JPH0453497A - Blood cell-treating agent - Google Patents

Abstract

PURPOSE:To provide the subject treating agent useful for producing useful substances such as cytokinin and not causing trouble such as elution by immobilizing N-glycolylneuraminic acid on the surface of an insoluble carrier with an amide bond. CONSTITUTION:An insoluble carrier (preferably having a thickness of >=100mu) such as a Schale, pipe, film or bottle made of the homopolymer, etc., of styrene or alpha-methylstyrene preferably after aminomethyl groups are introduced on the surface of the carrier is immersed in the solution of a mixture of Nglycolylneuraminic acid and a carbodiimide, etc., to immobilize the glycolylneuraminic acid on the carrier for providing the objective treating agent. Blood cells are cultured in the presence of the treating agent to produce cytokinin.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a blood cell processing agent capable of acting on blood cells to produce useful substances, and a method for producing various useful substances using the processing agent.

(Prior technology) Physiologically active substances immobilized on insoluble carriers are widely used as adsorbents for affinity chromatography, therapeutic blood processing agents, antibacterial materials, and analytical reagents, and will be used in an even wider range of applications in the future. It is expected to be an important substance.

Among therapeutic blood treatment agents, those targeting blood cells include those with immobilized lipopolysaccharide derived from the cell wall of Durum-negative bacteria (Japanese Patent Application Laid-open No. 59-21145), and those with immobilized plant lectins such as po and clad mitogen. [Haka Sugahara, Artificial Organs 18(3).

1401 (1989): ] are known, but these immobilize highly toxic substances.

Therefore, there should be no elution of these ligands when they are used, but since they are immobilized natural products with complex chemical structures, there is a problem that there is no compensation for this possibility. However, since the ligand is a polymer, it has multiple active sites within the molecule, and therefore its action is complicated.

(Problems to be Solved by the Invention) In view of the problems of the prior art, the present inventors have immobilized a non-toxic low-molecular compound on an insoluble carrier and brought it into contact with blood cells. As a result of investigating whether it is possible to produce substances, we found that immobilized N-glycolylneuraminic acid, a component of cell membranes, can be used to induce interleukin-1, interleukin-6, TNFSG-C8F, and mononuclear cells into mononuclear cells. It was discovered that GM-C8F etc. can be produced, and the present invention was achieved.

That is, an object of the present invention is to provide a novel blood cell treatment agent that can act on blood cells to produce useful substances such as cytokines.

Furthermore, the present invention aims to provide a method for producing cytokines using such a blood treatment agent.

(Means for Solving the Problems) The configuration of the present invention for achieving such an object is comprised of the following technical means (1) to (6).

(1) A blood cell treatment agent comprising N-glycolylneuraminic acid immobilized on the surface of an insoluble carrier via an amide bond.

(2) The blood cell treatment agent according to (1) above, wherein the insoluble carrier is a petri dish, bottle, membrane, fiber, hollow fiber, granular material, or an assembly using these.

(3) A method for producing cytokines, which comprises bringing blood cells into contact with the blood cell treatment agent according to claim (1).

(4) The method for producing a cytokine according to claim (3), wherein the blood cells are leukocytes.

(5) The method for producing a cytokine according to claim (3) or (4), wherein the cytokine is interleukin-1.

(6) The insoluble carrier is a vinyl polymer molded product mainly composed of styrene or α-methylstyrene with aminomethyl groups introduced onto the surface.
The blood cell treatment agent described in section 1).

The insoluble carrier as used in the present invention means a carrier that is substantially insoluble, has a primary or secondary amino group, and is capable of immobilizing N-glycolylneuraminic acid through an amide bond. The shape of the insoluble carrier is a petri dish, a bottle, a tube, a membrane,
Any of fibers, hollow fibers, granules, or assembled products using these may be used, but it is particularly preferable that these materials have light transparency, since this makes it easier to observe cells and manage cells.

Specific examples of the insoluble carrier referred to in the present invention include: (1) A styrene or α-methylstyrene homopolymer, or an aromatic vinyl copolymer containing these as the main component, in a petri dish;
Test tubes, tubes, films, bottles, syringes, catheters, etc. that have aminomethyl groups introduced onto their surfaces;
■ Styrene/ethylene-butylene ABA block copolymer membrane with aminomethyl groups introduced, ■ Polypropylene-reinforced polystyrene fibers with aminomethyl groups introduced on the surface, ■ Polystyrene or styrene-divinylbenzene copolymer beads, Examples include, but are not limited to, those in which an aminomethyl group is introduced as an aromatic nucleus substituent, and (2) aminopropylated glass beads. Moreover, among these, 1 and 2 are particularly preferably used because they have high light transmittance and are easy to handle.

