JPH07242553A - Method for production plasma or serum having immunodepressive action and device for prodcing the same - Google Patents
Method for production plasma or serum having immunodepressive action and device for prodcing the sameInfo
- Publication number
- JPH07242553A JPH07242553A JP6037133A JP3713394A JPH07242553A JP H07242553 A JPH07242553 A JP H07242553A JP 6037133 A JP6037133 A JP 6037133A JP 3713394 A JP3713394 A JP 3713394A JP H07242553 A JPH07242553 A JP H07242553A
- Authority
- JP
- Japan
- Prior art keywords
- blood
- group
- manufactured
- plasma
- serum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 230000035322 succinylation Effects 0.000 description 1
- 238000010613 succinylation reaction Methods 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-O sulfonium group Chemical group [SH3+] RWSOTUBLDIXVET-UHFFFAOYSA-O 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
Landscapes
- External Artificial Organs (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、材料と血液とを接触さ
せることにより、免疫抑制作用を有する血漿並びに血清
を製造する方法及び装置に関する。本発明は、慢性関節
リウマチ、全身性エリテマトーデスのような自己免疫性
疾患やアレルギー性疾患等の治療に利用される。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for producing plasma and serum having an immunosuppressive action by bringing a material into contact with blood. INDUSTRIAL APPLICABILITY The present invention is used for treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, and allergic diseases.
【０００２】[0002]
【従来の技術】慢性リウマチや全身性エリテマトーデス
のような自己免疫性疾患やアレルギー性疾患の原因は、
一般的に、いずれも異常な免疫亢進によって、非自己あ
るいは自己抗原に対して免疫反応が起こり、それが炎症
反応に進み、自己臓器破壊に向かうものと考えられてい
る。BACKGROUND OF THE INVENTION The causes of autoimmune and allergic diseases such as chronic rheumatism and systemic lupus erythematosus are
Generally, it is considered that an abnormal immune enhancement causes an immune reaction to non-self or self-antigen, which progresses to an inflammatory reaction to destroy self-organs.
【０００３】そのため、これらの臨床的な治療では、異
常な免疫状態を是正するために、従来から、種々のステ
ロイド剤や免疫抑制剤による薬剤療法が行われている。
しかしながら、これらの薬剤療法では免疫抑制作用を期
待し得る量を用いると副作用が起こる。Therefore, in these clinical treatments, in order to correct an abnormal immune state, conventionally, drug therapy with various steroid agents and immunosuppressive agents is performed.
However, in these drug therapies, side effects occur when an amount which can be expected to have an immunosuppressive action is used.
【０００４】そのため、これらの薬剤療法とは別のアプ
ローチとして、流血中に存在する自己抗体、免疫複合体
及び病因関連物質を除去することを目的にした、次のよ
うな治療方法が試みられているが、異常な免疫状態の根
本的な解決には至っていない。また、下記の何れの方法
にも固有の問題点があった。Therefore, as an alternative approach to these drug therapies, the following therapeutic methods aimed at removing autoantibodies, immune complexes and pathogenic substances present in bloodstream have been attempted. However, it has not been the fundamental solution to the abnormal immune status. Further, there are problems inherent in any of the following methods.
【０００５】（１）血漿交換療法…この方法は、血漿の
入手が困難であり、感染の危険性が高いという問題を有
する。 （２）ＤＮＡやプロテインＡのようなリガンドを種々の
材料に固定化し、免疫吸着反応を利用して、抗ＤＮＡ抗
体や免疫複合体を吸着除去する治療方法。しかしなが
ら、この方法は、リガンドの精製の際の不純物の混入
や、リガンドの脱離が問題である。(1) Plasma exchange therapy: This method has a problem that it is difficult to obtain plasma and the risk of infection is high. (2) A therapeutic method in which a ligand such as DNA or protein A is immobilized on various materials and the immunoadsorption reaction is utilized to adsorb and remove anti-DNA antibody or immune complex. However, this method is problematic in that impurities are mixed in during purification of the ligand and desorption of the ligand.
【０００６】（３）特定のリガンドを使用せず、高分子
材料に特定の官能基を固定した吸着材料を用いた病因物
質の吸着除去療法が考案され、特公平２−５０９８号公
報、特公平２−５０９７号公報、特開昭６４−６８２７
３号公報に開示されている。しかしながら、これらの方
法は、選択性に乏しいことが問題になっている。(3) A method for adsorbing and removing a pathogenic substance using an adsorbent material in which a specific functional group is immobilized on a polymeric material without using a specific ligand has been devised, and is disclosed in Japanese Examined Patent Publication No. 2-5098. 2-5097, JP-A-64-6827
It is disclosed in Japanese Patent No. However, these methods suffer from poor selectivity.
【０００７】一方、血液や白血球と種々の高分子材料と
を反応させて、血液細胞や白血球からインターロイキン
２、ＴＮＦなどのサイトカインが産生されることが報告
されている（人工臓器２０(1),ｐ２３５−２４０，１９
９１、同２２(5),ｐ１２３３−１２３７））。しかしな
がら、これらの因子は免疫増強作用や抗癌作用を持つ因
子であり、リウマチやアレルギー性疾患、自己免疫疾患
の治療に適する免疫抑制作用は有していない。On the other hand, it has been reported that blood cells and leukocytes are reacted with various polymer materials to produce cytokines such as interleukin 2 and TNF from blood cells and leukocytes (artificial organ 20 (1)). , p235-240, 19
91, ibid. 22 (5), p1233-1237)). However, these factors have an immunopotentiating action and an anticancer action, and do not have an immunosuppressive action suitable for treating rheumatism, allergic diseases, and autoimmune diseases.
【０００８】[0008]
【発明が解決しようとする課題】本発明の目的は、上述
した従来技術の諸欠点を解消し、従来法に比べて簡便に
免疫抑制作用を有する血漿もしくは血清を製造する方法
及びそのための製造装置を提供することにある。SUMMARY OF THE INVENTION The object of the present invention is to solve the above-mentioned drawbacks of the prior art and to easily produce plasma or serum having an immunosuppressive action as compared with the conventional method, and a production apparatus therefor. To provide.
【０００９】[0009]
【課題を解決するための手段】本発明者らは、かかる実
情に鑑み、鋭意研究を重ねた結果、血液を特定の高分子
材料と接触させることによって、血液中に免疫抑制作用
を誘導することができることを見いだし、本発明を完成
するに至った。本発明は、従来よりも、簡単に、免疫抑
制作用を有する血漿並びに血清を製造する方法及び装置
を提供するものである。[Means for Solving the Problems] In view of such circumstances, the present inventors have conducted intensive studies, and as a result, induce an immunosuppressive action in blood by bringing blood into contact with a specific polymer material. As a result, they have completed the present invention. The present invention provides a method and a device for producing plasma and serum having an immunosuppressive action more easily than ever before.
【００１０】すなわち、請求項１に記載の発明は、水酸
基、アミド基及びエステル基からなる群から選択した少
なくとも１つの官能基を有する高分子材料と、血液とを
接触させることを特徴とする免疫抑制作用を有する血漿
もしくは血清の製造方法である。That is, the invention according to claim 1 is characterized in that blood is brought into contact with a polymeric material having at least one functional group selected from the group consisting of a hydroxyl group, an amide group and an ester group. A method for producing plasma or serum having an inhibitory action.
【００１１】また、請求項２に記載の発明は、カチオン
性官能基を有する高分子材料と、血液とを接触させるこ
とを特徴とする免疫抑制作用を有する血漿もしくは血清
の製造方法である。The second aspect of the present invention is a method for producing plasma or serum having an immunosuppressive action, which comprises contacting a polymeric material having a cationic functional group with blood.
【００１２】請求項３に記載の発明は、血液の導出入口
を有する容器と、前記容器内に充填された請求項１また
は２に記載の高分子材料とを備えることを特徴とする免
疫抑制作用を有する血漿もしくは血清の製造装置であ
る。なお、本発明でいう免疫抑制作用とは、ヒト末梢血
から分離したリンパ球のマイトゲン刺激による幼若化反
応を抑制する作用をいう。An invention according to claim 3 is characterized by comprising a container having a blood inlet / outlet port and the polymer material according to claim 1 or 2 filled in the container. It is an apparatus for producing plasma or serum having a. The immunosuppressive action referred to in the present invention refers to the action of suppressing the blastogenic reaction of lymphocytes separated from human peripheral blood due to mitogen stimulation.
【００１３】請求項１，２の発明では上記特定の高分子
材料が用いられる。後述の実施例から明らかなように、
水酸基、アミド基、エステル基から選ばれる１つあるい
は１つ以上の官能基を有する高分子材料や、カチオン性
官能基を有する高分子材料と血液とを接触させることに
よって、顕著な免疫抑制作用の誘導が認められたのに対
して、ポリスチレンやポリエチレン、ポリプロピレン、
ポリ塩化ビニルのような高分子材料では、免疫抑制作用
の誘導がほとんど認められなかった。また、スルホン基
やカルボキシル基のようなアニオン性基だけを有する高
分子材料では、低いレベルの免疫抑制作用の誘導を認め
たに過ぎなかった。In the inventions of claims 1 and 2, the above-mentioned specific polymer material is used. As is clear from the examples described below,
By bringing a polymer material having one or more functional groups selected from a hydroxyl group, an amide group and an ester group, or a polymer material having a cationic functional group into contact with blood, a remarkable immunosuppressive action can be obtained. Whereas induction was observed, polystyrene, polyethylene, polypropylene,
Induction of immunosuppressive effect was hardly observed with polymeric materials such as polyvinyl chloride. In addition, with a polymer material having only an anionic group such as a sulfone group or a carboxyl group, only a low level of induction of immunosuppressive action was observed.
【００１４】請求項１の発明で用いられる、水酸基、ア
ミド基、エステル基から選ばれる１つあるいは１つ以上
の官能基を有する高分子材料には、アガロース、セルロ
ース、デンプン、プルラン、デキストラン、グリコーゲ
ン、マンナン、グルコマンノグリカン、ガラクトマンノ
グリカン、ペクチン、アルギン酸、ヒアルロン酸、コン
ドロイチン硫酸、キチン、キトサンのような天然多糖類
やそれらをカルボキシメチル化、スクシニル化、糖側鎖
グラフト、ペプチド側鎖グラフト、グリコール化、アシ
ル化、アミノ化によって修飾した種々の誘導体が挙げら
れる。The polymer material having one or more functional groups selected from a hydroxyl group, an amide group and an ester group used in the invention of claim 1 includes agarose, cellulose, starch, pullulan, dextran and glycogen. , Natural saccharides such as mannan, glucomannoglycan, galactomannoglycan, pectin, alginic acid, hyaluronic acid, chondroitin sulfate, chitin, chitosan and their carboxymethylation, succinylation, sugar side chain grafting, peptide side chain Examples include various derivatives modified by grafting, glycolation, acylation and amination.
