differentiation-inducing substances,
, a human-derived differentiation-inducing substance characterized by culturing human-derived cells contaminated with Microplasma, or adding inactivated Microplasma cells and fragments to a human-derived cell culture system. This is the manufacturing method.

In the present invention, a substance having a cell differentiation-inducing effect is a substance produced by a human-derived normal cell or cell line, and is a substance produced by a cell line Tl-I P-1 which corresponds to at least human Mφ progenitor cells in vitro. , HL-60 and U93
7. It is a substance that differentiates mouse Ml cells and has the ability to induce n-benign function, and is a glycoprotein having the above-mentioned physicochemical properties.

THP-1 is the International Journal of
Cancer (Int, J, Cancer) 26 volumes, 17+ pages (1980), HL-60 is Fichi+
-(Nature).

Volume 270, page 347 (1977), U937 is the International Journal of Canosa Inl.
J, Cancer Vol. 17, p. 565 (197
6 years), Ml is ma hominis), orale (M, orale), arginini (M, ar
ginini) is preferred. Also, pneumoniae (M, pneumoniae), salivarium (M,
salivarium), fermentans (M, f
erientans), ribophyllum (M, 1ipo
philum), brimasome (M, prima L)
ump, Bcoides, M, axanthum, Bactoclasticium, Acholaplasma 1aidlavii
) etc., it is difficult to prepare well using any species. On the other hand, cell lines can proliferate cells indefinitely and are easy to use. Although there are no particular restrictions on cell lines, it is preferable to use leukemic cell lines that correspond to Mφ progenitor cells that exhibit the ability to produce large amounts of differentiation-inducing substances.
This includes cell lines such as P-1°HL-60 and U937.

Mycoplasma used in the present invention includes Mycobraz 7
H (Mycoplasma), Acholeplasma (A
choleplasma), Ureaplasma (Ure
There are some that belong to aplas+a). In carrying out the present invention, Mike Plasma's fl! Class Ii is not particularly limited, but includes hominis (Mycoplas esters, diterpene compounds represented by meserein, and teleo/
Examples include gin. In addition, among phorbol esters, 4β-hydroxy/isomer is preferable, and among them, 12-0
-Tetradecanoylphorbol-13-acetate (hereinafter abbreviated as TPA) is suitable.

In addition, vitamin A derivatives that are activators of Mφ, such as vitamin A acid, vitamin A alcohol, vitamin A acetate, and vitamin A palmitate, dimethyl sulfoxide, sodium butyrate, hydrofutisone, and Noriboborisanoka derived from Durum-negative bacteria. Bacterial walls such as Ride, Lipinod A, and BCG bacteria, and muramyl dipeptide are used alone or in appropriate combinations to activate Mφ and promote the production of cell differentiation-inducing substances.

A method for contaminating cultured cells with mycoplasma is to contaminate cultured cells by adding a culture solution of mycoplasma that has been cultured alone or a culture supernatant of infected cells to the culture solution of cultured cells. The method for culturing mycoplasma was based on the method of Chanock et al. Alternatively, THP-1 cells can be contaminated with THP-1.
1 cells.

In carrying out the present invention, the method is not limited to contaminating cells with mycoplasma or adding inactivated mycoplasma cells and fragments to a cell culture system. The types of mycoplasma inactivation methods are not particularly limited, but biochemical methods include lysis of bacteria using a bifacial active agent, sodium lauryl sulfate (hereinafter abbreviated as SDS), physical methods include crushing bacterial cells by ultrasonic vibration, Simple inactivation by heating is preferred. Yet another biochemical method of inactivation is Nonidenoto P-40 (NP
-40), TWEEN-20, TWEEN-80, and the like can be used, and may be inactivated under strong alkali or strong acid conditions.

Other physical methods include inactivation of bacteria by UV irradiation;
Any method can be used, such as crushing the bacterial cells using a high flow rate using a French press, using a low-osmotic solution, rupturing the bacterial cells by freezing and thawing, or crushing the bacteria using a mortar and pestle under freezing.

A solution containing is obtained. Using a solution containing this cell differentiation inducer, biochemical isolation and purification operations such as concentration and desalting by ultrafiltration, ion exchange chromatography with a cation exchanger and anion exchanger, affinity with lectin or specific antibody, etc. By performing purification using an appropriate combination of chromatography, gel filtration, electrophoresis, etc., a highly purified purified sample of the differentiation-inducing substance can be obtained.

