JP2006271809A - Irradiated modified base material - Google Patents
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本発明は、プロテインＣ活性化能を有し、かつ放射線照射された改質基材に関する。活性化プロテインＣは血栓形成を阻害するため、抗血栓性が必要な材料に幅広く用いることができる。具体的には、人工血管、カテーテル、血液バッグ、血液浄化用モジュール、人工腎臓、人工肺、手術用補助器具などの医用材料に好適に用いられる。 The present invention relates to a modified substrate having protein C activation ability and irradiated. Since activated protein C inhibits thrombus formation, it can be widely used for materials that require antithrombogenicity. Specifically, it is suitably used for medical materials such as artificial blood vessels, catheters, blood bags, blood purification modules, artificial kidneys, artificial lungs, and surgical aids.
医用材料において生体適合性は重要な問題であり、特に血栓形成を抑制する性質、即ち抗血栓性が要求される。 Biocompatibility is an important problem in medical materials, and in particular, a property of inhibiting thrombus formation, that is, antithrombogenicity is required.
材料に抗血栓性を付与するために、ヒト・トロンボモジュリンを固定化する方法が開示されている。例えば、特開平４−１５０６３にはスペーサーを介してヒト・トロンボモジュリンを不溶性担体に共有結合により固定化する方法が、特開平９−２９１０４３にはヒト・トロンボモジュリンを疎水化し、有機溶媒中で疎水性基材に被覆させる方法が開示されている。これらは基材にヒト・トロンボモジュリンを固定化することで、プロテインＣを活性化し、材料に抗血栓性を付与させるものである。しかしながら、医療用途に用いる場合には滅菌処理が必要である。滅菌方法としては、放射線滅菌、蒸気滅菌、エチレンオキサイドガス滅菌が挙げられる。近年では、エチレンオキサイドガス滅菌は、残留毒性の問題からあまり用いられておらず、現在は放射線滅菌処理か蒸気滅菌が主流となっている。蒸気滅菌は基材の制限を受け、ポリメチルメタクリレートや塩化ビニルなどは適用することができない。一方で、ヒト・トロンボモジュリンは、放射線に対する耐性の低さが問題であった。すなわち、これまでに、放射線照射され、かつプロテインＣ活性化能をもつ抗血栓性材料は得られていなかった。 A method for immobilizing human thrombomodulin to impart antithrombogenic properties to the material is disclosed. For example, Japanese Patent Laid-Open No. 4-15063 discloses a method of immobilizing human thrombomodulin to an insoluble carrier through a spacer, and Japanese Patent Application Laid-Open No. 9-291043 hydrophobizes human thrombomodulin to form a hydrophobic group in an organic solvent. A method of coating a material is disclosed. These immobilize human thrombomodulin on a substrate to activate protein C and impart antithrombogenic properties to the material. However, sterilization is required for use in medical applications. Examples of the sterilization method include radiation sterilization, steam sterilization, and ethylene oxide gas sterilization. In recent years, ethylene oxide gas sterilization has not been used much due to the problem of residual toxicity, and at present, radiation sterilization or steam sterilization has become the mainstream. Steam sterilization is subject to substrate limitations, and polymethyl methacrylate, vinyl chloride, etc. cannot be applied. On the other hand, human thrombomodulin has a problem of low resistance to radiation. That is, until now, no antithrombotic material that has been irradiated and has the ability to activate protein C has not been obtained.
本発明の目的は、かかる従来技術の欠点を改良し、放射線照射により滅菌処理され、かつプロテインＣ活性化能をもつ抗血栓性の改質基材を提供することにある。 An object of the present invention is to improve the disadvantages of the prior art and provide an antithrombogenic modified base material that is sterilized by irradiation and has the ability to activate protein C.
本発明は上記課題を達成するため、以下の構成を有する。
(1) 基材にプロテインＣを活性化させる生理活性物質が存在した状態で放射線照射され、プロテインＣ活性化能を持つ改質基材。
(2) 前記の放射線照射線量が５ｋＧｙから１００ｋＧｙであること特徴とする(1)に記載の改質基材。
(3) 前記のプロテインＣを２０％以上活性化させること特徴とする(1)または(2)のいずれかに記載の改質基材。
(4) 前記の生理活性物質がヒト・トロンボモジュリンもしくはその一部を有するペプチドであることを特徴とする(1)〜(3)のいずれかに記載の改質基材。
(5) 前記の生理活性物質が基材と炭素数３００以下の有機基からなるスペーサーを介して結合していることを特徴とする(1)〜(4)のいずれかに記載の改質基材。
(6) 前記の改質基材からの生理活性物質の溶出濃度が１０ｎｇ／ｍｌ以下であることを特徴とする(1)〜(5)のいずれかに記載の改質基材。
(7) 前記生理活性物質の水溶液を基材と接触させた状態で放射線照射することにより得られた(1)〜(6)のいずれかに記載の改質基材。
(8) 前記の基材が分離膜であることを特徴とする(1)〜(7)のいずれかに記載の改質基材。
(9) 前記の分離膜が中空糸膜であることを特徴とする(8)の改質基材。
(10) (9)に記載の中空糸膜が内蔵された人工腎臓。
In order to achieve the above object, the present invention has the following configuration.
(1) A modified base material that is irradiated with radiation in the presence of a physiologically active substance that activates protein C on the base material and has the ability to activate protein C.
(2) The modified base material according to (1), wherein the radiation dose is 5 kGy to 100 kGy.
(3) The modified base material according to either (1) or (2), wherein the protein C is activated by 20% or more.
(4) The modified base material according to any one of (1) to (3), wherein the physiologically active substance is human thrombomodulin or a peptide having a part thereof.
(5) The modified group according to any one of (1) to (4), wherein the physiologically active substance is bonded to a base material through a spacer composed of an organic group having 300 or less carbon atoms. Wood.
(6) The modified substrate according to any one of (1) to (5), wherein the elution concentration of the physiologically active substance from the modified substrate is 10 ng / ml or less.
(7) The modified base material according to any one of (1) to (6), which is obtained by irradiating an aqueous solution of the physiologically active substance in contact with the base material.
(8) The modified substrate according to any one of (1) to (7), wherein the substrate is a separation membrane.
(9) The modified base material according to (8), wherein the separation membrane is a hollow fiber membrane.
(10) An artificial kidney incorporating the hollow fiber membrane according to (9).
本発明によって、放射線により滅菌処理され、かつプロテインＣ活性化能をもつ抗血栓性に優れた改質基材を提供することができる。 According to the present invention, it is possible to provide a modified base material that is sterilized by radiation and has protein C activation ability and excellent antithrombotic properties.