【００１５】例えば、本発明者らが得た知見では、キチ
ン及びそのＮ−脱アセチル化物であるキトサンやキトサ
ンのアミノ基に１、２、３、４級アミンを導入したキト
サン誘導体、アルキル基を導入した誘導体、また水酸基
にスルホン基、カルボキシメチル基を導入した誘導体な
どは血液との接触によって、免疫抑制作用の大きな誘導
を示した。For example, according to the findings obtained by the present inventors, chitin and its N-deacetylated product, chitosan, and chitosan derivatives and alkyl groups in which 1, 2, 3, 4 primary amines are introduced into the amino groups of chitosan. The introduced derivative, the derivative having a sulfone group or a carboxymethyl group introduced into the hydroxyl group, etc., showed a large induction of immunosuppressive action upon contact with blood.
【００１６】また、アガロースやアガロースに２、３−
ジブロモプロパノールを強アルカリ条件下で作用させて
架橋することで強度を高めた架橋型アガロースや、それ
にジエチルアミノエチル（ＤＥＡＥ）基等のイオン交換
基をエーテル結合させたアガロース誘導体、４級アミン
等で修飾したアガロース誘導体よりなるゲルビーズと血
液との接触によっても、免疫抑制作用の顕著な誘導がみ
られた。[0016] In addition, agarose and agarose are mixed with 2,3-
Cross-linked agarose with increased strength by cross-linking dibromopropanol under strong alkaline conditions, and modified with agarose derivatives in which ion exchange groups such as diethylaminoethyl (DEAE) groups are ether-bonded, quaternary amines, etc. A significant induction of immunosuppressive action was also observed by contacting blood with gel beads composed of the agarose derivative.
【００１７】また、アルギン酸ナトリウムなどを水に溶
解させ、これらと多価金属イオンを含む水溶液とを接触
させることにより作製したアルギン酸ゲルビーズと血液
との接触によっても、免疫抑制作用の顕著な誘導がみら
れた。Further, a remarkable induction of immunosuppressive action is also observed by contacting blood with alginic acid gel beads prepared by dissolving sodium alginate or the like in water and bringing them into contact with an aqueous solution containing polyvalent metal ions. Was given.
【００１８】また、請求項１の発明で用いられる水酸
基、アミド基、エステル基を有する高分子材料には、ポ
リビニルアルコール、ポリヒドロキシエチルアクリレー
ト、ポリヒドロキシメチルアクリレート、ポリフェノー
ル、ポリビニルピロリドン、ポリアクリルアミド、ポリ
リジン、ポリビニルピロリドン、ポリアクリル酸エステ
ル、ポリメタクリル酸エステル等の合成高分子材料やそ
の種々の誘導体や架橋物及びそれらとスチレン、メタク
リル酸エステル等のビニルモノマーとの種々の共重合体
が挙げられる。The polymeric material having a hydroxyl group, an amide group and an ester group used in the invention of claim 1 includes polyvinyl alcohol, polyhydroxyethyl acrylate, polyhydroxymethyl acrylate, polyphenol, polyvinylpyrrolidone, polyacrylamide and polylysine. Examples thereof include synthetic polymer materials such as polyvinylpyrrolidone, polyacrylic acid ester, and polymethacrylic acid ester, various derivatives and cross-linked products thereof, and various copolymers thereof with vinyl monomers such as styrene and methacrylic acid ester.
【００１９】例えば、本発明者らが得た知見では、ポリ
ビニルアルコールゲルは、血液との接触によって免疫抑
制作用の顕著な誘導を示した。このポリビニルアルコー
ルゲルは、ほう酸添加により架橋を導入したり、側鎖に
光感応性の官能基であるスチリルピリジニウム基やスチ
リルキノリニウム基を有する光架橋性ポリビニルアルコ
ールを用いたり、ゲル化剤と称する種々の有機及び無機
化合物を添加したり、該水溶液の真空凍結乾燥や凍結融
解の反復操作によって作製することができる。また、本
発明者らが得た知見では、上記官能基を有するメタクリ
ル酸エステルモノマーを重合及び、各種のビニル・モノ
マーなどと共重合することによって水酸基を導入した修
飾体や、アルキル基、フェニル基を導入した材料でも、
免疫抑制作用の顕著な誘導が確認された。For example, according to the findings obtained by the present inventors, polyvinyl alcohol gel showed a remarkable induction of immunosuppressive action by contact with blood. This polyvinyl alcohol gel introduces crosslinking by adding boric acid, or uses a photocrosslinkable polyvinyl alcohol having a styrylpyridinium group or a styrylquinolinium group which is a photosensitive functional group in a side chain, or as a gelling agent. It can be prepared by adding various organic and inorganic compounds to be referred to, or by repeating the freeze-drying or freeze-thawing of the aqueous solution. In addition, the findings obtained by the present inventors are that a methacrylic acid ester monomer having the above functional group is polymerized and modified with a hydroxyl group introduced by copolymerization with various vinyl monomers, an alkyl group or a phenyl group. With the material that introduced
A remarkable induction of immunosuppressive action was confirmed.
【００２０】また、請求項２の発明で用いられるカチオ
ン性官能基を有する高分子材料は、１、２、３、４級ア
ミノ基、スルホニウム基、ホスホニウム基等を有する高
分子材料であり、例えば、ポリビニルピリジン及びその
塩、イオネンポリマー、Ｎ−トリアルキルアミノメチル
ポリスチレン、アミノアセタール化ポリビニルアルコー
ル、ポリビニルイミダゾール、ポリエチレンイミン、ポ
リジアルキルジアリルアンモニウム塩、ポリジアルキル
ジアリルアンモニウム塩−ＳＯ2 共重合体、ポリビニル
ベンジルスルホニウム塩、ポリビニルベンジルホスホニ
ウム塩等が挙げられる。Further, the polymeric material having a cationic functional group used in the invention of claim 2 is a polymeric material having a 1, 2, 3, quaternary amino group, sulfonium group, phosphonium group, etc. , Polyvinylpyridine and its salts, ionene polymer, N-trialkylaminomethylpolystyrene, aminoacetalized polyvinyl alcohol, polyvinylimidazole, polyethyleneimine, polydialkyldiallylammonium salt, polydialkyldiallylammonium salt-SO 2 copolymer, polyvinyl Examples thereof include benzylsulfonium salt and polyvinylbenzylphosphonium salt.
【００２１】上記カチオン性官能基を有する高分子材料
は、天然多糖類及びポリスチレン等の合成高分子に種々
の化学修飾方法を用いて、上記カチオン性官能基を導入
したり、これらの官能基を有するビニルモノマー間の共
重合や架橋反応により得ることができる。The polymeric material having the above-mentioned cationic functional group is introduced into the synthetic polymer such as natural polysaccharides and polystyrene by various chemical modification methods, or the above-mentioned cationic functional group is introduced into the polymer. It can be obtained by copolymerization or cross-linking reaction between the vinyl monomers.
【００２２】例えば、スチレンとジビニルベンゼンを共
重合し、Ｆｒｉｅｄｉｌ−Ｃｒａｆｔｓ反応を介して、
クロロメチル基をベンゼン核に導入し、クロロメチル基
をアミンで処理することによってアミノ化し、しかる後
にアルキル置換を行うことにより、ポリスチレンにカチ
オン性官能基を導入することができる。For example, styrene and divinylbenzene are copolymerized, and Friedil-Crafts reaction is carried out to
A cationic functional group can be introduced into polystyrene by introducing a chloromethyl group into the benzene nucleus, amination of the chloromethyl group by treatment with an amine, and subsequent alkyl substitution.
【００２３】また、他の方法として、例えば、エピクロ
ルヒドリンのような分子内にクロルメチル基とオキシラ
ン環とを有する化合物にイミダゾール類を反応させ、変
性イミダゾールを合成し、これを多官能性エポキシ化合
物で樹脂化することによっても得ることができる。ま
た、他にもカチオン性官能基を有する種々の合成法が考
えられるが、本発明はその方法によって限定されるもの
ではない。As another method, for example, a compound having a chloromethyl group and an oxirane ring in the molecule such as epichlorohydrin is reacted with an imidazole to synthesize a modified imidazole, which is then treated with a resin having a polyfunctional epoxy compound. It can also be obtained by converting. In addition, various synthetic methods having a cationic functional group are conceivable, but the present invention is not limited to these methods.
【００２４】本発明者らが得た知見では、カチオン性基
として、４級アンモニウムのトリアルキル置換窒素原子
をもつトリメチルアンモニウム基やジアルキルエタノー
ルであるジメチルエタノールアンモニウム基を導入した
ポリスチレン修飾体は、未修飾のポリスチレンやアニオ
ン性基で修飾したポリスチレンからなる材料に比較し
て、著しく大きな免疫抑制作用の誘導を示した。また、
カチオン性基として、１級または２級アミノ基をもつも
のや３級アミノ基をもつものでも、免疫抑制作用の大き
な誘導が確認された。一方、アニオン性基であるスルホ
ン酸基を導入したポリスチレン誘導体では、ほとんど免
疫抑制作用の誘導が見られなかった。According to the knowledge obtained by the present inventors, a polystyrene modified product in which a trimethylammonium group having a quaternary ammonium trialkyl-substituted nitrogen atom or a dimethylethanolammonium group which is a dialkylethanol is introduced as a cationic group is not known. Compared with the material composed of modified polystyrene or polystyrene modified with anionic group, it showed significantly greater induction of immunosuppressive effect. Also,
A large induction of immunosuppressive action was confirmed even with a cationic group having a primary or secondary amino group or a tertiary amino group. On the other hand, almost no induction of immunosuppressive action was observed with the polystyrene derivative having a sulfonic acid group as an anionic group introduced therein.