αI determination of the activity of the differentiation-inducing substance was performed in vitro in mouse M1.
This was done based on the effect of inducing phagocytosis in cells.

The method used by the present inventors is an improved method of Hayashi's method described in Toxicology Forum, Vol. 7, p. 50 (1984). That is, 10,000 M1 cells in the proliferation phase were added to a medium (Eagle MEM, twice the amount of normal amino acids and vitamin supplements + 10% beef tallow serum),
Mix the test solution (containing differentiation-inducing activity) and set it to 0.51,
Place in a 24-well culture plate and place in a carbon dioxide incubator.
Incubate at 37°C for 2 days. Subsequently, centrifugation treatment (100
0 rpm for 10 minutes), discard the supernatant liquid, and remove the blood l^
Add 0.5 liters of the above-mentioned culture solution containing no chlorine and resuspend the cells, and add polystyrene chloride (+nnl + rm・I 1 liter) to a final concentration of 2 μm/11). Wash and centrifuge to remove extracellular latex particles. After repeating this operation twice, the cells precipitated at the bottom of the centrifuge tube are sucked up with a pipette and dropped 1 iai onto a glass slide. Add 1 drop of 05% eosin solution to this,
Place a cover glass and observe under a microscope. Excluding dead cells that are stained red, only living cells are counted, including cells that have phagocytosed 10 or more latex particles and cells that have not phagocytosed, to determine the ratio of phagocytosed cells. Dilute the test solution appropriately and perform measurement under the above conditions, and the ratio of hypophagic cells is 10.
% of the differentiation-inducing substance activity] is defined as 1 unit.

Hereinafter, the activity amount of the differentiation-inducing substance in the present invention is shown in units measured by this method for measuring poultry performance.

The produced physiologically active substances act on human and mouse myeloid leukemia cells, which correspond to Mφ precursor cells, to
It induces differentiation into Mφ-like cells, brings about enhancement of various functions specific to Mφ, and increases the molecular weight in solution, that is, molecule 1 at the time of gel filtration, is 51,000 ± s, ooo. The molecular weight in electrophoresis must be 50,000±s, ooo, and various synthetic media suitable for culturing Funcanapa cells are used.

As a typical medium, for example, RP M l-1640
Medium, Eagle's MEM medium, Dulbecco's modified MEM
A medium such as α-MEM medium, Ham's medium, 199 medium, Manokoi 5A medium, Isufu's medium, etc. may be used alone or in an appropriate mixture. The compositions of these media are described in ``Cell and Tissue Culture Manual'' by Sokoki, Kodansha, 1982. Serum-derived proteins such as albumin, insulin, and transferrin, and animal serums such as human serum, fetal bovine serum, bovine serum, and lactose may be added to these media alone or in appropriate combinations. The material of the culture container is not particularly limited, but it is preferably plastic, glass, or metal, which allows cell proliferation and has excellent cell adhesion.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

In the following description, % represents volume percentage (V/V) % unless otherwise specified. Unless otherwise specified, culturing was carried out at 37°C, humidity 90-100%, and air containing 5% carbon dioxide.

Example 1 A) Collection of culture supernatant The culture supernatant of cultured mycoplasma was added to the culture solution of human monocytic leukemia cell line THP-1 and cultured to obtain THP-1 contaminated with mycoplasma. Ta.

The THP-1 was dissolved in RPM containing 10% fetal bovine serum.
After culturing for 7 days in l-1640 medium (2i!! containing twice the normal amount of amino acids and vitamin supplements), the above medium without serum (12-0-tetradecanoylform, a differentiation inducer) was cultured. Ball-13-acetate-) (1'FA) O
, Lu g/ml and the Mφ activator vitamin A acid (
RA) containing lμg/ml) to a cell density of 60
1 million pieces/Peng 1. 10 ml of isothermal KN was added to 90 m Dinonko (Falcon! 2) and cultured for 72 hours. After the culture was completed, the Tsuchiura were collected and centrifuged at 2000 rpm for 10 minutes to remove the cell layer. Polyethylene glycol aooo (hereinafter referred to as PEG)
(abbreviated as 600Q) O.01% was added. When the activity of the differentiation-inducing substance in the supernatant was measured by the method shown in B), it showed a differentiation-inducing activity of 898 units/ml.