本発明は、基材にプロテインＣを活性化させる生理活性物質が存在した状態で放射線照射されたことを特徴とする改質基材である。活性化されたプロテインＣは第Ｖ因子と第VIII因子の不活性化により血液凝固を阻害する。プロテインＣを活性化させる生理活性物質（以下、単に生理活性物質という）としては、合成ペプチドやプロテインＣを活性化させる作用のあるヒト・トロンボモジュリンなどのタンパク質もしくはその一部を有するペプチドなどが好適に用いられる。 The present invention is a modified base material characterized by being irradiated with a physiologically active substance that activates protein C in the base material. Activated protein C inhibits blood clotting by inactivating Factor V and Factor VIII. As a physiologically active substance that activates protein C (hereinafter simply referred to as a physiologically active substance), a synthetic peptide, a protein such as human thrombomodulin having an action of activating protein C, or a peptide having a part thereof is preferably used. Used.
プロテインＣの活性化はヒト・トロンボモジュリンにトロンビンが結合することによって数千倍に増幅される。ヒト・トロンボモジュリンは、血管内皮細胞膜上の糖タンパク質であり、血管内および体外の血液凝固を制御している。また、トロンビンはヒト・トロンボモジュリンと結合することで、凝固因子であるトロンビンのフィブリン形成作用および血小板凝集作用等を消失される。したがって、トロンビンの凝固作用を消失できる機能を有していれば、抗血栓性にさらに有利であるため、ヒト・トロンボモジュリンもしくはその構造の一部分を有するペプチドが好ましい。 Protein C activation is amplified thousands of times by binding of thrombin to human thrombomodulin. Human thrombomodulin is a glycoprotein on the vascular endothelial cell membrane and regulates blood clotting both inside and outside the blood vessels. In addition, thrombin binds to human thrombomodulin to eliminate the fibrin forming action and platelet aggregation action of thrombin, which is a coagulation factor. Therefore, if it has a function capable of eliminating the coagulation action of thrombin, it is further advantageous for antithrombogenicity. Therefore, human thrombomodulin or a peptide having a part of its structure is preferable.
本発明でいうところの基材とは、プロテインＣを活性化させる生理活性物質が存在している担体のことを指し、高分子材料が好ましい。高分子材料の例としては、ポリスルホンやポリスチレン、ポリウレタン、ポリエチレン、ポリプロピレン、ポリ塩化ビニルや、これらの誘導体などが挙げられる。基材の形状としては、繊維、フィルム、樹脂、分離膜などが挙げられるが、これらに限定されるものではない。具体的には、抗血栓性が必要な材料、例えば、人工腎臓用の中空糸膜や、ケース、人工血管、カテーテル、血液バッグ、血液浄化用モジュール、人工肺、手術用補助器具などの医用材料として好適に用いられる。 The term “substrate” as used herein refers to a carrier in which a physiologically active substance that activates protein C is present, and a polymer material is preferred. Examples of the polymer material include polysulfone, polystyrene, polyurethane, polyethylene, polypropylene, polyvinyl chloride, and derivatives thereof. Examples of the shape of the substrate include, but are not limited to, fibers, films, resins, and separation membranes. Specifically, materials that require antithrombogenic properties, such as hollow fiber membranes for artificial kidneys, medical materials such as cases, artificial blood vessels, catheters, blood bags, blood purification modules, artificial lungs, and surgical aids Is preferably used.
また、本発明でいうところの基材に生理活性物質が存在した状態とは、基材と生理活性物質が相互作用している状態を指す。相互作用としては化学結合、吸着などが挙げられる。例えば、基材を成型後、化学反応を用いて生理活性物質を架橋させても良い。このとき、生理活性物質と基材の間に有機基からなるスペーサーを介しても良い。スペーサーとしては、ポリエチレングリコールやポリプロピレングリコールおよびそれらの誘導体などを例示することができる。なお、スペーサーの末端部分はアミド結合やエステル結合などが好適に用いられる。化学反応を用いて架橋させる例としては、基材および生理活性物質にアミノ基やカルボキシル基がある場合には、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸などの縮合剤を用いて生理活性物質と基材に化学結合を形成させることができる。また、基材成型原液に生理活性物質を混練させ、物理的に生理活性物質を基材に内包させる形にしても良い。さらには、基材に生理活性物質を吸着させても良い。吸着は、間接的な相互作用であってもよい。例えば、生理活性物質が基材と水分子を介した水素結合によって吸着されていても良いし、基材上に電荷をもつ物質を導入し、静電相互作用によって吸着されていてもよい。具体的には、下記のいずれかよって、生理活性物質の基材への吸着が達成できるが、これに限定されるわけではない。１）基材を生理活性物質溶液に浸漬させる。２）基材を生理活性物質溶液に浸漬させた後、溶液からに抜き出し、湿潤状態で保持させる。３）基材を生理活性物質溶液に浸漬させた後、溶液から抜き出し、水に浸漬させる。下記実施例では、生理活性物質水溶液を基材である中空糸膜内を還流させた後、すなわち、生理活性物質水溶液を基材と接触させた状態で放射線を照射しており、このような簡便な方法で本発明の改質基材を得ることができる。この場合、放射線を照射することにより、滅菌と同時に、生理活性物質の固定化も行なわれる。また、放射線を照射する際に用いる生理活性物質水溶液の濃度は、所望のプロテインＣの活性化度を達成できる量が適宜選択され、特に限定されるものではないが、通常、１μｇ／ｍｌ〜１００μｇ／ｍｌ程度の濃度の濃度の水溶液が用いられる。 In addition, the state where the physiologically active substance is present on the base material in the present invention refers to a state where the base material and the physiologically active substance interact. Examples of the interaction include chemical bonding and adsorption. For example, after the base material is molded, the physiologically active substance may be crosslinked using a chemical reaction. At this time, a spacer made of an organic group may be interposed between the physiologically active substance and the substrate. Examples of the spacer include polyethylene glycol, polypropylene glycol, and derivatives thereof. In addition, an amide bond, an ester bond, etc. are used suitably for the terminal part of a spacer. As an example of crosslinking using a chemical reaction, when the substrate and the physiologically active substance have an amino group or a carboxyl group, a condensing agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is used. Thus, a chemical bond can be formed between the physiologically active substance and the substrate. Alternatively, a physiologically active substance may be kneaded into the base material forming stock solution, and the physiologically active substance may be physically encapsulated in the base material. Furthermore, a physiologically active substance may be adsorbed on the substrate. Adsorption may be an indirect interaction. For example, a physiologically active substance may be adsorbed by hydrogen bonding via a base material and water molecules, or a substance having a charge may be introduced onto the base material and adsorbed by electrostatic interaction. Specifically, the adsorption of the physiologically active substance to the substrate can be achieved by any of the following, but the present invention is not limited to this. 1) The substrate is immersed in the physiologically active substance solution. 2) After immersing the substrate in the physiologically active substance solution, the substrate is extracted from the solution and held in a wet state. 3) After the substrate is immersed in the physiologically active substance solution, the substrate is extracted from the solution and immersed in water. In the following examples, radiation is irradiated after the physiologically active substance aqueous solution is refluxed in the hollow fiber membrane that is the base material, that is, while the physiologically active substance aqueous solution is in contact with the base material. The modified substrate of the present invention can be obtained by a simple method. In this case, the physiologically active substance is immobilized simultaneously with sterilization by irradiation with radiation. In addition, the concentration of the physiologically active substance aqueous solution used when irradiating with radiation is appropriately selected in an amount capable of achieving the desired degree of protein C activation, and is not particularly limited, but usually 1 μg / ml to 100 μg An aqueous solution having a concentration of about / ml is used.