【００２５】また、ジメチルエタノールアンモニウム
基、ジエチルアミノ基、末端に１級アミノ基のようなカ
チオン性基を有するメタクリル酸エステルモノマーの共
重合により得られたポリメタクリル酸エステル系材料
は、顕著な免疫抑制作用の誘導を示した。一方、アニオ
ン性基であるカルボキシル基を有するメタクリル酸エス
テル系材料では、ほとんど免疫抑制作用の誘導が見られ
なかった。Further, the polymethacrylic acid ester-based material obtained by the copolymerization of the methacrylic acid ester monomer having a cationic group such as a dimethylethanol ammonium group, a diethylamino group and a primary amino group at the terminal is remarkable for immunosuppression. Induction of action was shown. On the other hand, in the case of the methacrylic acid ester-based material having a carboxyl group which is an anionic group, almost no induction of immunosuppressive action was observed.
【００２６】また、キチン及びそのＮ−脱アセチル化物
であるキトサンやキトサンのアミノ基に１、２、３、４
級アミンを導入したキトサン誘導体、ジエチルアミノエ
チル（ＤＥＡＥ）基等のイオン交換基をエーテル結合さ
せたアガロース誘導体、４級アミン等で修飾したアガロ
ース誘導体は、上述したように、特に高い免疫抑制作用
の誘導を示した。In addition, chitin and its N-deacetylated product, chitosan, and amino groups of chitosan have 1, 2, 3, 4
As described above, a chitosan derivative having a primary amine introduced thereinto, an agarose derivative having an ether-exchanged ion-exchange group such as a diethylaminoethyl (DEAE) group, and an agarose derivative modified with a quaternary amine induce a particularly high immunosuppressive action. showed that.
【００２７】請求項１，２の発明において用いられる種
々の免疫抑制作用誘導材料の形状は、粒子状、繊維状、
中空糸状、膜状等のいずれの公知の形状のものであって
もよい。例えば、スチレンモノマーにジビニルベンゼン
とラジカル開始剤としてのベンゾイルパーオキサイドを
加えて、水中で６０℃、５時間、懸濁攪拌を続けるとス
チレン−ジビニルベンゼン共重合体の球状粒子を容易に
得ることができる。粒子径は攪拌速度、水中に加える安
定剤の種類と濃度、モノマーと水の容量比などで制御で
きる。また、希釈剤とモノマーの混液の懸濁重合を行う
ことで、多孔質化を行うことも容易である。The various immunosuppressive action-inducing materials used in the first and second aspects of the present invention are particles, fibers,
It may have any known shape such as a hollow fiber shape or a film shape. For example, spherical particles of a styrene-divinylbenzene copolymer can be easily obtained by adding divinylbenzene and benzoyl peroxide as a radical initiator to a styrene monomer and continuing suspension stirring at 60 ° C. for 5 hours in water. it can. The particle size can be controlled by the stirring speed, the type and concentration of the stabilizer added to water, the volume ratio of the monomer to water, and the like. Further, it is also easy to make it porous by carrying out suspension polymerization of a mixed liquid of a diluent and a monomer.
【００２８】また、各種メタクリル酸エステルモノマー
の重合をモノマーに対する良溶媒であり、かつポリマー
に対して貧溶媒中で懸濁重合を行うと、ポリメタクリル
酸エステルの粒状粒子を容易に得ることができる。粒子
径は攪拌速度、添加する安定剤の種類と濃度などで制御
することができる。このようにして合成した粒状粒子
に、上記したような化学修飾反応を行うことにより、種
々の免疫抑制作用誘導材料が得られる。Further, when the polymerization of various methacrylic acid ester monomers is a good solvent for the monomers and the suspension polymerization is carried out for the polymer in a poor solvent, granular particles of polymethacrylic acid ester can be easily obtained. . The particle size can be controlled by the stirring speed, the type and concentration of the stabilizer added, and the like. By subjecting the thus-synthesized granular particles to the chemical modification reaction as described above, various immunosuppressive action induction materials can be obtained.
【００２９】本発明において、血液と上記材料とを接触
させる方法については、血液と上記材料が十分に接触さ
れ得る限り、任意の方法を用いることができる。例え
ば、繊維状の上記材料をカラムに充填し、該カラムに血
液を循環させる方法や、粒径５０μｍ〜５ｍｍのビーズ
状の材料をカラムに充填し、血液を循環させる方法を用
いることができる。さらに、適当な容器に採取した血液
中に種々の形状の上記材料を懸濁浮遊させることによ
り、血液と上記材料を接触させてもよい。また、容器内
壁自体を上記表面粗さを有するように加工し、この容器
内で血液を振盪してもよい。In the present invention, any method can be used as a method of bringing blood into contact with the above material as long as blood can be brought into sufficient contact with the above material. For example, a method in which the above fibrous material is packed in a column and blood is circulated in the column, or a method in which a bead-shaped material having a particle size of 50 μm to 5 mm is packed in the column and blood is circulated can be used. Further, the blood may be brought into contact with the material by suspending and suspending the material having various shapes in the blood collected in an appropriate container. Alternatively, the inner wall of the container itself may be processed to have the above-mentioned surface roughness, and the blood may be shaken in the container.
【００３０】本発明による免疫抑制作用を有する血漿も
しくは血清の製造装置としては、血液の導出入口を有
し、容器内表壁が上記表面粗さを有する容器、もしく
は、上記特定の表面粗さの材料を充填した容器を備える
ものであれば、いかなる装置も用いることができる。例
えば、市販の体外循環システムや血液バッグ等の上記操
作過程を可能にする装置であれば任意のものを用いるこ
とができる。The apparatus for producing plasma or serum having an immunosuppressive effect according to the present invention has a blood inlet / outlet and a container inner surface having the above surface roughness, or a container having the above specific surface roughness. Any device can be used as long as it has a container filled with material. For example, a commercially available extracorporeal circulation system, a blood bag, or the like can be used as long as it is a device that enables the above operation process.
【００３１】[0031]
【作用】請求項１，２に記載の発明では、水酸基、アミ
ド基及びエステル基の少なくとも１つを有する高分子材
料やカチオン性官能基を有する高分子材料が血液と接触
されるため、後述の実施例から明らかなように、上記高
分子材料の性質によるためか、血液中の免疫抑制作用を
産生する細胞と上記高分子材料との相互作用が促進さ
れ、あるいは上記高分子材料と何らかの因子との相互作
用により誘導された別の因子により、免疫抑制作用が効
率良く誘導されるものと考えられる。In the invention described in claims 1 and 2, since the polymer material having at least one of the hydroxyl group, the amide group and the ester group and the polymer material having the cationic functional group are brought into contact with blood, As is clear from the examples, the interaction between the cells producing the immunosuppressive action in blood and the polymeric material is promoted, probably because of the properties of the polymeric material, or the polymeric material and some factor It is considered that the immunosuppressive action is efficiently induced by another factor induced by the interaction of
【００３２】[0032]
【実施例】以下、本発明の非限定的な実施例及び比較例
を挙げることにより、本発明を詳細に説明する。The present invention will now be described in detail by giving non-limiting examples and comparative examples of the present invention.
【００３３】まず、後述の実施例及び比較例の評価方法
を説明する。 〔評価方法−血漿中の免疫抑制活性の測定〕健常人から
末梢血をヘパリン添加にて採血し、Ｆｉｃｏｌｌ／Ｐａ
ｑｕｅ（Ｐｈａｒｍａｃｉａ社製）比重分離法にて、末
梢血単核球を分離した。この末梢血単核球を２×１０5
細胞／１００μｌ／ｗｅｌｌになるようにマイクロプレ
ートに巻き込んだ。Ｐｏｋｅｗｅｅｄ Ｍｙｔｏｇｅｎ
（Ｓｉｇｍａ社製）（最終濃度２μｇ／ｍｌ）と血漿サ
ンプル（最終濃度１０重量％）を、最終２００μｌにな
るように上記マイクロプレートに添加した。５％Ｃ
Ｏ2 、３７℃で７２時間培養後、培養終了の４時間前に
１μＣｉ／ｗｅｌｌのトリチウムサイミジン（3 Ｈ−Ｔ
ｄＲ）を添加し、その取り込みを液体シンチレーション
カウンターにて測定した。測定の結果は、平均値±標準
偏差で表した。また、対照の血漿非添加群の測定値は、
１２５，０００ｄ．ｐ．ｍであった。抑制率は次の式か
ら計算した。First, the evaluation methods of Examples and Comparative Examples described later will be described. [Evaluation Method-Measurement of Immunosuppressive Activity in Plasma] Peripheral blood was collected from a healthy person by adding heparin, and Ficoll / Pa
Peripheral blood mononuclear cells were separated by que (Pharmacia) specific gravity separation method. This peripheral blood mononuclear cell is 2 × 10 5
The cells / 100 μl / well were rolled up into a microplate. Pokeweed Mytogen
(Manufactured by Sigma) (final concentration 2 μg / ml) and plasma sample (final concentration 10% by weight) were added to the above microplate so that the final concentration was 200 μl. 5% C
After culturing for 72 hours at 37 ° C. in O 2 , 4 hours before the end of culturing, 1 μCi / well of tritium thymidine ( 3 HT
dR) was added, and its uptake was measured by a liquid scintillation counter. The result of the measurement is represented by the average value ± standard deviation. In addition, the measured values of the control plasma-free group are
125,000 d. p. It was m. The inhibition rate was calculated from the following formula.
【００３４】[0034]
【数１】 [Equation 1]
【００３５】〔キトサンまたはキトサン誘導体による免
疫抑制作用の誘導〕実施例１ キトサンのアミノ基がそのまま残っているキトサンゲル
粒子Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３
（富士紡績社製）を１５ｍｌ用ポリプロピレンチューブ
（岩城硝子社製）に、かさ体積で１ｍｌ入れた。これに
注射用生理食塩水（大塚製薬社製）を１２ｍｌ添加して
軽く攪拌した。５００ｒｐｍで５分間遠心し、上澄みを
吸引して捨て、同様に注射用生理食塩水（大塚製薬社
製）を１２ｍｌ加えて攪拌し、遠心して上澄みを吸引し
て捨てた。この洗浄操作を３回行い、一晩４℃にて放置
した。その後、同じ洗浄操作を５回行い、最後にできる
だけ生理食塩水を取り除いた。[Induction of Immunosuppressive Action by Chitosan or Chitosan Derivative] Example 1 Chitosan gel particles Chitosan basic AL-03 in which the amino group of chitosan remains as it is
(Fuji Spinning Co., Ltd.) was placed in a polypropylene tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 1 ml. To this, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred. The mixture was centrifuged at 500 rpm for 5 minutes, and the supernatant was aspirated and discarded. Similarly, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and stirred, and the mixture was centrifuged and the supernatant was aspirated and discarded. This washing operation was performed 3 times and left overnight at 4 ° C. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.