B) Activity measurement method for differentiation-inducing substances 100,000 Ml cells in the proliferation phase were cultured in a medium (Eagle M
E M + a Double the usual amount of amino acids and vitamin U
The mixture was mixed with a test solution (containing differentiation-inducing activity) and a test solution (containing differentiation-inducing activity), and the mixture was placed in a 24-well culture plate and cultured in a carbon dioxide gas incubator for 2 days. Subsequently, centrifugation was performed (+000 rpm for 10 minutes), the supernatant was discarded, and 0.5 ml of the above-mentioned culture medium without serum was added to resuspend the cells, and the cells were suspended in polystyrene latex at a concentration of 2 μm/liter. Particles (1, H4μ Yong: manufactured by Block Chemical Co., Ltd.)
After adding and stirring, the mixture was further cultured for 4 hours. The cells were thoroughly washed with 111 phosphate buffered saline (hereinafter referred to as PBS) and centrifuged to remove the extracellular fluid.

The dust particles were removed. After repeating this operation twice, the cells precipitated at the bottom of the culture plate were sucked up with a pipette (raised 1.1 cm), dropped 1.1 cm onto a slide glass, and added 0.5%
Eosin solution 1a was added, a glass plate was placed on the plate, and observation was made using a microscope. Excluding the dead cells that are stained red, count only the live cells that have phagocytosed 10 or more latex particles and the cells that have not phagocytosed, and calculate the ratio of n-phagocytic cells by appropriately diluting the analyte solution. The above measurements were carried out, and the amount of activity of the differentiation-inducing substance required for the proportion of phagocytic cells to reach 10% was defined as 1 unit.

I met.

C) Isolation and purification of the differentiation-inducing substance The differentiation-inducing substance was isolated and purified from the obtained culture supernatant. The culture supernatant obtained in step A) was passed through a Zetablenobu 15 (QAE) disk (manufactured by AMF) to remove solid content and acidic impurities. Subsequently, the solution was filtered through a polysulfone ultrafiltration module (capto-off molecule ff1
10,000, concentrated at Asahi Kasei Corporation).

Next, EG fa a was added to a column (501 volume ff1) packed with lentil lectin-Sepharose (Pharmacia).
) and eluted with buffer A containing 0.5M α-methyl-d-mannose.

Next, the crudely purified sample was purified by cation exchange chromatography. As a liquid chromatograph/stem, FPl
, C/Stem (Pharma/A) was used.

0.01% (V/V) PEG6000 and 0.01% (V/V). [
50mM female (2(N-Morphorino)ethanesu) containing 12% (v/v) sodium azide
pl+ was added to a MonoS column (5 x 5, manufactured by Farmana) equilibrated with a buffer solution (pt15.0, hereinafter abbreviated as buffer B) with hydrochloric acid for 5 minutes.
After adding the crudely purified sample adjusted to 0 and washing with mild III solution B, washing again with buffer B adjusted to pH 60 (hereinafter abbreviated as buffer C), and adding IM sodium chloride. An active fraction was obtained by gradient elution with buffer C containing .

The active fraction obtained by repeating this operation and treating the entire amount of the crudely purified sample was further applied to a MonoS column (
5X5 (Pharma/A) to obtain a concentrated and purified sample.

Next, the sample was mixed with O, 01% (v/v) PEG600.
10 mM Tris (hydroxymeL) containing 0 and 0.02% (v/V) sodium azide.
hyl) aminomethane) buffer (pH 8,
5. It was added to a desalting column (PD-10, manufactured by Pharmacia) equilibrated with a buffer solution (hereinafter abbreviated as buffer solution), and the buffer solution was exchanged for the next anion chromatography operation.

The sample obtained in the above operation was added to a MonoQ column (5X5, manufactured by Farmana) equilibrated with a buffer solution,
After washing with a buffer solution, gradient elution was performed with an IM buffer solution containing sodium chloride to obtain a fraction showing differentiation-inducing activity. The active fraction was used as the final purified preparation of the differentiation-inducing substance.

Properties such as activity, molecular weight, and lectin binding properties of the obtained final purified sample were measured, and the molecular weight was determined by analyzing the amino acid sequence of the amino acid/terminus of non-reduced 5DS-polyacrylamide electrophoresis Example 3. It was the same as the differentiation-inducing substance shown above.

Example 2 The culture supernatant obtained by culturing mycoplasma (Mycoplasma orale) was added to THP-1 cells and cultured,
TI(P-1) contaminated with mycoplasma was obtained.