ここで、湿潤状態とは、基材を浸漬していた溶液を除去して乾燥させない状態のことを言う。特に限定されるものではないが、基材の乾燥重量に対して３重量％以上の水分を含んでいることが好ましい。ここでいう、乾燥重量とは改質基材を乾燥させて、乾燥中の１時間での重量変化率が３％以内になった状態の重量をいう。 Here, the wet state refers to a state where the solution in which the substrate is immersed is removed and not dried. Although not particularly limited, it preferably contains 3% by weight or more of moisture with respect to the dry weight of the substrate. As used herein, the dry weight means a weight in a state where the modified base material is dried and the rate of change in weight within one hour during drying is within 3%.
本発明の改質基材は、上記のような、基材に生理活性物質が存在した状態で放射線照射して得られる。放射線としてはα線、β線、γ線、Ｘ線、紫外線、電子線などが用いられる。 The modified base material of the present invention is obtained by irradiation with radiation in the state where the physiologically active substance is present on the base material as described above. As the radiation, α rays, β rays, γ rays, X rays, ultraviolet rays, electron beams and the like are used.
放射線照射線量としては、基材の材質や形状にも依存するが、５ｋＧｙ以上、多くの基材は１５ｋＧｙ以上の放射線を照射することで、滅菌することができる。 Although the radiation irradiation dose depends on the material and shape of the base material, it can be sterilized by irradiating 5 kGy or more, and many base materials with 15 kGy or more.
また、放射線によって発生するラジカル反応により、生理活性物質と基材に化学結合が形成される。このため、生理活性物質を基材に吸着させただけでも、放射線照射によって生理活性物質が基材に固定化され、溶出を低減させることができる。つまり、放射線照射によって、滅菌と生理活性物質の基材への固定化を同時に行うことも可能である。 Further, a chemical bond is formed between the physiologically active substance and the substrate by a radical reaction generated by radiation. For this reason, even if the physiologically active substance is simply adsorbed to the base material, the physiologically active substance is immobilized on the base material by irradiation and the elution can be reduced. That is, sterilization and immobilization of a physiologically active substance on a base material can be simultaneously performed by irradiation.
一方で、放射線照射線量が高くなると、プロテインＣを活性化させる生理活性物質の変性が起こり、１００ｋＧｙを越える線量を照射すると、活性化能の低下が顕著になる。 On the other hand, when the radiation irradiation dose becomes high, denaturation of the physiologically active substance that activates protein C occurs, and when the dose exceeding 100 kGy is irradiated, the decrease in activation ability becomes remarkable.
ここで、生理活性物質はなるべく基材近傍に存在したほうが、放射線照射による活性の低下を低く抑えることができる。具体的には、上記のように生理活性物質が基材に吸着しているか、基材に内包させているか、生理活性物質と基材が化学結合していても、生理活性物質と基材の間のスペーサーが無いか、もしく炭素数３００以下の有機基という短いものが好ましい。 Here, when the physiologically active substance is present in the vicinity of the substrate as much as possible, the decrease in activity due to irradiation can be suppressed to a low level. Specifically, as described above, even if the physiologically active substance is adsorbed on the base material, is encapsulated in the base material, or is chemically bonded to the physiologically active substance and the base material, A short one having no intervening spacer or an organic group having 300 or less carbon atoms is preferred.
この理由は、明らかではないが、放射線照射時に水分子から発生する水酸基ラジカルが関与しているものと考えられる。すなわち、放射線照射による生理活性物質の変性は、生理活性物質に直接生じるラジカルよりも、周囲の水分子から発生する水酸基ラジカルによって攻撃されるためと考えられている。生理活性物質が基材近傍に存在するような状態の場合は、発生した水酸基ラジカルの攻撃は、基材と生理活性物質の双方に分散されるために、生理活性物質へのラジカル攻撃が少なくなったものと推測される。すなわち、基材が生理活性物質に対して放射線保護的な作用を発揮していることが考えられる。ただし、その効果も１００ｋＧｙを越えると顕著に低下することから、限界があると思われる。 The reason for this is not clear, but it is thought that hydroxyl radicals generated from water molecules upon irradiation are involved. That is, it is considered that denaturation of a physiologically active substance by radiation irradiation is attacked by hydroxyl radicals generated from surrounding water molecules rather than radicals directly generated in the physiologically active substance. In the state where the physiologically active substance exists in the vicinity of the base material, the attack of the generated hydroxyl radical is dispersed in both the base material and the physiologically active substance, so that the radical attack on the physiologically active substance is reduced. Presumed to have been. That is, it is conceivable that the base material exerts a radiation protective action on the physiologically active substance. However, it seems that there is a limit because the effect is remarkably reduced when it exceeds 100 kGy.
以上のことから、放射線照射線量は５ｋＧｙ以上、１００ｋＧｙ未満が好ましく、さらには１５ｋＧｙ以上、８０ｋＧｙ以下、さらに好ましくは２０ｋＧｙ以上、５０ｋＧｙ以下である。なお線量の測定は線量計にて測定することができる。 From the above, the radiation irradiation dose is preferably 5 kGy or more and less than 100 kGy, more preferably 15 kGy or more and 80 kGy or less, and further preferably 20 kGy or more and 50 kGy or less. The dose can be measured with a dosimeter.
本発明の改質基材においては、前述したように、放射線照射されているため、生理活性物質の基材の導入法の如何に関わらず、生理活性物質の溶出量が少ないことが特長である。溶出量が多いと、患者によっては出血傾向を助長するなど、安全性に問題が生じる可能性がある。そのため、溶出は少ないことが望まれる。溶出量の測定方法の詳細については後述するが、体液と基材の接触部分の面積１ｍ２あたり４Ｌ以上の水で洗浄した後、室温で５００ｍＬ以上の水を用いて２時間抽出した水中の濃度が、１０ｎｇ／ｍｌ以下であることを言う。ここで、水の代わりに生理食塩水もしくは緩衝液を用いても良い。 Since the modified base material of the present invention is irradiated with radiation as described above, it is characterized in that the amount of the bioactive substance eluted is small regardless of the method of introducing the base material of the bioactive substance. . If the amount of elution is large, there may be a problem in safety, such as promoting a bleeding tendency in some patients. Therefore, it is desired that the elution is small. The details of the method for measuring the elution amount will be described later. After washing with 4 L or more of water per 1 m 2 of the contact area between the body fluid and the substrate, the concentration in water extracted for 2 hours with 500 mL or more of water at room temperature Is 10 ng / ml or less. Here, physiological saline or buffer may be used instead of water.