【００３６】次に、注射用生理食塩水を充填して滅菌洗
浄した２ｍｌ用チューブ（Ｅｐｐｅｎｄｏｒｆ社製）
に、上記のようにして洗浄されたキトサンゲル粒子をか
さ体積で５００μｌ充填した。Next, a 2 ml tube (manufactured by Eppendorf) that was filled with physiological saline for injection and sterilized and washed.
Then, 500 μl of the bulk volume of the chitosan gel particles washed as described above was filled.
【００３７】実施例２ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンのアミノ基がアセチル化されたＣｈｉｔｏｐｅａ
ｒｌ ｂａｓｉｃ ＢＬ−０３（富士紡績社製）に変更
した以外はすべて実施例１と同様にして行った。 Example 2 Chitopearl basic AL-03 was prepared by using Chitopea in which the amino group of chitosan was acetylated.
rl basic BL-03 (manufactured by Fuji Spinning Co., Ltd.) was used, and the same procedure as in Example 1 was performed.
【００３８】実施例３ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンのアミノ基に芳香族アルキル基を介して１級アミ
ンが導入されたＣｈｉｔｏｐｅａｒｌ ＢＣＷ−３５０
３（富士紡績社製）に変更した以外はすべて実施例１と
同様にして行った。 Example 3 Chitopearl basic AL-03 was prepared by introducing a primary amine into the amino group of chitosan via an aromatic alkyl group.
The same procedure as in Example 1 was performed except that the number was changed to 3 (manufactured by Fuji Spinning Co., Ltd.).
【００３９】実施例４ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンのアミノ基に直鎖アルキル基を介して１級アミン
が導入されたＣｈｉｔｏｐｅａｒｌ ＢＣＷ−３００３
（富士紡績社製）に変更した以外はすべて実施例１と同
様にして行った。 Example 4 Chitopearl basic AL-03 was prepared by introducing a primary amine into the amino group of chitosan via a linear alkyl group to obtain Chitopearl BCW-3003.
The same procedure as in Example 1 was carried out except that the product was changed to (Fuji Spinning Co., Ltd.).
【００４０】実施例５ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンのアミノ基に３級アミンが導入されたＣｈｉｔｏ
ｐｅａｒｌ ＢＣＷ−２６０３（富士紡績社製）に変更
した以外はすべて実施例１と同様にして行った。 Example 5 Chitopearl basic AL-03 was prepared using Chitosan in which a tertiary amine was introduced into the amino group of chitosan.
The same procedure as in Example 1 was carried out except that the weight was changed to Pearl BCW-2603 (manufactured by Fuji Spinning Co., Ltd.).
【００４１】実施例６ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンのアミノ基に４級アミンが導入されたＣｈｉｔｏ
ｐｅａｒｌ ＢＣＷ−２５０３（富士紡績社製）に変更
した以外はすべて実施例１と同様にして行った。 Example 6 Chitopearl basic AL-03 was prepared by introducing Chitosan in which a quaternary amine was introduced into the amino group of chitosan.
All were performed in the same manner as in Example 1 except that the color was changed to Pearl BCW-2503 (manufactured by Fuji Spinning Co., Ltd.).
【００４２】実施例７ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンの６位の水酸基にカルボキシメチル基が導入され
たＣｈｉｔｏｐｅａｒｌ ＣＭ−０３（富士紡績社製）
に変更した以外はすべて実施例１と同様にして行った。 Example 7 Chitopearl basic AL-03 was prepared by using Chitopearl CM-03 (manufactured by Fuji Spinning Co.) in which a carboxymethyl group was introduced into the hydroxyl group at the 6-position of chitosan.
The procedure of Example 1 was repeated except that
【００４３】実施例８ Ｃｈｉｔｏｐｅａｒｌ ｂａｓｉｃ ＡＬ−０３を、キ
トサンの６位の水酸基にスルホン基が導入されたＣｈｉ
ｔｏｐｅａｒｌ ＳＵ−０３（富士紡績社製）に変更し
た以外はすべて実施例１と同様にして行った。 Example 8 Chitopearl basic AL-03 was prepared by introducing a sulfo group into the hydroxyl group at the 6-position of chitosan.
All were performed in the same manner as in Example 1 except for changing to topear SU-03 (manufactured by Fuji Spinning Co., Ltd.).
【００４４】実施例９ ＣＨＩＴＯＳＡＮ １０Ｂ（ヒゲタ醤油社製）粉末５ｇ
を注射用生理食塩水（大塚製薬社製）３０ｍｌに懸濁
し、高速遠心し、上澄みを吸引して捨てた。この操作を
３回行い、一晩４℃にて放置した。その後、同じ洗浄操
作を５回行い、最後にできるだけ生理食塩水を取り除い
た。注射用生理食塩水を充填して、滅菌洗浄した２ｍｌ
用チューブ（Ｅｐｐｅｎｄｏｒｆ社製）に、上記のよう
にして洗浄されたキトサンゲル粉末をかさ体積で５００
μｌ充填した。 Example 9 5 g of powder of CHITOSAN 10B (manufactured by Higeta Shoyu Co., Ltd.)
Was suspended in 30 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), centrifuged at high speed, and the supernatant was aspirated and discarded. This operation was repeated 3 times and left overnight at 4 ° C. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible. 2 ml that was filled with physiological saline for injection and sterilized and washed
In a tube (made by Eppendorf), the chitosan gel powder washed as described above in a bulk volume of 500
μl was filled.
【００４５】実施例１〜９の各ビーズを充填した各チュ
ーブにヘパリン採血した健常人新鮮血１．６ｍｌを加え
て回転円盤に取り付けて、３７℃にて２時間、回転数２
６ｒｐｍで転倒混和した。To each tube filled with each bead of Examples 1 to 9, 1.6 ml of heparinized fresh blood of a healthy person was added and attached to a rotating disk, and the rotation speed was 2 at 37 ° C. for 2 hours.
Mix by inverting at 6 rpm.
【００４６】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を前述の方法に従って測定し
た。また、採血直後の血液を遠心分離して血漿を採取し
て、同様に免疫抑制活性を測定した。結果を表１に示
す。The mixed blood was centrifuged to collect plasma, and the immunosuppressive activity in the plasma was measured according to the method described above. Immediately after blood collection, blood was centrifuged to collect plasma, and immunosuppressive activity was similarly measured. The results are shown in Table 1.
【００４７】[0047]
【表１】 [Table 1]
【００４８】〔アガロース及びアガロース誘導体による
免疫抑制作用の誘導〕実施例１０ アガロース（ナカライ化学社製、電気泳動用特製試薬
ＧＰ−３６）を５重量％濃度で蒸留水に溶解させ、１２
１℃で２０分間オートクレーブを行った。この溶液を６
０℃に保温しておき、冷蒸留水中にマイクロシリンジを
用いて滴下して、アガロースゲルビーズ（粒径５ｍｍ）
を作製した。[Induction of Immunosuppressive Action by Agarose and Agarose Derivatives] Example 10 Agarose (Nakarai Chemical Co., Ltd., special reagent for electrophoresis)
GP-36) was dissolved in distilled water at a concentration of 5% by weight, and
Autoclave was performed at 1 ° C for 20 minutes. 6 this solution
Keep the temperature at 0 ℃ and drop it in cold distilled water using a microsyringe to agarose gel beads (particle size 5mm).
Was produced.
【００４９】このビーズを注射用生理食塩水（大塚製薬
社製）で洗浄後、同じく注射用生理食塩水で洗浄した２
ｍｌ用チューブ（Ｅｐｐｅｎｄｏｒｆ社製）に充填し
た。充填量はアガロースゲルビーズ２０個とした。The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection.
It was filled in a ml tube (manufactured by Eppendorf). The filling amount was 20 agarose gel beads.
【００５０】実施例１１ 約２重量％濃度のアガロースよりなる、Ｓｅｐｈａｒｏ
ｓｅ ２Ｂ（Ｐｈａｒｍａｃｉａ ＬＫＢ Ｂｉｏｔｅ
ｃｈｎｏｌｏｇｙ社製）の懸濁液３ｍｌを１５ｍｌポリ
プロピレンチューブ（岩城硝子社製）に入れた。これを
１０００ｒｐｍで５分間遠心して、上澄みを吸引して捨
て、注射用生理食塩水（大塚製薬社製）を１２ｍｌ加え
て攪拌し、同様に遠心して上澄みを吸引して捨てた。こ
の洗浄操作を３回行い、一晩４℃にて放置した。 Example 11 Sepharo consisting of agarose in a concentration of about 2% by weight
se 2B (Pharmacia LKB Biote
3 ml of a suspension of chnology) was placed in a 15 ml polypropylene tube (manufactured by Iwaki Glass). This was centrifuged at 1000 rpm for 5 minutes, and the supernatant was aspirated and discarded, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and stirred, and similarly centrifuged, the supernatant was aspirated and discarded. This washing operation was performed 3 times and left overnight at 4 ° C.
【００５１】このゲル担体５００μｌ（かさ体積）を実
施例１と同様に、２ｍｌ用ポリプロピレンチューブ（Ｅ
ｐｐｅｎｄｏｒｆ社製）に入れて、注射用生理食塩水に
て洗浄した。500 μl (bulk volume) of this gel carrier was added to a polypropylene tube for 2 ml (E) in the same manner as in Example 1.
(made by Ppendorf) and washed with physiological saline for injection.
【００５２】実施例１２ Ｓｅｐｈａｒｏｓｅ ２Ｂに代えて、約６重量％濃度の
架橋型アガロースよりなる、Ｓｅｐｈａｒｏｓｅ ＣＬ
−６Ｂ（Ｐｈａｒｍａｃｉａ ＬＢＫ Ｂｉｏｔｅｃｈ
ｎｏｌｏｇｙ社製）を用いて、実施例１１と同様に操作
した。 Example 12 Instead of Sepharose 2B, Sepharose CL consisting of about 6% by weight of cross-linked agarose.
-6B (Pharmacia LBK Biotech
The operation was performed in the same manner as in Example 11 using the product Noology).
