JPH048295A - Treating agent of blood cell - Google Patents

Abstract

PURPOSE:To stably obtain an atoxic blood cell-treating agent by immobilizing N-acylueuramin lactose to the surface of an insoluble carrier by amide bond. CONSTITUTION:A homopolymer of styrene or alpha-methylstyrene or an aromatic vinyl-based monomer consisting essentially of styrene or alpha-methylstyrene is formed to give a molded article (A) such as laboratory dish, bottle, membrane, yarn, hollow yarn, granule or assembled material thereof having >=100mu thickness and <=3 light absorbance at 500-700 nanometer wavelength. Then, an N- acylneuramin lactose containing acetyl group as the acyl group is immobilized to the surface of the component A by amide bond to give a blood cell-treating agent (B). Then the component B is brought into contact with a blood cell such as leukocyte to give cytokinin as interleukin.

Description

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

(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 that immobilize lipopolysaccharide derived from the cell wall of Durham-negative bacteria (Japanese Patent Application Laid-Open No. 59-21145) and those that immobilize plant lectins such as Balkwood mitogen. [Haka Sugahara, Artificial Organs 18(3).

1401 (19'89)E and the like are known, but these are those in which highly toxic substances are immobilized.

Therefore, when using them, these ligands must not elute, but since they are immobilized natural products with complex chemical structures, there is no compensation for this possibility. Since it is a polymer, it has multiple active sites within its molecule, which has the disadvantage that its action is complicated.

(Problems to be Solved by the Invention) In view of the problems of the prior art, the present inventors immobilized a non-toxic, low-molecular-weight compound on an insoluble carrier and brought it into contact with blood cells to produce useful cytokines, etc. As a result of investigating whether N-acetylneuramine lactose, which is contained in mammalian colostrum, can be immobilized, mononuclear cells can produce interleukin-1, interleukin-6, TNF, and G. -C5F and GM-C5
We have discovered that F and the like can be produced, and have arrived at the present invention.

(Means for Solving the Problems) The present invention is comprised of the following technical means (1) to (7).

(1) A blood cell treatment agent comprising N-acylneuramine lactose 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), wherein the cytokine is interleukin-1.

(6) The acyl group of N-acylneuramine lactose is
The method for producing a cytokine according to claim (3), wherein the cytokine is an acetyl group.

(7) 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 2.

N-acylneuramine lactose as used in the present invention refers to N-acylneuramine lactose.
- It means lactose bonded to the 2-position of acylneuraminic acid through an ether bond, and examples of N-acyl groups include acyl groups of short-chain carboxylic acids such as acetyl, glycolyl, propyl, and butyl groups. . Specific examples include N-acylneuraminic acid nucnosyl (1→4)-D-glucobylanose, N-acetylneuramyl (2-6) β-galactopyranosyl (1-
4) -D-glucobylanose and the like.

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-acylneuramine lactose through an amide bond. The shape of the insoluble carrier may be a petri dish, a bottle, a tube, a membrane, a fiber, a hollow fiber, a granular material, or an assembly using these materials, but in particular, if the carrier is transparent, cells can be observed. This is preferred because it is easy to perform and the cells are easy to manage.

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 is 3 or less. The smaller the absorbance, the better the light transmittance of the molded product (and it is more preferable if the absorbance is 1 or less).

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

The cytokines used in the present invention include interleukin-1,
Interleukins represented by interleukin-2, interleukin-6, etc., GM-C8F, G-C8F
colony-stimulating factors, interferon, etc.
Means TNF etc.

N-acylneuramine lactose used in the present invention is a substance 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. As shown in Example 2, this product has almost no ability to induce cytokines without being immobilized. It was found that activity can be obtained only after immobilization 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
, M-C8F, etc. are known.

It is also known that these are produced by monocytes, macrophages, fibroblasts, vascular endothelial cells, bone marrow stroma, and the like. GM-C5F is known to not only increase the number of white blood cells, but also have advanced effects on granulocyte function, and is used for the treatment of myeloid leukemia, leukopenia after AIDS and cancer chemotherapy. It is also used (Experimental Medicine E (15) (Special Edition) 198-203 (1989)
).