In the case of (2), there is no particular restriction on the thickness of the molded product, but in order to maintain strength sufficient for practical use, a molded product having a thickness of 100 microns or more is usually preferably used. In addition, it is desirable that the molded product has sufficient light transmittance to enable confirmation of the shape of the substance present on the opposite side of the bottom or side surface of the molded product. Since the light transmittance of the molded article is determined primarily by scattering on the surface, the surface must be completely smooth. In the case of the molded product, 500 to 70
It is necessary that the absorbance for light with a wavelength of 0 nanometers be 3 or less, especially 1 or less. The smaller the second absorbance, the better the light transmittance of the molded product, and the absorbance is 0.1.
It is more preferable if it is below.

The blood cell treatment agent of the present invention can be easily produced by adding an insoluble carrier to a mixed solution of N-glycolylneuraminic acid and carbodiimide, or by immersing it in a solution of an active ester such as N-hydroxysuccinimide ester. will be achieved.

The cytokine referred to in the present invention is interleukin-1
, interleukins represented by interleukin-2, interleukin-6, etc., GM-C3FSG-C8
Colony stimulating factors such as F, interferon,
Means TNF etc.

N-glycolylneuraminic acid used in the present invention is an α-keto acid with 9 carbon atoms that widely exists in the animal kingdom, but it can also be obtained by means of organic synthesis.

A compound in which the hydroxyl group is converted into a carboxylic acid ester can also be used in the same manner since it is easily deesterified by a hydrolase secreted by cells. Note that this substance has almost no ability to induce cytokines in a state where it is not immobilized. It becomes active only after it is immobilized on an insoluble carrier.

Colony stimulating factor is a glycoprotein known as a specific stimulating factor that stimulates granulocyte and macrophage stem cells in soft agar to form colonies consisting of mature granulocytes and macrophages.

Among the colony stimulating factors, GM-C8FSG-C8F
It is known that there are products such as SM-C8F.

In addition, these include monocytes, macrophages, fibroblasts,
It is known to be produced by vascular endothelial cells, bone marrow stroma, etc. GM-C8F is known to not only increase the number of white blood cells, but also act as a light guide for granulocyte function, and is useful in the treatment of myeloid leukemia, AIDS, and leukopenia after cancer chemotherapy. (Experimental Medicine 7 (15) (Special issue > 198-203 (1989)
)).

In addition, G-C5F is also used for the prevention and treatment of severe infectious diseases (Experimental Medicine 7(15) (Special Edition) 191-19
7 (1989)).

Interleukin-1 has been known for a long time as a pyrogen produced by white blood cells, but recently it has been known to have various effects, especially its antitumor effect and hematopoiesis-promoting effect, and its clinical use is increasing. being attempted (experimental medicine)
7 (15) (special issue) 170-177 (1989)).

It has been found that when blood cells are cultured while being brought into contact with the blood cell treatment agent of the present invention, the blood cells are activated and produce various cytokines at high concentrations. In this case, it is possible to extract only monocytes from blood cells and process them, or it is also possible to process a mixture of various blood cells. Cytokines produced in vivo and in vitro.

It can be used as a white blood cell increase agent.

Examples of the use of the blood cell treatment agent of the present invention include use in blood circuits, cell culture instruments, column packing materials for extracorporeal circulation, and the like.

(Effects of the Invention) The blood cell treatment agent of the present invention can be used for the production of useful substances such as cytokines; ■ Since it is immobilized with a chemically stable amide bond, there is no need to worry about elution. It has the following advantages: (1) Even if it were to elute, it is a non-toxic compound.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Note that the evaluation method in the examples was as follows.

1. Chemical analysis of aromatic vinyl polymer molded products When a molded product is Soxhlet-extracted with methylene chloride, an insoluble film-like material is obtained, which is vacuum-dried, weighed, and subjected to infrared absorption spectroscopy, etc. Ta.

2. Infrared absorption spectrum Shimadzu Fourier transform infrared spectrophotometer FT I R-430
It was measured by the KBr tablet method using 0.

3. Measurement of light transmittance of molded product Using a Shimadzu spectrophotometer UV2100, absorbance in the vertical direction of the plate-shaped molded product was measured at 500 nanometers. As a control, the absorbance of a polystyrene plate molded product of the same thickness was measured, and the difference between the two was defined as A300. Therefore, the smaller the value, the higher the light transmittance of the molded product.

4. Determination of N-glycolylneuraminic acid Based on the determination method of sialic acid (Biochemistry Experimental Methods 23 Glycoprotein Experimental Methods p. 17 Gakkai Publishing Center), fluorescently labeled 1,2-diamino-4,5- React with methylenedioxybenzene,
It was quantified by high performance liquid chromatography.

5. Quantification of cytokines Interleukin-1 was determined using the MTT method (T, Mo5n+
An Journal of the Immunological Methods 6555-63 (1983)).

TNF was quantified by cell killing ability using L929 as an indicator cell.

Example 1 A mixed solution of 500 ml of 1-nitropropane and 272 ml of sulfuric acid was cooled to 0°C, and 20.4 g of N-methylol-α
- Chloracetamide was added and dissolved at 0-10°C.