【００５３】実施例１３ Ｓｅｐｈａｒｏｓｅ ２Ｂに代えて、約６重量％濃度の
架橋型アガロースにジエチルアミノエチル（ＤＥＡＥ）
基をエーテル結合で導入した、ＤＥＡＥ Ｓｅｐｈａｒ
ｏｓｅ ＣＬ−６Ｂ（Ｐｈａｒｍａｃｉａ ＬＢＫ Ｂ
ｉｏｔｅｃｈｎｏｌｏｇｙ社製）を用いて、実施例１１
と同様に操作した。 Example 13 Instead of Sepharose 2B, about 6% by weight of cross-linked agarose was added to diethylaminoethyl (DEAE).
DEAE Sephare introduced with ether bond
ose CL-6B (Pharmacia LBK B
Example 11 using iotechnology)
The same operation was performed.
【００５４】実施例１４ Ｓｅｐｈａｒｏｓｅ ２Ｂに代えて、約４重量％濃度の
架橋型アガロースにエーテル結合を介してＰｈｅｎｙｌ
基を導入した、Ｐｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ Ｃ
Ｌ−４Ｂ（Ｐｈａｒｍａｃｉａ ＬＢＫ Ｂｉｏｔｅｃ
ｈｎｏｌｏｇｙ社製）を用いて、実施例１１と同様に操
作した。 Example 14 Instead of Sepharose 2B, Phenyl was added to cross-linked agarose at a concentration of about 4% by weight through an ether bond.
Phenyl Sepharose C introduced with a group
L-4B (Pharmacia LBK Biotec
Hnology) was used and the same operation as in Example 11 was performed.
【００５５】実施例１５ Ｓｅｐｈａｒｏｓｅ ２Ｂに代えて、約６重量％濃度の
架橋型アガロースに４級アミンを導入した、Ｑ Ｓｅｐ
ｈａｒｏｓｅ ＦＦ（Ｐｈａｒｍａｃｉａ ＬＢＫ Ｂ
ｉｏｔｅｃｈｎｏｌｏｇｙ社製）を用いて、実施例１１
と同様に操作した。 Example 15 Q Sep, in which a quaternary amine was introduced into a cross-linked agarose having a concentration of about 6% by weight, in place of Sepharose 2B.
harose FF (Pharmacia LBK B
Example 11 using iotechnology)
The same operation was performed.
【００５６】実施例１０〜１５の各ビーズを充填した各
チューブにヘパリン採血した健常人新鮮血１．６ｍｌを
加えて回転円盤に取り付けて、３７℃にて２時間、回転
数２６ｒｐｍで転倒混和した。To each tube filled with each bead of Examples 10-15, 1.6 ml of heparinized fresh blood of a healthy person was added and attached to a rotating disk, and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm. .
【００５７】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を前述の方法にて測定した。
また、採血直後の血液を遠心分離して血漿を採取して、
同様に免疫抑制活性を測定した。結果を表２に示す。The mixed blood was centrifuged to collect plasma, and the immunosuppressive activity in plasma was measured by the above-mentioned method.
In addition, the blood immediately after blood collection is centrifuged to collect plasma,
Similarly, immunosuppressive activity was measured. The results are shown in Table 2.
【００５８】[0058]
【表２】 [Table 2]
【００５９】〔アルギン酸ゲルによる免疫抑制作用の誘
導〕実施例１６ ２重量％のアルギン酸ナトリウム（ＡＬ−１、中粘度タ
イプ、新田ゼラチン社製）を生理食塩水中に懸濁して、
オートクレーブにより１２１℃、２０分で処理すること
で、加熱滅菌と同時にアルギン酸ナトリウムを溶解させ
た。これを滅菌済み１．５重量％塩化カルシウム溶液中
へ滴下してゲル化させることにより、粒径約２．５ｍｍ
のアルギン酸カルシウムのゲルビーズを作製した。[Induction of Immunosuppressive Action by Alginate Gel] Example 16 2% by weight of sodium alginate (AL-1, medium viscosity type, manufactured by Nitta Gelatin Co., Ltd.) was suspended in physiological saline.
By treating with an autoclave at 121 ° C. for 20 minutes, sodium alginate was dissolved simultaneously with heat sterilization. Particle size of about 2.5 mm by dropping this into sterilized 1.5 wt% calcium chloride solution and gelling.
Calcium alginate gel beads were prepared.
【００６０】得られたゲルビーズを１５ｍｌ用ポリプロ
ピレンチューブ（岩城硝子社製）に、かさ体積で１ｍｌ
入れた。これに注射用生理食塩水（大塚製薬社製）を１
２ｍｌ添加して軽く攪拌した。５００ｒｐｍで１分間遠
心し、上澄みを吸引して捨て、同様に注射用生理食塩水
（大塚製薬社製）を１２ｍｌ加えて攪拌し、遠心して上
澄みを吸引して捨てた。この洗浄操作を３回行い、一晩
４℃にて放置した。その後、同じ洗浄操作を５回行い、
最後にできるだけ生理食塩水を取り除いた。The gel beads obtained were placed in a polypropylene tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 1 ml.
I put it in. Add saline for injection (Otsuka Pharmaceutical Co., Ltd.) to this 1
2 ml was added and stirred gently. The mixture was centrifuged at 500 rpm for 1 minute, and the supernatant was aspirated and discarded. Similarly, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and stirred, and the mixture was centrifuged and the supernatant was aspirated and discarded. This washing operation was performed 3 times and left overnight at 4 ° C. After that, perform the same washing operation 5 times,
Finally, the saline solution was removed as much as possible.
【００６１】注射用生理食塩水を充填して滅菌洗浄した
２ｍｌ用チューブ（Ｅｐｐｅｎｄｏｒｆ社製）に、上記
のようにして洗浄されたゲルビーズを７０個充填した。A 2 ml tube (manufactured by Eppendorf) that had been sterilized and filled with physiological saline for injection was filled with 70 gel beads washed as described above.
【００６２】実施例１７ ２重量％のアルギン酸ナトリウムを５重量％のアルギン
酸ナトリウムに変更した以外はすべて実施例１６と同様
にして行った。 Example 17 The procedure of Example 16 was repeated except that 2% by weight of sodium alginate was changed to 5% by weight of sodium alginate.
【００６３】実施例１８ ２重量％のアルギン酸ナトリウムを１０重量％のアルギ
ン酸ナトリウムに変更した以外はすべて実施例１６と同
様にして行った。 Example 18 The procedure of Example 16 was repeated except that 2% by weight of sodium alginate was changed to 10% by weight of sodium alginate.
【００６４】実施例１９ １．５重量％の塩化カルシウム溶液を３重量％の塩化カ
ルシウム溶液に変更した以外はすべて実施例１６と同様
にして行った。 Example 19 The procedure of Example 16 was repeated except that the 1.5 wt% calcium chloride solution was changed to a 3 wt% calcium chloride solution.
【００６５】実施例２０ １．５重量％の塩化カルシウム溶液を１．５重量％の塩
化バリウム溶液に変更した以外はすべて実施例１６と同
様にして行った。 Example 20 The procedure of Example 16 was repeated except that the 1.5 wt% calcium chloride solution was changed to a 1.5 wt% barium chloride solution.
【００６６】実施例２１ １．５重量％の塩化カルシウム溶液を１．５重量％の塩
化バリウム溶液に変更し、５重量％のアルギン酸ナトリ
ウムを３重量％のアルギン酸ナトリウムに変更した以外
はすべて実施例１６と同様にして行った。 Example 21 All examples except that the 1.5 wt% calcium chloride solution was changed to a 1.5 wt% barium chloride solution and the 5 wt% sodium alginate was changed to 3 wt% sodium alginate. It carried out similarly to 16.
【００６７】実施例２２ アルギン酸ナトリウムをアルギン酸ナトリウム（１００
〜１５０ｃｐｓ、和光純薬社製）に変更した以外はすべ
て実施例１６と同様にして行った。 Example 22 Sodium alginate was replaced with sodium alginate (100
.About.150 cps, manufactured by Wako Pure Chemical Industries, Ltd.).
【００６８】実施例２３ アルギン酸ナトリウムをアルギン酸ナトリウム（３００
〜４００ｃｐｓ、和光純薬社製）に変更した以外はすべ
て実施例１６と同様にして行った。 Example 23 Sodium alginate was replaced with sodium alginate (300
.About.400 cps, manufactured by Wako Pure Chemical Industries, Ltd.).
【００６９】実施例２４ アルギン酸ナトリウムをアルギン酸ナトリウム（５００
〜６００ｃｐｓ、和光純薬社製）に変更した以外はすべ
て実施例１６と同様にして行った。 Example 24 Sodium alginate was replaced with sodium alginate (500
.About.600 cps, manufactured by Wako Pure Chemical Industries, Ltd.).
【００７０】実施例１６〜２４の各ビーズを充填した各
チューブにヘパリン採血した健常人新鮮血１．６ｍｌを
加えて回転円盤に取り付けて、３７℃にて２時間、回転
数２６ｒｐｍで転倒混和した。To each tube filled with each bead of Examples 16 to 24, 1.6 ml of fresh blood of a healthy person from which heparin was collected was added and attached to a rotating disk, and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm. .
【００７１】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を測定した。また、採血直後
の血液を遠心分離して血漿を採取して、同様に免疫抑制
活性を測定した。結果を表３に示す。The blood after mixing was centrifuged to collect plasma, and the immunosuppressive activity in the plasma was measured. Immediately after blood collection, blood was centrifuged to collect plasma, and immunosuppressive activity was similarly measured. The results are shown in Table 3.
【００７２】[0072]
【表３】 [Table 3]
【００７３】〔ポリビニルアルコールゲルによる免疫抑
制作用の誘導〕実施例２５ 重合度；約１４００、けん化度；９９モル％のポリビニ
ルアルコール（キシダ化学社製）１０重量％の水溶液を
調製した。この水溶液はＦｅ3+を、Ｒ＝〔ポリビニルア
ルコールモノマー単位〕／〔金属イオン〕を定義して、
Ｒ＝１０となるように含んでいる。この粘ちょう溶液を
滅菌済み１ＭのＮａＯＨ水溶液中にシリンジを用いて滴
下して、約３０分放置した。これによって、粒径約２．
５ｍｍのポリビニルアルコールゲルビーズを作製した
〔横井弘：ＰＶＡ及びＰＡＡの錯体ゲル．高分子加工、
Ｖｏｌ．４０、Ｎｏ．１１、１９９１〕。[Induction of Immunosuppressive Action by Polyvinyl Alcohol Gel] Example 25 A 10% by weight aqueous solution of polyvinyl alcohol (Kishida Chemical Co., Ltd.) having a polymerization degree of about 1400 and a saponification degree of 99 mol% was prepared. This aqueous solution defines Fe 3+ as R = [polyvinyl alcohol monomer unit] / [metal ion],
It is included so that R = 10. This viscous solution was dropped into a sterilized 1M NaOH aqueous solution using a syringe and left for about 30 minutes. This results in a particle size of about 2.