なお、ここでいう表面積とは、体液との接触部分の面積をさす。基材の形状が多孔質体であり、体液との接触部分に血小板よりも小さい孔を有する場合、多孔質内部の面積は考慮しない。例えば人工腎臓用中空糸膜の場合は、中空糸内表面だけの面積をさし、中空糸膜厚部分の細孔面積は考慮しない。 In addition, the surface area here refers to the area of the contact portion with the body fluid. When the shape of the base material is a porous body and there are pores smaller than platelets at the contact portion with the body fluid, the area inside the porous body is not considered. For example, in the case of a hollow fiber membrane for an artificial kidney, it refers to the area of the hollow fiber inner surface only, and does not consider the pore area of the hollow fiber film thickness portion.
本発明の改質基材は、プロテインＣを５０％以上、好ましくは１００％以上、さらに好ましくは２００％以上活性化させる。改質基材のプロテインＣの活性化測定についての詳細は後述するが、405nmの吸光度で比較する。プロテインＣが５０％活性化しているとは、ブランクの吸光度の値よりも1.5倍の値を示していることをいう。ここで、ブランクとは、後述する測定方法のなかで、基材を用いずに、同様の測定を行った場合のことをいう。プロテインＣの活性化が５０％未満の場合は、十分な抗血栓性の効果が得られない場合が多い。 The modified base material of the present invention activates protein C by 50% or more, preferably 100% or more, more preferably 200% or more. The details of the protein C activation measurement of the modified substrate will be described later, but the comparison is made with the absorbance at 405 nm. “Protein C is 50% activated” means that the value is 1.5 times the absorbance value of the blank. Here, a blank means the case where the same measurement is performed without using a base material in the measurement method described later. When the activation of protein C is less than 50%, sufficient antithrombotic effect is often not obtained.
以下実施例を挙げて本発明を説明するが、本発明はこれらの例によって限定されるものではない。 EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
１．基材の作成
（１）ポリスルホンフィルム
抗血栓性を付与する基材にポリスルホンを用いた。実験はモデルとして行うため、フィルムに成形して行った。フィルムの成形方法は以下の通りである。まず、ポリスルホン（ソルベイ社製ユーデルポリスルホン（登録商標）Ｐ−３５００）１０部をジメチルアセトアミド９０部に加え室温にて溶解し、成型溶液とした。この溶液をホットプレートにて、表面温度が１００度になっているガラス板上で厚さ２０３μｍでキャストした。表面の温度は、接触式温度計により測定した。キャスト後、５分間ホットプレート上で放置し、溶媒を蒸発させた後、ガラス板ごと、水浴へ浸漬し透明フィルムを得た。（水浴に浸漬させるのは、フィルムをガラス板からはがしやすくさせるためである。）
1. Preparation of substrate (1) Polysulfone film Polysulfone was used as a substrate for imparting antithrombogenicity. Since the experiment was performed as a model, it was molded into a film. The method for forming the film is as follows. First, 10 parts of polysulfone (Udelpolysulfone (registered trademark) P-3500 manufactured by Solvay) was added to 90 parts of dimethylacetamide and dissolved at room temperature to obtain a molding solution. This solution was cast with a hot plate at a thickness of 203 μm on a glass plate having a surface temperature of 100 degrees. The surface temperature was measured with a contact thermometer. After casting, it was left on a hot plate for 5 minutes to evaporate the solvent, and then the glass plate was immersed in a water bath together to obtain a transparent film. (The reason why the film is immersed in a water bath is to make it easier to peel the film from the glass plate.)
（２）ポリスルホン／ポリビニルピロリドン混合中空糸膜ミニカラム
抗血栓性を付与する基材に、これは、人工腎臓のモデル実験として、ポリスルホン／ポリビニルピロリドン混合中空糸膜を用いたミニカラムを作成した。ポリスルホン（ソルベイ社製ユーデルポリスルホン（登録商標）Ｐ−３５００）１８重量部およびポリビニルピロリドン（ＢＡＳＦ社製Ｋ３０）９重量部をＮ，Ｎ’−ジメチルアセトアミド７２重量部および水１重量部の混合溶媒に加え、９０℃で１４時間加熱して溶解し、製膜原液を得た。この製膜原液を外径０．３ｍｍ、内径０．２ｍｍのオリフィス型二重円筒型口金の外側の管より吐出した。芯液としてＮ，Ｎ’−ジメチルアセトアミド５８重量部および水４２重量部からなる溶液を内側の管より吐出した。吐出された製膜原液は、乾式長３５０ｍｍを通過した後、水１００％の凝固浴に導かれ、中空糸が得られた。
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane mini-column A mini-column using a polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane as a model experiment of an artificial kidney was prepared as a base material imparting antithrombogenicity. A mixed solvent of 18 parts by weight of polysulfone (Udelpolysulfone (registered trademark) P-3500 manufactured by Solvay) and 9 parts by weight of polyvinylpyrrolidone (K30 manufactured by BASF) with 72 parts by weight of N, N′-dimethylacetamide and 1 part by weight of water In addition, the film was dissolved by heating at 90 ° C. for 14 hours to obtain a film forming stock solution. This film-forming stock solution was discharged from an outer tube of an orifice type double cylindrical die having an outer diameter of 0.3 mm and an inner diameter of 0.2 mm. A solution consisting of 58 parts by weight of N, N′-dimethylacetamide and 42 parts by weight of water was discharged from the inner tube as the core liquid. The discharged film-forming stock solution passed through a dry length of 350 mm, and was then introduced into a 100% water coagulation bath to obtain a hollow fiber.
中空糸膜を１００本束ね、直径約７ｍｍ、長さは１２ｃｍのプラスチック管ミニカラムケースに挿入した。中空糸膜の両末端を、中空糸膜中空部を閉塞しないようにウレタン系ポッティング剤で固定し、ミニカラムを作成した。該カラムの中空糸内表面積は７５ｃｍ２である。ミニカラムの中空糸膜内側および外側を３７℃の超純水を１ｍｌ／ｍｉｎの流速で３０分間洗浄した。 100 hollow fiber membranes were bundled and inserted into a plastic tube mini-column case having a diameter of about 7 mm and a length of 12 cm. Both ends of the hollow fiber membrane were fixed with a urethane potting agent so as not to block the hollow portion of the hollow fiber membrane, and a mini-column was prepared. The column has a hollow fiber inner surface area of 75 cm 2 . The inside and outside of the hollow fiber membrane of the mini column were washed with ultrapure water at 37 ° C. at a flow rate of 1 ml / min for 30 minutes.