After heating this solution to 10°C, φ3.5cm×1.
11 in a 2 mmH polystyrene culture dish (1 mm thick)
1 ml of the solution was added, and the reaction was carried out at room temperature for 1hr. The reaction solution was discarded, and the culture dish was immersed in methanol at -20°C, further washed with methanol and water, and then vacuum dried to obtain a molded product A in which only the internal surface was chloroacetamidomethylated. The A500 of this molded product was 0.050.

When one molded product A obtained above was subjected to Soxhlet extraction with methylene chloride, 3.1 g of a thin film-like insoluble material was obtained. Since the surface area of the reaction surface of the culture dish is 21 cd, the thickness of the reaction area is considered to be approximately 1.2μ. In addition, the infrared absorption spectrum of this insoluble material is 1659 cm-1 (
The structure was confirmed because strong absorption of the amide group was observed at amide-I), 1529 cm'' (amide-■), and 3297 cm'' (NH).

40 molded products A obtained above were immersed in 2000 ml of 10% dimethylaminoethanol/dimethylsulfoxide solution, left to stand at room temperature for 18 hours, and then immersed in 2000 ml of 10% dimethylaminoethanol aqueous solution at 70°C.
Heated for hr. After washing the molded product with water, it was dried to obtain an aminomethylated molded product B.

A of this molded product. . was 0.05.

When one molded product B of the present invention was subjected to Soxhlet extraction with methylene chloride, 2.3 cm of a thin film-like insoluble matter was obtained. In the infrared absorption spectrum of this insoluble material, molded product A
1659 cm-1 (amide-■) and 1
The structure was confirmed because the strong absorption Cm-1 of the primary amide group at 529 cm-' (amide-■) had completely disappeared.

810 pieces of the above molded product of the present invention were immersed in 500ml of water,
After adjusting the pH to 5 to 6 with N/100-hydrochloric acid, molded product B
0. 4 ml of a 2 g/ml N-glycolylneuraminic acid aqueous solution and 4 ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and the mixture was allowed to stand at room temperature for 48 hours. Discard the reaction solution, wash with water,
A molded product C, which is a blood cell treatment agent of the present invention, was obtained. 0.012 m from the concentration of N-glycolylneuraminic acid in the mother liquor
It was confirmed that g was immobilized. In addition, N on the molded product
It was confirmed that -glycolylneuraminic acid did not elute even when washed with N/4O-NaOH.

Example 2 Mouse macrophage strain TME9 (Yasuki Ogawa et al., Japanese Society of Immunology General Meeting/Academic Meeting Record, 17599 (198
7)) 3X10-5M 2-mercaptoethanol,
50U/ml penicillin (Banyu), 50μg/m
1 of streptomycin (Meiji Seika) and 3X in RPM11640 (Gibco) containing 10% fetal bovine serum.
The 10-' pieces/ml of the floating product was mixed with two molded products C (sterilized by ultraviolet irradiation) on which N-glycolylneuraminic acid was immobilized, obtained in Example 1, and N-glycolylneuraminic acid. Two unfixed molded products A and two molded products B
(both sterilized by ultraviolet irradiation) and cultured in an incubator (37°C, 5% CO2) for 20 hours. When the culture supernatant was taken and the cytokines in it were examined, interleukin 1 was found to be 4 and 5 U/m in the two molded products immobilized with N-glycolylneuraminic acid.
High activity was observed for both 1 and 9.8 U/ml for TNF. On the other hand, in molded product A and molded product B in which N-glycolylneuraminic acid is not immobilized, interleukin-1 is 1.5 U/m1 or less and TNF is 0.5 U/m1 or less.
No activity was observed at 3 U/ml or less. Also, N-
A molded product in which lactobionic acid was immobilized instead of glycolylneuraminic acid in the same manner as in Example 1 (immobilized amount 0°068
mg: ultraviolet irradiation sterilization), but interleukin-1 is 1
．． 5 U/ml or less, TNF was less than 0.3 U/ml, and no activity was observed.

Similarly, interleukin-6, G-C8F.

It was confirmed that cytokines such as GM-C8F were produced at high concentrations.

Claims (6)

[Claims] (1) A blood cell treatment agent comprising N-glycolylneuraminic acid immobilized on the surface of an insoluble carrier via an amide bond. (2) The shape of the insoluble carrier is petri dish, bottle, membrane, fiber,
The blood cell treatment agent according to claim 1, which is a hollow fiber, a granular material, or an assembled product using these. (3) A method for producing cytokines, which comprises bringing blood cells into contact with the blood cell treatment agent according to claim (1). (4) The method for producing a cytokine according to claim (3), wherein the blood cells are leukocytes. (5) The method for producing a cytokine according to claim (3) or (4), wherein the cytokine is interleukin-1. (6) The insoluble carrier is a vinyl polymer molded product mainly composed of styrene or α-methylstyrene with aminomethyl groups introduced onto the surface.
The blood cell treatment agent described in section 1).