5 mm polyvinyl alcohol gel beads were prepared [Yokoi Hiroshi: PVA and PAA complex gel. Polymer processing,
Vol. 40, No. 11, 1991].
【００７４】得られたゲルビーズを１５ｍｌ用ポリプロ
ピレンチューブ（岩城硝子社製）に、かさ体積で１ｍｌ
入れた。これに注射用生理食塩水（大塚製薬社製）を１
２ｍｌ添加して軽く攪拌した。５００ｒｐｍで１分間遠
心し、上澄みを吸引して捨て、同様に注射用生理食塩水
（大塚製薬社製）を１２ｍｌ加えて攪拌し、遠心して上
澄みを吸引して捨てた。この洗浄操作を３回行い、一晩
４℃にて放置した。その後、同じ洗浄操作を５回行い、
最後にできるだけ生理食塩水を取り除いた。The obtained gel beads were placed in a polypropylene tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 1 ml.
I put it in. Add saline for injection (Otsuka Pharmaceutical Co., Ltd.) to this 1
2 ml was added and stirred gently. The mixture was centrifuged at 500 rpm for 1 minute, and the supernatant was aspirated and discarded. Similarly, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and stirred, and the mixture was centrifuged and the supernatant was aspirated and discarded. This washing operation was performed 3 times and left overnight at 4 ° C. After that, perform the same washing operation 5 times,
Finally, the saline solution was removed as much as possible.
【００７５】注射用生理食塩水を充填して滅菌洗浄した
２ｍｌ用チューブ（Ｅｐｐｅｎｄｏｒｆ社製）に、上記
のようにして洗浄されたゲルビーズを７０個充填した。A 2 ml tube (manufactured by Eppendorf) that had been sterilized and filled with physiological saline for injection was filled with 70 gel beads washed as described above.
【００７６】実施例２６ 用いたポリビニルアルコールを、重合度；約２０００、
けん化度；９８．５〜９９．４モル％のポリビニルアル
コール（キシダ化学社製）に変更した以外はすべて実施
例２５と同様にして行った。The polyvinyl alcohol used in Example 26 had a degree of polymerization of about 2000,
The saponification degree was the same as in Example 25 except that polyvinyl alcohol (manufactured by Kishida Chemical Co., Ltd.) having a degree of saponification of 98.5 to 99.4 mol% was used.
【００７７】実施例２７ 用いたポリビニルアルコールの濃度を１５重量％に変更
した以外はすべて実施例２５と同様にして行った。 Example 27 The procedure of Example 25 was repeated except that the concentration of polyvinyl alcohol used was changed to 15% by weight.
【００７８】実施例２８ 用いたポリビニルアルコールの濃度を１５重量％に変更
した以外はすべて実施例２６と同様にして行った。 Example 28 The procedure of Example 26 was repeated except that the concentration of polyvinyl alcohol used was changed to 15% by weight.
【００７９】実施例２９ 用いた金属イオン量をＲ＝２０に変更した以外はすべて
実施例２５と同様にして行った。 Example 29 The procedure of Example 25 was repeated except that the amount of metal ions used was changed to R = 20.
【００８０】実施例３０ 用いた金属イオン量をＲ＝２０に変更した以外はすべて
実施例２６と同様にして行った。 Example 30 The same procedure as in Example 26 was carried out except that the amount of metal ions used was changed to R = 20.
【００８１】実施例３１ 用いた金属イオンをＣｕ2+に変更した以外はすべて実施
例２５と同様にして行った。 Example 31 The same procedure as in Example 25 was carried out except that the metal ion used was changed to Cu 2+ .
【００８２】実施例３２ 用いた金属イオンをＣｕ2+に変更した以外はすべて実施
例２６と同様にして行った。 Example 32 The procedure of Example 26 was repeated except that the metal ion used was changed to Cu 2+ .
【００８３】実施例３３ 光架橋性ポリビニルアルコール（重合度；１７００、け
ん化度；８８モル％及びスチリルピリジニウム置換基の
割合；１．３％）を、５重量％となるように注射用生理
食塩水に加え、オートクレーブにより１２１℃、２０分
で処理することで加熱滅菌と同時に光架橋性ポリビニル
アルコールを溶解させ、光架橋性ポリビニルアルコール
溶液を得た。 Example 33 Photocrosslinkable polyvinyl alcohol (degree of polymerization: 1700, degree of saponification: 88 mol% and ratio of styrylpyridinium substituents: 1.3%) was added to physiological saline for injection to be 5% by weight. In addition, the photocrosslinkable polyvinyl alcohol was dissolved simultaneously with heat sterilization by treating it at 121 ° C. for 20 minutes in an autoclave to obtain a photocrosslinkable polyvinyl alcohol solution.
【００８４】得られた溶液を流動パラフィン中に加えて
充分に攪拌し、溶液を懸濁させた。この後に３００Ｗの
ハロゲンランプを装着したスライドプロジェクターを用
いて、３０分間攪拌しながら光を照射することにより、
光架橋性ポリビニルアルコールをゲル化し、粒径２．５
ｍｍのゲルビーズを作製した。このゲルビーズを注射用
生理食塩水を用いて充分に洗浄し、付着していた流動パ
ラフィンを除いた。上記ゲルビーズ作製法以外は実施例
２５と同様にして行った。The obtained solution was added to liquid paraffin and stirred sufficiently to suspend the solution. After that, using a slide projector equipped with a 300 W halogen lamp, by irradiating light while stirring for 30 minutes,
Photo-crosslinkable polyvinyl alcohol gelled, particle size 2.5
mm gel beads were prepared. The gel beads were thoroughly washed with physiological saline for injection to remove the attached liquid paraffin. The same procedure as in Example 25 was carried out except for the above method for producing gel beads.
【００８５】実施例３４ 用いた光架橋性ポリビニルアルコールを５重量％から１
０重量％に変更した以外はすべて実施例３３と同様にし
て行った。 Example 34 The photocrosslinkable polyvinyl alcohol used was from 5% by weight to 1%.
The procedure of Example 33 was repeated except that the amount was changed to 0% by weight.
【００８６】実施例３５ 用いた光架橋性ポリビニルアルコールを５重量％から１
５重量％に変更した以外はすべて実施例３３と同様にし
て行った。 Example 35 The photocrosslinkable polyvinyl alcohol used was 5% by weight to 1%.
The same procedure as in Example 33 was carried out except that the amount was changed to 5% by weight.
【００８７】実施例２５〜３５の各ビーズを充填した各
チューブにヘパリン採血した健常人新鮮血１．６ｍｌを
加えて回転円盤に取り付けて、３７℃にて２時間、回転
数２６ｒｐｍで転倒混和した。To each tube filled with each bead of Examples 25-35, 1.6 ml of heparinized fresh blood of a healthy person was added and attached to a rotating disk, and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm. .
【００８８】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を測定した。また、採血直後
の血液を遠心分離して血漿を採取して、同様に免疫抑制
活性を測定した。結果を表４に示す。The blood after mixing was centrifuged to collect plasma, and the immunosuppressive activity in the plasma was measured. Immediately after blood collection, blood was centrifuged to collect plasma, and immunosuppressive activity was similarly measured. The results are shown in Table 4.
【００８９】[0089]
【表４】 [Table 4]
【００９０】〔ポリスチレン修飾体による免疫抑制作用
の誘導〕実施例３６ ４級アンモニウム基であるトリメチルアンモニウム基を
もつ、スチレン−ジビニルベンゼン共重合体である、Ｄ
ｉａｉｏｎ ＳＡ１１Ａ（三菱化成社製）を５０ｍｌ用
ポリプロピレンチューブ（岩城硝子社製）に、かさ体積
で３ｍｌ入れた。これにメタノール（和光純薬社製 液
体クロマトグラム用グレード）４０ｍｌを添加して、軽
く攪拌した。静置後、上澄みを吸引して除去した。これ
を３回行った後、同様にメタノール４０ｍｌを添加し
て、室温にて一晩静置した。その後、メタノールを吸引
して除き、同様にメタノールを用いて２回洗浄した。こ
れに、滅菌済み蒸留水を４０ｍｌ添加して軽く攪拌し
た。５００ｒｐｍで１分間遠心し、上澄みを吸引して除
き、同様に滅菌済み蒸留水を用いて３回洗浄した。その
後、滅菌済み蒸留水４０ｍｌを加えて、室温にて３時間
静置した。その後、同じ洗浄操作を３回行い、最後にで
きるだけ蒸留水を取り除いた。これに、注射用生理食塩
水（大塚製薬社製）を４０ｍｌ添加して軽く攪拌した。
５００ｒｐｍで１分間遠心し、上澄みを吸引して除き、
同様に注射用生理食塩水（大塚製薬社製）にて３回洗浄
を行い、最後に注射用生理食塩水４０ｍｌを加えて、一
晩室温にて静置した。その後、同じ洗浄操作を５回行
い、最後にできるだけ生理食塩水を取り除いた。[Induction of Immunosuppressive Action by Modified Polystyrene] Example 36 D, which is a styrene-divinylbenzene copolymer having a trimethylammonium group which is a quaternary ammonium group
iaion SA11A (manufactured by Mitsubishi Kasei) was placed in a polypropylene tube for 50 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 3 ml. To this, 40 ml of methanol (liquid chromatogram grade manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently. After standing, the supernatant was removed by suction. After this was repeated 3 times, 40 ml of methanol was added in the same manner, and the mixture was allowed to stand overnight at room temperature. After that, the methanol was removed by suction, and the same washing with methanol was performed twice. To this, 40 ml of sterilized distilled water was added and gently stirred. The mixture was centrifuged at 500 rpm for 1 minute, the supernatant was aspirated and removed, and similarly washed with sterilized distilled water three times. Then, 40 ml of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 3 hours. Then, the same washing operation was performed three times, and finally distilled water was removed as much as possible. To this, 40 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred.