２．ヒト・トロンボモジュリン
プロテインＣを活性化させる生理活性物質として、ヒト・トロンボモジュリンを使用した。ヒト・トロンボモジュリンとしては、特開平１−６２１９号公報の記載に従い、遺伝子工学的手法によって得たヒト・トロンボモジュリンを用いた。
2. Human thrombomodulin Human thrombomodulin was used as a physiologically active substance that activates protein C. As human thrombomodulin, human thrombomodulin obtained by genetic engineering technique was used according to the description in JP-A-1-6219.
３．改質基材の作成
（１）ポリスルホンフィルム
ヒト・トロンボモジュリン１２５μｇをリン酸緩衝液（以下、ＰＢＳと略記）５ｍｌに溶解させたヒト・トロンボモジュリン水溶液に片面の面積が１ｃｍ２の前記１（１）で作成したポリスルホンフィルムを浸漬させた状態でγ線照射した。
3. Preparation of modified substrate (1) Polysulfone film 1 (1) of 1 cm 2 on one side of an aqueous solution of human thrombomodulin in which 125 μg of human thrombomodulin was dissolved in 5 ml of a phosphate buffer (hereinafter abbreviated as PBS) The prepared polysulfone film was irradiated with γ rays while immersed.
（２）ポリスルホン／ポリビニルピロリドン混合中空糸膜ミニカラム
前記１（２）で得られた中空糸膜ミニカラムの中空糸内側にヒト・トロンボモジュリンのＰＢＳと溶液（ヒト・トロンボモジュリン濃度は２５μｇ／ｍｌ）２．５ｍｌを１ｍｌ／ｍｉｎの流速で１０分間、室温で灌流させた後、γ線照射した。
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane mini-column 2.5 ml of human thrombomodulin PBS and solution (the concentration of human thrombomodulin is 25 μg / ml) inside the hollow fiber of the hollow fiber membrane mini-column obtained in 1 (2) above Was perfused at room temperature for 10 minutes at a flow rate of 1 ml / min and then irradiated with γ rays.
４．ヒト・トロンボモジュリンの溶出実験
（１）ポリスルホンフィルム
前記３（１）で得られたフィルムを１０ｍｌのＰＢＳにて２回リンスした。この後、１ｍｌのＰＢＳに室温で２時間浸漬させ、このＰＢＳ中に溶出したヒト・トロンボモジュリン溶出濃度を測定した。ヒト・トロンボモジュリンの濃度は、ＥＬＩＳＡ法にて測定した。
4). Elution experiment of human thrombomodulin (1) Polysulfone film The film obtained in 3 (1) was rinsed twice with 10 ml of PBS. Thereafter, the sample was immersed in 1 ml of PBS at room temperature for 2 hours, and the elution concentration of human thrombomodulin eluted in this PBS was measured. The concentration of human thrombomodulin was measured by ELISA.
（２）ポリスルホン／ポリビニルピロリドン混合中空糸膜ミニカラム
前記３（２）で得られた中空糸膜ミニカラムについて、ヒト・トロンボモジュリン溶出試験を始める直前に、ＰＢＳ３０ｍｌ（中空糸内表面１ｍ２換算でＰＢＳ４Ｌに相当する）を室温にて１ｍｌ／ｍｉｎで３０ｍｉｎ循環し、洗浄した。ＰＢＳ５ｍｌ（中空糸内表面１ｍ２換算で６６７ｍＬに相当する）を０．５ｍｌ／ｍｉｎの流速で、室温で２時間循環させた。なお、後述する血液循環試験と対応させるために、循環開始後の最初の１ｍｌは廃棄した後、循環した。２時間循環させた液に溶出したヒト・トロンボモジュリンの濃度は、ＥＬＩＳＡ法にて測定した。
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane mini-column Immediately before starting the human thrombomodulin elution test for the hollow fiber membrane mini-column obtained in 3 (2) above, 30 ml of PBS (equivalent to PBS 4L in terms of 1 m 2 of the inner surface of the hollow fiber) Was circulated at room temperature for 30 min at 1 ml / min and washed. 5 ml of PBS (corresponding to 667 mL in terms of 1 m 2 of the inner surface of the hollow fiber) was circulated at room temperature for 2 hours at a flow rate of 0.5 ml / min. In order to correspond to the blood circulation test described later, the first 1 ml after the start of circulation was discarded and then circulated. The concentration of human thrombomodulin eluted in the solution circulated for 2 hours was measured by ELISA.
５．ヒト・トロンボモジュリン濃度測定
ヒト・トロンボモジュリンの濃度は、ヒトＣＤ１４１ ＥＬＩＳＡキット（ＤＩＡＣＬＯＮＥ社製造）を使用した。実験手順は、以下の通りである。ＥＬＩＳＡプレートのウェルにサンプルを１００μｌずつ加えた。ビオチン化ａｎｔｉ−ＣＤ１４１を調整し、５０μｌずつウェルに加えた。プレートカバーでカバーし、１時間室温下で定温放置した。カバーを外し、プレートをマイクロプレートウオッシャー（ＢＩＯ−ＲＡＤ ＩｍｍｕｎｏＷａｓｈ Ｍｏｄｅｌ １５７５）にて、以下のように洗浄した。
5. Measurement of Human Thrombomodulin Concentration The human CD141 ELISA kit (manufactured by DIACLONE) was used for the concentration of human thrombomodulin. The experimental procedure is as follows. 100 μl of sample was added to each well of the ELISA plate. Biotinylated anti-CD141 was prepared and 50 μl was added to each well. Covered with a plate cover and allowed to stand at room temperature for 1 hour. The cover was removed, and the plate was washed with a microplate washer (BIO-RAD ImmunoWash Model 1575) as follows.
１） 全てのウェルから溶液を吸い出した。
２） 全てのウェルに洗浄液を３００μｌずつ満たした。
３） 再度全てのウェルから溶液を吸い出した。
４） 手順２）と３）を二度繰り返した。
1) Solution was aspirated from all wells.
2) All wells were filled with 300 μl of washing solution.
3) The solution was aspirated again from all wells.
4) Procedures 2) and 3) were repeated twice.