In addition, G-C3F 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 antitumor effects and hematopoiesis promoting effects, and attempts are being made to use it clinically. (Experimental Medicine 7)
(15) (Special Edition) 170-177 (1989)).

It was found that when blood cells were cultured while being brought into contact with the blood cell treatment agent of the present invention, the blood cells were activated and produced high concentrations of the above-mentioned cytokines in the same manner as in Example 3. 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. The cytokines produced can be used, for example, in vivo and in vtt.
It can be used as a leukocyte increase agent in RO.

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 advantage of being a non-toxic compound even if it were to be eluted.

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-acylneuramine lactose In addition to the determination method of sialic acid (Biochemistry Experimental Methods 23 Glycoprotein Experimental Methods p. 17, Gakkai Publishing Center), the fluorescent label 1,2-
Diamino-4,5-methylenedioxybenzene was reacted and quantified by high performance liquid chromatography.

5. Quantification of cytokines Interleukin-1 was determined using the MTT method (T, Mosma
n 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.
1 in a styrene culture dish (1 mm thick) used for 2 mrnH.
1ml each was added and reacted for 1hr at room temperature. 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.

A of this molded product. . was 0.050.

When one molded product A obtained above was subjected to Soxhlet extraction with methylene chloride, a thin film-like insoluble material 3,1a+g was obtained. Since the surface area of the reaction surface of the culture dish is 21 cd,
The thickness of the reaction part is thought to be approximately 1.2μ. In addition, the infrared absorption spectrum of this insoluble material is 1659 cm" (
The structure was confirmed because strong absorption of the amide group was observed at amide-I), 1529cm, '(amide-■), and 3297crrr' (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 the 81 molded products of the present invention were subjected to Soxhlet extraction with methylene chloride, 2.3 mg of thin film-like insoluble matter was obtained. The infrared absorption spectrum of this insoluble material shows 1659 cm' (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.

Soak 10 molded products B of the present invention in 500 ml of water,
After adjusting the pH to 5 to 6 with N/100-hydrochloric acid, molded product B
Add 4 ml of 0.2 g/ml N-acetylneuramine lactose (Sigma Chemical Co.) aqueous solution and 5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the inside of the container, and leave it at room temperature for 48 hours. did. The reaction solution was discarded and washed with water to obtain a molded product C, which is a blood cell treatment agent of the present invention. It was confirmed from the concentration of N-acetylneuramine lactose in the mother liquor that 0.019 mg was immobilized. Furthermore, it was confirmed that N-acetylneuramine lactose on the molded product did not elute even when washed with N/4O-NaOH.

Example 2 Mouse macrophage strain TME9. (Yasuki Ogawa et al.
Records of the Japanese Society of Immunology General Meeting and Academic Meeting, 17599 (19
87)) in 3 x 10-5 M 2-mercaptoethanol, 50 U/ml penicillin (Banyu), 50 μg
/ml streptomycin (Meiji Seika) and RPM11640 containing 10% fetal bovine serum (Gibco
2 ml each of 3×10-5 cells/ml suspended in the molded products B and C obtained in Example 1 (sterilized by ultraviolet irradiation) were placed in an incubator (37°C, 5% C
02) for 20 hours. When the culture supernatant was taken and the cytokines in it were examined, IL-1
High activity was observed for both 3.7 U/ml and 67.2 U/ml for TNF. On the other hand, in the product in which N-acetylneuramine lactose was not immobilized (molded product B), IL-1 was less than IU/ml and TNF was 2 U/m
1 or less, no activity was observed.

In addition, a commercially available culture dish (FALCON1008)
20 μg of N-acetylneuramine lactose using
In the system cultured in the same manner as above with the addition of 5 μg/ml and 5 μg/ml, IL-1 and TNF were each 1. OU/m1
Below, 2. No activity was observed below OU/ml.

Claims (7)

[Claims] (1) A blood cell treatment agent comprising N-acylneuramine lactose 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), wherein the cytokine is interleukin-1. (6) The acyl group of N-acylneuramine lactose is
The method for producing a cytokine according to claim (3), wherein the cytokine is an acetyl group. (7) 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).