Centrifuge at 500 rpm for 1 minute, remove the supernatant by aspiration,
Similarly, physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was washed three times, and finally 40 ml of physiological saline for injection was added, and the mixture was allowed to stand overnight at room temperature. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.
【００９１】注射用生理食塩水を充填して滅菌洗浄した
２ｍｌ用チューブ（Ｅｐｐｅｎｄｏｒｆ社製）に、上記
のようにして洗浄されたポリスチレン粒子をかさ体積で
５００μｌ充填した。A 2 ml tube (manufactured by Eppendorf) that had been sterilized and filled with physiological saline for injection was filled with 500 μl of the polystyrene particles washed as described above in a bulk volume.
【００９２】実施例３７ 用いたポリスチレン粒子を、４級アンモニウム基である
ジメチルエタノールアンモニウム基をもつ、Ｄｉａｉｏ
ｎ ＳＡ２１Ａ（三菱化成社製）に変更した以外はすべ
て実施例３６と同様にして行った。The polystyrene particles used in Example 37 had a quaternary ammonium group, dimethylethanolammonium group, and Diaio.
n SA21A (manufactured by Mitsubishi Kasei Co., Ltd.) was carried out in the same manner as in Example 36 except that the same was used.
【００９３】実施例３８ 用いたポリスチレン粒子を、１、２級アミノ基〔−ＣＨ
2 ＮＨ（ＣＨ2 ＣＨ2ＮＨ）n Ｈ（ｎ＝１〜３）〕をも
つ、Ｄｉａｉｏｎ ＷＡ２１（三菱化成社製）に変更し
た以外はすべて実施例３６と同様にして行った。The polystyrene particles used in Example 38 were mixed with primary and secondary amino groups [-CH
2 NH (CH 2 CH 2 NH) n H (n = 1 to 3)], and was changed to Diaion WA21 (manufactured by Mitsubishi Kasei Co.).
【００９４】実施例３９ 用いたポリスチレン粒子を、３級アミノ基〔−（Ｃ
Ｈ2 ）n Ｎ（ＣＨ3 ）2 （ｎ＝１〜３）〕をもつ、Ｄｉ
ａｉｏｎ ＷＡ３０（三菱化成社製）に変更した以外は
すべて実施例３６と同様にして行った。The polystyrene particles used in Example 39 were treated with a tertiary amino group [-(C
H 2 ) n N (CH 3 ) 2 (n = 1 to 3)], and
The same procedure as in Example 36 was performed except that the aion WA30 (manufactured by Mitsubishi Kasei Co., Ltd.) was used.
【００９５】比較例１ 用いたポリスチレン粒子を、未修飾のポリスチレン粒子
であるテクポリマー：ＳＢ−１００Ｓ（積水化成品工業
社製）に変更した以外はすべて実施例３６と同様にして
行った。 Comparative Example 1 The same procedure as in Example 36 was carried out except that the polystyrene particles used in Comparative Example 1 were changed to techpolymer: SB-100S (manufactured by Sekisui Plastics Co., Ltd.) which was an unmodified polystyrene particle.
【００９６】比較例２ 用いたポリスチレン粒子をスルホン酸基をもつスチレン
−ジビニルベンゼン共重合体である、Ｄｉａｉｏｎ Ｓ
Ｋ１Ｂ（三菱化成社製）に変更した以外はすべて実施例
３６と同様にして行った。 Comparative Example 2 The polystyrene particles used were Diaion S, which is a styrene-divinylbenzene copolymer having sulfonic acid groups.
The same procedure as in Example 36 was performed except that K1B (manufactured by Mitsubishi Kasei Co., Ltd.) was used.
【００９７】実施例３６〜３９及び比較例１，２の各ビ
ーズを充填した各チューブにヘパリン採血した健常人新
鮮血１．６ｍｌを加えて回転円盤に取り付けて、３７℃
にて２時間、回転数２６ｒｐｍで転倒混和した。To each tube filled with the beads of Examples 36 to 39 and Comparative Examples 1 and 2, 1.6 ml of heparinized fresh blood of a healthy person was added and attached to a rotating disk, and the temperature was set to 37 ° C.
The mixture was mixed by inversion at 26 rpm for 2 hours.
【００９８】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を測定した。また、採血直後
の血液を遠心分離して血漿を採取して、同様に免疫抑制
活性を測定した。結果を表５に示す。The blood after mixing was centrifuged to collect plasma, and the immunosuppressive activity in the plasma was measured. Immediately after blood collection, blood was centrifuged to collect plasma, and immunosuppressive activity was similarly measured. The results are shown in Table 5.
【００９９】[0099]
【表５】 [Table 5]
【０１００】〔ポリメタクリル酸エステル誘導体による
免疫抑制作用の誘導〕実施例４０ ジメチルエタノールアミンで修飾されている、ポリメタ
クリル酸エステル系材料である、ＳＥＰＡＢＥＡＤＳ
ＦＰ−ＱＡ１３（三菱化成社製）を５０ｍｌ用ポリプロ
ピレンチューブ（岩城硝子社製）に、かさ体積で３ｍｌ
入れた。これにメタノール（和光純薬社製 液体クロマ
トグラム用グレード）４０ｍｌを添加して、軽く攪拌し
た。静置後、上澄みを吸引して除去した。これを３回行
った後、同様にメタノール４０ｍｌを添加して、室温に
て一晩静置した。[Induction of Immunosuppressive Action by Polymethacrylate Derivative] Example 40 SEPABEADS, which is a polymethacrylate ester material modified with dimethylethanolamine
FP-QA13 (manufactured by Mitsubishi Kasei) in polypropylene tube for 50 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 3 ml
I put it in. To this, 40 ml of methanol (liquid chromatogram grade manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently. After standing, the supernatant was removed by suction. After this was repeated 3 times, 40 ml of methanol was added in the same manner, and the mixture was allowed to stand overnight at room temperature.
【０１０１】次に、メタノールを吸引して除き、同様に
メタノールを用いて２回洗浄した。これに、滅菌済み蒸
留水を４０ｍｌ添加して軽く攪拌した。５００ｒｐｍで
１分間遠心し、上澄みを吸引して除き、同様に滅菌済み
蒸留水を用いて３回洗浄した。その後、滅菌済み蒸留水
４０ｍｌを加えて、室温にて３時間静置した。その後、
同じ洗浄操作を３回行い、最後にできるだけ蒸留水を取
り除いた。Next, the methanol was removed by suction, and the same washing was performed twice with methanol. To this, 40 ml of sterilized distilled water was added and gently stirred. The mixture was centrifuged at 500 rpm for 1 minute, the supernatant was aspirated and removed, and similarly washed with sterilized distilled water three times. Then, 40 ml of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 3 hours. afterwards,
The same washing operation was performed three times, and finally distilled water was removed as much as possible.
【０１０２】これに、注射用生理食塩水（大塚製薬社
製）を４０ｍｌ添加して軽く攪拌した。５００ｒｐｍで
１分間遠心し、上澄みを吸引して除き、同様に注射用生
理食塩水（大塚製薬社製）にて３回洗浄を行い、最後に
注射用生理食塩水４０ｍｌを加えて、一晩室温にて静置
した。その後、同じ洗浄操作を５回行い、最後にできる
だけ生理食塩水を取り除いた。To this, 40 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred. Centrifuge at 500 rpm for 1 minute, remove the supernatant by suction, and similarly wash with physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.) three times, and finally add 40 ml of physiological saline for injection, and overnight at room temperature. It was left still at. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.
【０１０３】注射用生理食塩水を充填して滅菌洗浄した
２ｍｌ用チューブ（Ｅｐｐｄｎｄｏｒｆ社製）に、上記
のようにして洗浄されたポリメタクリル酸エステル系材
料の粒子をかさ体積で５００μｌ充填した。A 2 ml tube (manufactured by Eppdndorf) that had been sterilized and filled with physiological saline for injection was filled with 500 μl of particles of the polymethacrylate ester material washed as described above in a bulk volume.
【０１０４】実施例４１ 用いたポリメタクリル酸エステル系材料を、ジエチルア
ミノ基をもつＳＥＰＡＢＥＡＤＳ ＦＰ−ＤＡ１３（三
菱化成社製）に変更した以外はすべて実施例４０と同様
にして行った。 Example 41 The procedure of Example 40 was repeated except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-DA13 (manufactured by Mitsubishi Kasei) having a diethylamino group.
【０１０５】実施例４２ 用いたポリメタクリル酸エステル系材料を、末端に１級
アミノ基をもつＳＥＰＡＢＥＡＤＳ ＦＰ−ＨＡ１３
（三菱化成社製）に変更した以外はすべて実施例４０と
同様にして行った。The polymethacrylic acid ester-based material used in Example 42 was prepared by using SEPABEADS FP-HA13 having a primary amino group at the terminal.
The same procedure as in Example 40 was carried out except that the product was manufactured by Mitsubishi Kasei.
【０１０６】実施例４３ 用いたポリメタクリル酸エステル系材料を、水酸基をも
つＳＥＰＡＢＥＡＤＳＦＰ−ＨＧ１３（三菱化成社製）
に変更した以外はすべて実施例４０と同様にして行っ
た。The polymethacrylic acid ester material used in Example 43 was prepared from SEPABEA SFP-HG13 (manufactured by Mitsubishi Kasei) having a hydroxyl group.
Example 40 was performed in the same manner as in Example 40 except that
【０１０７】実施例４４ 用いたポリメタクリル酸エステル系材料を、疎水性基
〔−ＯＣ6 Ｈ5 〕をもつＳＥＰＡＢＥＡＤＳ ＰＨ−１
３（三菱化成社製）に変更した以外はすべて実施例４０
と同様にして行った。 Example 44 The polymethacrylic acid ester-based material used was SEPABEADS PH-1 having a hydrophobic group [-OC 6 H 5 ].
Example 40 except that the number was changed to 3 (manufactured by Mitsubishi Kasei)
I went in the same way.
【０１０８】比較例３ 用いたポリメタクリル酸エステル系材料を、カルボキシ
ル基をもつＳＥＰＡＢＥＡＤＳ ＦＰ−ＣＭ１３（三菱
化成社製）に変更した以外はすべて実施例４０と同様に
して行った。 Comparative Example 3 The procedure of Example 40 was repeated except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-CM13 (manufactured by Mitsubishi Kasei) having a carboxyl group.