洗浄操作後、ｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ溶液を調整し、ウェルに１００μｌ加えた。プレートカバーでカバーし、３０分間室温下で定温放置した。カバーを外してウェルを、上記洗浄操作１）〜４）と同様に洗浄を行った。ｒｅａｄｙ−ｕｓｅ ＴＭＢ基質溶液をウェルに１００μｌ加えた。プレートをアルミホイルで包んで光を遮断し１５分間室温で定温放置した。ウェルに１００μｌの停止試薬Ｈ２ＳＯ４を加えて、酵素基質反応を停止した。停止試薬Ｈ２ＳＯ４を加えた後に、速やかにマイクロプレートリーダー（ＢＩＯ−ＲＡＤ マイクロプレートリーダー モデル６８０）を用いて４５０ｎｍの吸光度を読み取った。既知濃度の吸光度の値から検量線を引き、サンプルのヒト・トロンボモジュリン濃度を算出した。 After the washing operation, a streptavidin-HRP solution was prepared, and 100 μl was added to the well. The plate was covered with a plate cover and allowed to stand at room temperature for 30 minutes. After removing the cover, the wells were washed in the same manner as in the washing operations 1) to 4). 100 μl of ready-use TMB substrate solution was added to the wells. The plate was wrapped with aluminum foil to block the light and left at room temperature for 15 minutes. The enzyme substrate reaction was stopped by adding 100 μl of stop reagent H 2 SO 4 to the wells. After adding the stop reagent H 2 SO 4 , the absorbance at 450 nm was immediately read using a microplate reader (BIO-RAD microplate reader model 680). A calibration curve was drawn from the absorbance value at a known concentration to calculate the human thrombomodulin concentration of the sample.
６．プロテインＣ活性化度の測定
（１）ポリスルホンフィルム
フィルムを１０ｍｌのＰＢＳにて２回リンスした。その後、混合緩衝液Ａ（２０ｍＭ トリス−塩酸緩衝液（ｐＨ７．５）、０．１５Ｍ ＮａＣｌ、０．５％Ｂｏｖｉｎｅ Ｓｅｒｕｍ Ａｌｂｍｉｎ（フラクションＶ、和光純薬工業(株)、以下、ＢＳＡと略記）、２．５ｍＭ ＣａＣｌ２）１０ｍｌを用いて洗浄した。フィルムを５ｕｎｉｔ／ｍｌのトロンビン溶液（５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）、０．１％ＮａＣｌ、０．１％ＢＳＡ、２ｍＭ ＣａＣｌ２）１ｍｌ中３７℃で１時間インキュベートした。フィルムを混合緩衝液Ｂ（５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）、０．１％ＮａＣｌ、０．１％ＢＳＡ、２ｍＭ ＣａＣｌ２）１０ｍｌを用いて洗浄した。フィルムを１００ｎMのプロテインC溶液（５０ｍＭトリス−塩酸緩衝液（ｐＨ８．０）、０．１％ＮａＣｌ、０．１％ＢＳＡ、２ｍＭ ＣａＣｌ２）０．５ｍｌ中３７℃で１時間インキュベートした。この上清５０μｌに１ｍＭのＳ−２２３８（２０ｍＭトリス−塩酸緩衝液（ｐＨ７．４）、０．１％ＮａＣｌ、０．１％ＢＳＡの混合溶液）１００μｌを加えて４０５ｎｍの吸光度を測定した。吸光度の値が大きいほど、プロテインＣが活性化されていることを示す。
6). Measurement of protein C activation degree (1) Polysulfone film The film was rinsed twice with 10 ml of PBS. Thereafter, mixed buffer A (20 mM Tris-HCl buffer (pH 7.5), 0.15 M NaCl, 0.5% Bovine Serum Albamin (Fraction V, Wako Pure Chemical Industries, Ltd., hereinafter abbreviated as BSA), Washing with 10 ml of 2.5 mM CaCl 2 ). The film was incubated at 37 ° C. for 1 hour in 1 ml of 5 unit / ml thrombin solution (50 mM Tris-HCl buffer (pH 8.0), 0.1% NaCl, 0.1% BSA, 2 mM CaCl 2 ). The film was washed with 10 ml of Mixing Buffer B (50 mM Tris-HCl buffer (pH 8.0), 0.1% NaCl, 0.1% BSA, 2 mM CaCl 2 ). The film was incubated at 37 ° C. for 1 hour in 0.5 ml of 100 nM protein C solution (50 mM Tris-HCl buffer (pH 8.0), 0.1% NaCl, 0.1% BSA, 2 mM CaCl 2 ). 100 μl of 1 mM S-2238 (a mixed solution of 20 mM Tris-HCl buffer (pH 7.4), 0.1% NaCl, 0.1% BSA) was added to 50 μl of this supernatant, and the absorbance at 405 nm was measured. The larger the absorbance value, the more protein C is activated.
（２）ポリスルホン／ポリビニルピロリドン混合中空糸膜ミニカラム
中空糸膜ミニカラムについて、ＰＢＳ３０ｍｌを室温にて１ｍｌ／ｍｉｎで３０ｍｉｎ循環し、洗浄した。５（１）と同様の液を用いた。すなわち、中空糸膜カラムの中空糸内側を、混合緩衝液Ａ １０ｍｌを用いて洗浄した。中空糸内側を５ｕｎｉｔ／ｍｌのトロンビン溶液５ｍｌで３７℃、１時間、０．５ｍｌ／ｍｉｎで循環させた。その後、中空糸内側を混合緩衝液Ｂ １０ｍｌを用いて洗浄した。中空糸内側を１００ｎMのプロテインC溶液２．５ｍｌで３７℃、１時間０．５ｍｌ／ｍｉｎで循環させた。この循環後の液、５０μｌに１ｍＭのＳ−２２３８ １００μｌを加えて４０５ｎｍの吸光度を測定した。吸光度の値が大きいほど、プロテインＣが活性化されていることを示す。
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane mini-column With respect to the hollow fiber membrane mini-column, 30 ml of PBS was circulated at room temperature for 30 min at 1 ml / min and washed. The same liquid as 5 (1) was used. That is, the hollow fiber inner side of the hollow fiber membrane column was washed with 10 ml of the mixed buffer A. The inside of the hollow fiber was circulated with 5 ml of 5 unit / ml thrombin solution at 37 ° C. for 1 hour at 0.5 ml / min. Thereafter, the inside of the hollow fiber was washed with 10 ml of the mixed buffer B. The inside of the hollow fiber was circulated with 2.5 ml of 100 nM protein C solution at 37 ° C. for 1 hour at 0.5 ml / min. 100 μl of 1 mM S-2238 was added to 50 μl of the solution after circulation, and the absorbance at 405 nm was measured. The larger the absorbance value, the more protein C is activated.
７．抗血栓性の評価
（１）ポリスルホンフィルム
フィルムを１０ｍｌのＰＢＳにて２回リンスした。フィルムについたＰＢＳをよく切った後、Ｆａｌｃｏｎ（登録商標）チューブ（１８ｍｍφ、Ｎｏ．２０５１）の中に移した。健常者ボランティアから静脈血を採血後、すみやかに５ｍｌを該ファルコンチューブに入れた。３０分間、室温で静置した後、フィルムを取り出し、ＰＢＳ１０ｍｌで軽くリンスし、フィルムに付着した血栓の状態を観察した。
7). Evaluation of antithrombogenicity (1) Polysulfone film The film was rinsed twice with 10 ml of PBS. After the PBS attached to the film was cut well, it was transferred into a Falcon (registered trademark) tube (18 mmφ, No. 2051). After collecting venous blood from healthy volunteers, 5 ml was immediately put into the falcon tube. After leaving still at room temperature for 30 minutes, the film was taken out and rinsed lightly with 10 ml of PBS, and the state of the thrombus adhering to the film was observed.