To the obtained cells, add 12-〇-tetradecamethyl-13-acetate (TPA) at 0.1 μg/ml and M
Production of differentiation-inducing substances and measurement of differentiation-inducing substances were carried out in the same manner as in Example 1, with a culture time of 96 hours after addition of 1 μg/ml of vitamin A acid (RA), a φ-activating substance.

As a result, a culture supernatant having a differentiation-inducing activity of 1031ヰ1/ml was obtained. Furthermore, a glycoprotein exhibiting the same differentiation-inducing activity as in Example 1 was isolated from the culture solution, and a differentiation-inducing substance was produced using a human promyelocytic leukemia cell line (HL-60). THP contaminated with mycoplasma
1 culture supernatant was added to the HL-60 culture solution, and HL-6
0 was contaminated with mycoplasma. Mycoplasma-contaminated HL-60 cells were transferred to RPM containing 0% fetal bovine serum.
After culturing in l-1640 medium (containing twice the normal amount of amino acids and vitamin supplements), the above-mentioned serum-free medium (differentiation inducing agent 12-0-tetradecamylphorbol-13-acetate) was cultured. ) (TPA) 0.1μg/ml
and Mφ activator vitamin A acid (RA) lμg/m
The cells were suspended in 100ml (containing 1) to give a cell density of 600,000 cells/1.

901m day of the same solution 101.

(manufactured by Falcon) and cultured for 72 hours. After completion of the culture, the supernatant was collected and centrifuged at 2000 rpm for 10 minutes to remove the cell layer. Add to this the anti-adsorption agent PEG60.
Q00.01% was added. The activity of the differentiation-inducing substance in the supernatant was measured by the method shown in Example I, B).

As a result, from the culture supernatant of mycoplasma-contaminated cells, 46
Production of differentiation-inducing active substance at 0 units/ml was observed.

Further, from the culture solution, a differentiation-inducing substance having the same physicochemical and physiological properties as in Example 1 was obtained.

Example 4 Culture supernatant 201 of Mycoplasma hominis was centrifuged at +00000 x g for 30 minutes using an ultracentrifuge, and the precipitate was suspended in 1 ml of physiological saline. Sodium lauryl sulfate (SDS) was added to the suspension to a final concentration of 0.05% to dissolve mycoplasma. The obtained mycoplasma lysate was added 5x, and a differentiation-inducing substance was produced in the same manner as in It'll 1 using THP-1 cells.

When the differentiation-inducing activity was measured by the method shown in B) of Example 1, an activity of 465 units/ml was observed in the obtained THP-1 supernatant.

Furthermore, using the same purification method as in Example 1, a differentiation-inducing substance having the same physicochemical properties as in Example 1 was obtained.

Comparative Example 1 Using the T HP-1 cells used in Example 1, a differentiation-inducing substance was produced in the same manner as in Example 1, except for contamination with mycoplasma.

As a result, a culture supernatant containing 133 units/ml of the differentiation-inducing substance was obtained.

Comparative Example 2 Using HL-60, a differentiation-inducing substance was produced in the same manner as in Example 3 except for mycoplasma contamination. As a result, a differentiation-inducing activity of 70 units/ml was observed in the culture supernatant.

As explained in detail above, the method for culturing a differentiation-inducing substance of the present invention allows the production amount to be significantly increased compared to conventional production methods.

Patent applicant: Director of Industrial Technology Iho 1

Claims (1)

[Claims] Proline residue is Pro, leucine residue is Leu, isoleucine residue is IleN, valine residue is Val, alanine residue is Ala, and cannot be identified or cannot be identified by Edman degradation method. When a difficult amino acid is written as x,
The amino acid sequence of the amino terminal is shown as [There is an amino acid sequence], and it is a glycoprotein physiologically active substance that has the following properties: a) Molecular weight 51,000 ± 5,000 (gel filtration method) 5
0,000±5,000 (non-reducing SDS-polyacrylamide electrophoresis) b) Isoelectric point 8.9±0.3 (isoelectric focusing) c) Lectin-binding concanavalin A and lentil lectin When producing a human-derived differentiation-inducing substance that has a cell differentiation-inducing effect on human and mouse leukemia cells that have binding properties, it is necessary to cultivate human-derived cells contaminated with mycoplasma or to use inactivated myplasma cells. 1. A method for producing a human-derived differentiation-inducing substance, which comprises adding bacterial cells and a crushed material to a culture system of human-derived cells.