【０１０９】実施例４０〜４４及び比較例３の各ビーズ
を充填した各チューブにヘパリン採血した健常人新鮮血
１．６ｍｌを加えて回転円盤に取り付けて、３７℃にて
２時間、回転数２６ｒｐｍで転倒混和した。To each tube filled with each bead of Examples 40 to 44 and Comparative Example 3, 1.6 ml of heparinized fresh blood of a healthy person was added and attached to a rotating disc, and the rotation speed was 26 rpm at 37 ° C. for 2 hours. It mixed with the fall.
【０１１０】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を測定した。また、採血直後
の血液を遠心分離して血漿を採取して、同様に免疫抑制
活性を測定した。結果を表６に示す。The mixed blood was centrifuged to collect plasma, and the immunosuppressive activity in the plasma was measured. Immediately after blood collection, blood was centrifuged to collect plasma, and immunosuppressive activity was similarly measured. The results are shown in Table 6.
【０１１１】[0111]
【表６】 [Table 6]
【０１１２】〔疎水性材料による免疫抑制作用の誘導〕比較例４ 注射用生理食塩水（大塚製薬社製）で洗浄した２ｍｌ用
チューブ（Ｅｐｐｅｎｄｏｒｇｆ社製）を用いた。[Induction of Immunosuppressive Action by Hydrophobic Material] Comparative Example 4 A 2 ml tube (manufactured by Eppendorff) washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was used.
【０１１３】比較例５ ポリエチレンのビーズ（粒径２．５ｍｍ）を射出成形に
より作製した。これらをメタノールで洗浄後乾燥した。
このビーズを注射用生理食塩水（大塚製薬社製）で洗浄
後、同じく注射用生理食塩水で洗浄した２ｍｌ用チュー
ブ（Ｅｐｐｅｎｄｏｒｆ社製）に充填した。充填量はビ
ーズ７０個とした。 Comparative Example 5 Polyethylene beads (particle diameter 2.5 mm) were produced by injection molding. These were washed with methanol and dried.
The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and then filled in a 2 ml tube (manufactured by Eppendorf) similarly washed with physiological saline for injection. The filling amount was 70 beads.
【０１１４】比較例６ ポリエチレンのビーズ（粒径２．５ｍｍ）を射出成形に
より作製した。これらをメタノールで洗浄後乾燥した。
このビーズを注射用生理食塩水（大塚製薬社製）で洗浄
後、同じく注射用生理食塩水で洗浄した２ｍｌ用チュー
ブ（Ｅｐｐｅｎｄｏｒｆ社製）に充填した。充填量はビ
ーズ７０個とした。 Comparative Example 6 Polyethylene beads (particle diameter 2.5 mm) were produced by injection molding. These were washed with methanol and dried.
The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and then filled in a 2 ml tube (manufactured by Eppendorf) similarly washed with physiological saline for injection. The filling amount was 70 beads.
【０１１５】比較例７ ポリ塩化ビニルのビーズ（粒径２．５ｍｍ）を射出成形
により作製した。これらをメタノールで洗浄後乾燥し
た。このビーズを注射用生理食塩水（大塚製薬社製）で
洗浄後、同じく注射用生理食塩水で洗浄した２ｍｌ用チ
ューブ（Ｅｐｐｅｎｄｏｒｆ社製）に充填した。充填量
はビーズ７０個とした。 Comparative Example 7 Polyvinyl chloride beads (particle size 2.5 mm) were prepared by injection molding. These were washed with methanol and dried. The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and then filled in a 2 ml tube (manufactured by Eppendorf) similarly washed with physiological saline for injection. The filling amount was 70 beads.
【０１１６】比較例８ ポリプロピレンのビーズ（粒径２．５ｍｍ）を射出成形
により作製した。これらをメタノールで洗浄後乾燥し
た。このビーズを注射用生理食塩水（大塚製薬社製）で
洗浄後、同じく注射用生理食塩水で洗浄した２ｍｌ用チ
ューブ（Ｅｐｐｅｎｄｏｒｆ社製）に充填した。充填量
はビーズ７０個とした。 Comparative Example 8 Polypropylene beads (particle diameter 2.5 mm) were produced by injection molding. These were washed with methanol and dried. The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and then filled in a 2 ml tube (manufactured by Eppendorf) similarly washed with physiological saline for injection. The filling amount was 70 beads.
【０１１７】比較例９ ポリテトラフルオロエチレンプロピレン共重合体のビー
ズ（粒径２．５ｍｍ）を射出成形により作製した。これ
らをメタノールで洗浄後乾燥した。このビーズを注射用
生理食塩水（大塚製薬社製）で洗浄後、同じく注射用生
理食塩水で洗浄した２ｍｌ用チューブ（Ｅｐｐｅｎｄｏ
ｒｆ社製）に充填した。充填量はビーズ７０個とした。 Comparative Example 9 Polytetrafluoroethylene propylene copolymer beads (particle size 2.5 mm) were prepared by injection molding. These were washed with methanol and dried. The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then washed with physiological saline for injection (Eppendo).
rf). The filling amount was 70 beads.
【０１１８】比較例４〜９の各ビーズを充填した各チュ
ーブにヘパリン採血した健常人新鮮血１．６ｍｌを加え
て回転円盤に取り付けて、３７℃にて２時間、回転数２
６ｒｐｍで転倒混和した。To each tube filled with each bead of Comparative Examples 4 to 9 was added 1.6 ml of heparinized fresh blood of a healthy person, which was attached to a rotating disk and rotated at 37 ° C. for 2 hours at a rotation speed of 2
Mix by inverting at 6 rpm.
【０１１９】混和後の血液を遠心分離して血漿を採取
し、血漿中の免疫抑制活性を測定した。また、採血直後
の血液を遠心分離して血漿を採取して、同様に免疫抑制
活性を測定した。結果を表７に示す。The mixed blood was centrifuged to collect plasma, and the immunosuppressive activity in the plasma was measured. Immediately after blood collection, blood was centrifuged to collect plasma, and immunosuppressive activity was similarly measured. The results are shown in Table 7.
【０１２０】[0120]
【表７】 [Table 7]
【０１２１】採血直後の血漿や注射用生理食塩水（大塚
製薬社製）で洗浄した２ｍｌ用チューブに血液を充填し
ただけのもの（比較例４）の免疫抑制活性は１０％以下
であった。The immunosuppressive activity of a 2 ml tube washed with plasma or physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) immediately after blood collection only was filled with blood (Comparative Example 4) was 10% or less.
【０１２２】また、表１〜７の結果から水酸基、アミド
基、エステル基を有する高分子材料、及びカチオン性官
能基を有する高分子材料は、血液との接触によって特に
高い免疫抑制作用を誘導することが明らかである。他
方、未修飾のポリスチレン、ポリエチレン、ポリ塩化ビ
ニルなどの疎水性高分子材料やアニオン性官能基だけを
有する高分子材料では免疫抑制作用をほとんど誘導しな
かった。From the results shown in Tables 1 to 7, the polymeric material having a hydroxyl group, an amide group, an ester group and the polymeric material having a cationic functional group induce a particularly high immunosuppressive action upon contact with blood. It is clear. On the other hand, hydrophobic polymer materials such as unmodified polystyrene, polyethylene, and polyvinyl chloride and polymer materials having only anionic functional groups hardly induced immunosuppressive action.
【０１２３】[0123]
【発明の効果】以上のように、請求項１に記載の発明に
よれば、水酸基、アミド基及びエステル基のうち少なく
とも１種を有する高分子材料と血液とが、また請求項２
に記載の発明によればカチオン性官能基を有する高分子
材料と血液とが接触されるため、免疫抑制因子の誘導が
効果的に行われる。従って、患者自身の血液を用いるこ
とにより、患者由来の内因性の免疫抑制因子を含む血漿
または血清を容易に得ることができ、このような血漿ま
たは血清を患者に与えることが可能となる。As described above, according to the invention described in claim 1, the polymeric material having at least one of a hydroxyl group, an amide group and an ester group and blood are also present.
According to the invention described in (1), since the polymeric material having a cationic functional group is brought into contact with blood, the immunosuppressive factor can be effectively induced. Therefore, by using the patient's own blood, it is possible to easily obtain plasma or serum containing a patient-derived endogenous immunosuppressive factor, and to provide such plasma or serum to the patient.
【０１２４】また、請求項３に記載の発明では、それぞ
れ、上記特定の高分子材料により容器もしくは容器内に
充填された材料が構成されているため、請求項１，２に
記載の製造方法に用いるのに最適な血漿もしくは血清製
造装置を提供することができる。Further, in the invention described in claim 3, since the container or the material filled in the container is constituted by the specific polymer material, respectively, the manufacturing method according to claim 1 or 2 An optimal plasma or serum production device to be used can be provided.
Claims (3)
る群から選択した少なくとも１つの官能基を有する高分
子材料と、血液とを接触させることを特徴とする免疫抑
制作用を有する血漿もしくは血清の製造方法。1. Production of plasma or serum having an immunosuppressive action, which comprises contacting blood with a polymer material having at least one functional group selected from the group consisting of a hydroxyl group, an amide group and an ester group. Method.
と、血液とを接触させることを特徴とする免疫抑制作用
を有する血漿もしくは血清の製造方法。2. A method for producing plasma or serum having an immunosuppressive action, which comprises contacting blood with a polymeric material having a cationic functional group.
器内に充填された請求項１または２に記載の高分子材料
とを備えることを特徴とする免疫抑制作用を有する血漿
もしくは血清の製造装置。3. Production of plasma or serum having an immunosuppressive action, comprising a container having a blood inlet / outlet port and the polymer material according to claim 1 filled in the container. apparatus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6037133A JPH07242553A (en) | 1994-03-08 | 1994-03-08 | Method for production plasma or serum having immunodepressive action and device for prodcing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6037133A JPH07242553A (en) | 1994-03-08 | 1994-03-08 | Method for production plasma or serum having immunodepressive action and device for prodcing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07242553A true JPH07242553A (en) | 1995-09-19 |
Family
ID=12489123
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6037133A Pending JPH07242553A (en) | 1994-03-08 | 1994-03-08 | Method for production plasma or serum having immunodepressive action and device for prodcing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07242553A (en) |
-
1994
- 1994-03-08 JP JP6037133A patent/JPH07242553A/en active Pending
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