（２）ポリスルホン／ポリビニルピロリドン混合中空糸膜ミニカラム
中空糸膜ミニカラムの片側に、内径１ｍｍ、外径２ｍｍ、長さ５０ｃｍのシリコーンチューブ（製品名ＡＲＡＭ（登録商標））を接続した。中空糸膜ミニカラムについて、血液循環試験を始める直前に、ＰＢＳ３０ｍｌを室温にて１ｍｌ／ｍｉｎで３０ｍｉｎ循環し、洗浄した。
(2) Polysulfone / polyvinylpyrrolidone mixed hollow fiber membrane minicolumn A silicone tube (product name: ARAM (registered trademark)) having an inner diameter of 1 mm, an outer diameter of 2 mm, and a length of 50 cm was connected to one side of the hollow fiber membrane minicolumn. With respect to the hollow fiber membrane mini-column, immediately before starting the blood circulation test, 30 ml of PBS was circulated at 1 ml / min for 30 min at room temperature and washed.
健常者ボランティアから静脈血を採血後、十秒内にヘパリンを０．５Ｕ／ｍｌになるように添加した。採血後、実験開始までに、ある程度の時間が経過してしまうことなど、循環実験以外の要因で血液が活性化される。ヘパリンを0.5U/ml程度添加することで、そのような活性化を抑制できると考えられる。ヘパリン添加後、数分以内に、該血液をカラムで循環させた。循環は５ｍｌの血液を、垂直に立てた中空糸膜ミニカラムの上から下に、０．５ｍｌ／ｍｉｎの流速で、室温で行った。なお、ミニカラム内および回路内には、はじめはＰＢＳが充填してあるので、循環開始後の最初の１ｍｌは廃棄した後、循環した。該条件での、循環可能時間を測定した。なお、ここでいう循環可能時間とは、回路中もしくはミニカラム中に血栓などが生じて血液が流れなくなるまでの時間、もしくは血液が流れにくくなったため、回路接続部などから血液が漏れだしてくるまでの時間をいう。 After collecting venous blood from healthy volunteers, heparin was added to 0.5 U / ml within 10 seconds. After blood collection, blood is activated by factors other than the circulation experiment, such as a certain amount of time elapses before the start of the experiment. It is thought that such activation can be suppressed by adding about 0.5 U / ml of heparin. The blood was circulated through the column within minutes after the addition of heparin. Circulation was performed at room temperature at a flow rate of 0.5 ml / min from the top to the bottom of a hollow fiber membrane mini-column standing vertically. Since the minicolumn and the circuit were initially filled with PBS, the first 1 ml after the start of circulation was discarded and then circulated. Under this condition, the circulation possible time was measured. Note that the circulatorable time here is the time until blood clots occur in the circuit or mini-column and blood does not flow, or until blood leaks from the circuit connection etc. Of time.
実施例１
前記３（１）のフィルムにおいて２５ｋＧｙのγ線を照射した。該フィルムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。また、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．２６であり、ブランクは０．１１であった。すなわち、プロテインＣを１３６％活性化するものであった。さらにフィルムの抗血栓性を評価した結果、フィルムには血栓がほとんど付着していなかった。すなわち、ヒト・トロンボモジュリンの溶出は、ほとんどないうえに、プロテインＣを活性化することで抗血栓性を有するフィルムを得ることができた。
Example 1
The 3 (1) film was irradiated with 25 kGy of γ rays. As a result of human thrombomodulin elution experiments on the film, the human thrombomodulin concentration was 1 ng / ml or less. As a result of measuring the degree of protein C activation, the absorbance at 405 nm was 0.26, and the blank was 0.11. That is, it activated 136% of protein C. Furthermore, as a result of evaluating the antithrombogenicity of the film, almost no thrombus adhered to the film. That is, there was almost no elution of human thrombomodulin, and a film having antithrombogenicity could be obtained by activating protein C.
実施例２
前記３（１）のフィルムにおいて４０ｋＧｙのγ線を照射した。該フィルムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。また、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．２７であった。すなわち、プロテインＣを１４５％活性化するものであった。さらに該フィルムの抗血栓性を評価した結果、フィルムには血栓がほとんど付着していなかった。すなわち、ヒト・トロンボモジュリンの溶出は、ほとんどないうえに、プロテインＣを活性化することで抗血栓性を有するフィルムを得ることができた。
Example 2
The film of 3 (1) was irradiated with 40 kGy of γ rays. As a result of human thrombomodulin elution experiments on the film, the human thrombomodulin concentration was 1 ng / ml or less. As a result of measuring the degree of protein C activation, the absorbance at 405 nm was 0.27. That is, it activated 145% of protein C. Furthermore, as a result of evaluating the antithrombogenicity of the film, almost no thrombus was adhered to the film. That is, there was almost no elution of human thrombomodulin, and a film having antithrombogenicity could be obtained by activating protein C.
実施例３
前記３（２）の中空糸膜ミニカラムにおいて２５ｋＧｙのγ線を照射した。該中空糸膜ミニカラムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。また、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．３３であった。すなわち、プロテインＣを２００％活性化するものであった。さらに該中空糸膜ミニカラムの抗血栓性を評価した結果、循環可能時間は１時間５０分であった。すなわち、ヒト・トロンボモジュリンの溶出は、ほとんどないうえに、プロテインＣを活性化することで抗血栓性を有する中空糸膜ミニカラムを得ることができた。
Example 3
The 3 (2) hollow fiber membrane minicolumn was irradiated with 25 kGy of γ rays. As a result of the human thrombomodulin elution experiment on the hollow fiber membrane minicolumn, the human thrombomodulin concentration was 1 ng / ml or less. As a result of measuring the degree of protein C activation, the absorbance at 405 nm was 0.33. That is, it activated 200% of protein C. Furthermore, as a result of evaluating the antithrombogenicity of the hollow fiber membrane minicolumn, the circulation possible time was 1 hour 50 minutes. That is, there was almost no elution of human thrombomodulin, and a hollow fiber membrane minicolumn having antithrombogenicity could be obtained by activating protein C.
比較例１
前記３（１）のフィルムにおいて１５０ｋＧｙのγ線を照射した。該フィルムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。また、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．１５であった。さらに該フィルムの抗血栓性を評価した結果、フィルムには全面に血栓の形成が認められた。すなわち、該フィルムはヒト・トロンボモジュリンの溶出は、ほとんどないが、プロテインＣ活性能を有しておらず、抗血栓性もなかった。
Comparative Example 1
The film of 3 (1) was irradiated with 150 kGy of γ rays. As a result of human thrombomodulin elution experiments on the film, the human thrombomodulin concentration was 1 ng / ml or less. As a result of measuring the degree of protein C activation, the absorbance at 405 nm was 0.15. Furthermore, as a result of evaluating the antithrombogenicity of the film, thrombus formation was observed on the entire surface. That is, the film had almost no elution of human thrombomodulin, but did not have protein C activity ability and did not have antithrombotic properties.
比較例２
前記３（１）のフィルムにおいてヒト・トロンボモジュリン水溶液の代わりに、水にポリスルホンフィルムを浸漬させた状態で２５ｋＧｙのγ線を照射した。該フィルムについてプロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．１５であった。さらに該フィルムの抗血栓性を評価した結果、フィルムには全面に血栓の形成が認められた。すなわち、該フィルムはプロテインＣ活性能を有しておらず、抗血栓性もなかった。
Comparative Example 2
The film of 3 (1) was irradiated with 25 kGy of γ rays in a state where the polysulfone film was immersed in water instead of the human thrombomodulin aqueous solution. As a result of measuring the degree of protein C activation for the film, the absorbance at 405 nm was 0.15. Furthermore, as a result of evaluating the antithrombogenicity of the film, thrombus formation was observed on the entire surface. That is, the film did not have protein C activity ability and was not antithrombotic.
比較例３
前記３（１）のフィルムにおいてヒト・トロンボモジュリン水溶液に浸漬させただけで、γ線照射は行わなかった。該フィルムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は２１ｎｇ／ｍｌであった。また、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．２９であった。該フィルムの抗血栓性を評価した結果、フィルムには血栓がほとんど付着していなかった。すなわち、プロテインＣを活性化することで、抗血栓性を有するが、ヒト・トロンボモジュリンの溶出も多いフィルムであった。
Comparative Example 3
The film of 3 (1) was simply immersed in an aqueous human thrombomodulin solution, and no gamma irradiation was performed. As a result of the elution experiment of human thrombomodulin on the film, the human thrombomodulin concentration was 21 ng / ml. As a result of measuring the degree of protein C activation, the absorbance at 405 nm was 0.29. As a result of evaluating the antithrombogenicity of the film, almost no thrombus adhered to the film. That is, by activating protein C, the film has antithrombotic properties, but has a high elution of human thrombomodulin.
比較例４
前記３（２）の中空糸膜ミニカラムにおいてヒト・トロンボモジュリン水溶液の代わりに、水を用いて、２５ｋＧｙのγ線を照射した。該中空糸膜ミニカラムについてヒト・トロンボモジュリンの溶出実験の結果、ヒト・トロンボモジュリン濃度は１ｎｇ／ｍｌ以下であった。また、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．１４であった。さらに該中空糸膜ミニカラムの抗血栓性を評価した結果、循環可能時間は１時間２０分であった。すなわち、プロテインＣを活性化しないため、抗血栓性の低い中空糸膜ミニカラムであった。
Comparative Example 4
The hollow fiber membrane minicolumn of 3 (2) was irradiated with 25 kGy of γ rays using water instead of the human thrombomodulin aqueous solution. As a result of the human thrombomodulin elution experiment on the hollow fiber membrane minicolumn, the human thrombomodulin concentration was 1 ng / ml or less. As a result of measuring the degree of protein C activation, the absorbance at 405 nm was 0.14. Furthermore, as a result of evaluating the antithrombogenicity of the hollow fiber membrane minicolumn, the circulation possible time was 1 hour 20 minutes. That is, since it did not activate protein C, it was a hollow fiber membrane minicolumn with low antithrombotic properties.
比較例５
ヒト・トロンボモジュリンのＰＢＳ溶液（ヒト・トロンボモジュリン濃度は２５μｇ／ｍｌ）１０ｍｌを２５ｋＧｙのγ線を照射した。該溶液について、プロテインＣ活性化度を測定した結果、４０５ｎｍの吸光度は０．１３であり、放射線照射によって、活性が消失していた。すなわち、生理活性物質の近くに基材がないため、活性の保持ができなかったと考えられる。
Comparative Example 5
10 ml of PBS solution of human thrombomodulin (the concentration of human thrombomodulin is 25 μg / ml) was irradiated with γ-rays of 25 kGy. As a result of measuring the protein C activation degree of the solution, the absorbance at 405 nm was 0.13, and the activity was lost by irradiation. That is, it is considered that the activity could not be retained because there was no substrate near the physiologically active substance.
Claims (10)
An artificial kidney incorporating the hollow fiber membrane according to claim 9.
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|---|---|---|---|---|
| JPS6182760A (en) * | 1984-09-29 | 1986-04-26 | 三浦 喜温 | Antithrombotic medical material |
| JPS646219A (en) * | 1987-01-08 | 1989-01-10 | Asahi Chemical Ind | Peptide having promoting action on activation of protein c by thrombin |
| JPH0415063A (en) * | 1990-05-09 | 1992-01-20 | Mitsuru Akashi | Antithrombotic material having human thrombomoduline fixed |
| JPH07134127A (en) * | 1993-06-27 | 1995-05-23 | Masashi Funayama | Fixing method of ligand to carrier using photochemical reaction, and catching method and quantifying method of physiologically active material using this carrier |
| JPH09291043A (en) * | 1996-04-26 | 1997-11-11 | Mitsuru Akashi | Hydrophobic anti-thrombogenic physiologically active substance, anti-thrombogenic medical material and antithrombogenic medical tool |
| JP2006271839A (en) * | 2005-03-30 | 2006-10-12 | Toray Ind Inc | Auxiliary appliance for medical column for extracorporeal circulation |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6182760A (en) * | 1984-09-29 | 1986-04-26 | 三浦 喜温 | Antithrombotic medical material |
| JPS646219A (en) * | 1987-01-08 | 1989-01-10 | Asahi Chemical Ind | Peptide having promoting action on activation of protein c by thrombin |
| JPH0415063A (en) * | 1990-05-09 | 1992-01-20 | Mitsuru Akashi | Antithrombotic material having human thrombomoduline fixed |
| JPH07134127A (en) * | 1993-06-27 | 1995-05-23 | Masashi Funayama | Fixing method of ligand to carrier using photochemical reaction, and catching method and quantifying method of physiologically active material using this carrier |
| JPH09291043A (en) * | 1996-04-26 | 1997-11-11 | Mitsuru Akashi | Hydrophobic anti-thrombogenic physiologically active substance, anti-thrombogenic medical material and antithrombogenic medical tool |
| JP2006271839A (en) * | 2005-03-30 | 2006-10-12 | Toray Ind Inc | Auxiliary appliance for medical column for extracorporeal circulation |
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