JPS62243562A - Method and apparatus for stimulating leucocyte for remedy ofmalignant tumor - Google Patents
Method and apparatus for stimulating leucocyte for remedy ofmalignant tumorInfo
- Publication number
- JPS62243562A JPS62243562A JP61086084A JP8608486A JPS62243562A JP S62243562 A JPS62243562 A JP S62243562A JP 61086084 A JP61086084 A JP 61086084A JP 8608486 A JP8608486 A JP 8608486A JP S62243562 A JPS62243562 A JP S62243562A
- Authority
- JP
- Japan
- Prior art keywords
- leukocyte
- cells
- leukocytes
- malignant
- stimulator
- 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
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Abstract
(57)【要約】本公報は電子出願前の出願データであるた
め要約のデータは記録されません。(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は１体液中の白血球を活性化してｍｓ障害性細胞
を誘導する方法およびそれに用いられる装置に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for inducing ms-toxic cells by activating leukocytes in a body fluid, and a device used therefor.
周知の如く、生体の悪性腫瘍に対する免疫監視機構をに
なう抗腫瘍細胞としては、キラーＴ細胞。As is well known, killer T cells are anti-tumor cells that act as an immune surveillance mechanism against malignant tumors in the body.
ＮＫ細胞、活性化マクロファージ、に細胞等が重要な役
割をは友していることが報告とれている。It has been reported that NK cells, activated macrophages, and other cells play important roles.
したがって、悪性腫瘍に対する免疫学的療法としては、
癌患者免疫細胞（白血球）ｔ−活性化して。Therefore, as immunological therapy for malignant tumors,
Cancer patient immune cells (white blood cells) t-activated.
これらの抗腫膓細ｆｉＩを効率的に誘導することが考え
られる。しかしながら１Ｍｉ患者は一般的に１ＭＪの進
行とともに免疫能が低下することが報告されており、癌
患者生体中においては、免疫応答を抑制する免疫抑制因
子の存在あるいはサプレッサーＴ細胞、サプレッサーマ
クロファージの誘導活性化が報告されている。It is considered that these anti-tumor fiIs can be efficiently induced. However, it has been reported that 1Mi patients generally have a decline in their immune function as 1MJ progresses, and in cancer patients, the presence of immunosuppressive factors that suppress the immune response or the induction activity of suppressor T cells and suppressor macrophages. has been reported.
このような免疫能の抑制状態下にある癌患者生体中にお
いて、効率的な抗腫瘍細胞の誘導は困難であると言わな
ければならない。したがって、免疫抑制状態から解放さ
れ文体外に患者白血球を取シ出し、体外で効率的な抗腫
瘍細胞誘導活性化を行なうことは、効果の高め新しい癌
免疫療法になると考えられる。It must be said that it is difficult to efficiently induce antitumor cells in living cancer patients whose immune capacity is in such a suppressed state. Therefore, removing leukocytes from a patient outside the body after being released from the immunosuppressive state and efficiently inducing and activating antitumor cells outside the body is considered to be a new and highly effective cancer immunotherapy.
（従来の技術）
キラーＴ細胞は、抗腫勃細胞の中でも特に抗癌免疫にお
りで主役をはたしていると考えられてｂるが、これを体
外で誘導活性化しようとする研究が精力的になされてき
た。すなわち１体外に取り出しｆｃ癌思者末梢血白血球
に、通出した患者正常細胞を感作さぜ、患者白血球を活
性化して、特異的に患者稚Ｓ細胞だけを障害し、患者正
常細胞は障害しないキラーＴ細胞を誘導して、これを癌
患者体内にもどすことにより、癌を治療しようとする試
みである。(Prior art) Killer T cells are thought to play a major role in anti-tumor immunity, especially among anti-tumor cells, but research is being actively conducted to induce and activate them outside the body. It has been done. In other words, the patient's normal cells are sensitized to the peripheral blood leukocytes of the FC cancer patient taken out of the body, activating the patient's leukocytes and specifically damaging only the patient's young S cells, while damaging the patient's normal cells. This is an attempt to treat cancer by inducing killer T cells that do not respond to cancer and returning them to the cancer patient's body.
しかしながら、この方法で誘導し友キラーＴ細胞は、治
療効果を期待できるほど強力ではなめため、リンフ才力
インの１種であるＴ細胞増殖因子を用いて培養し、大量
に増殖させた後、患者に投与する方法が考えられてｂる
。この方法は、Ｔ細胞増殖因子が遺伝子操作の技術によ
り工業的大量生産が可能となり、大量のＴ細胞増殖因子
が使用できることが現実化してきたために実施可能では
あるが、キラーＴ細胞全体外で長期間培養することによ
る細胞の変質等の問題がある。ＩＬそのほかにも夾角化
するには困難な種々の問題点があり５例えば、キラーＴ
細胞の誘導のために患者正常細胞と手術が必要なこと、
試みた癌患者の一部にのみキラーＴ細胞の誘導が可能で
、全例で誘導されるわけではないこと、操作が非常に煩
雑であること等、解決されなければならない問題点が多
い。However, the Tomo Killer T cells induced by this method are not strong enough to be expected to have a therapeutic effect, so after culturing them using T cell growth factor, a type of lymphoid, and proliferating them in large quantities, Methods of administration to patients are contemplated. Although this method is possible because T cell growth factors can be produced in large quantities industrially through genetic engineering technology, and large amounts of T cell growth factors can be used, it is possible to use this method for long periods outside of the whole killer T cell. There are problems such as cell deterioration due to long-term culture. In addition to IL, there are various other problems that are difficult to eliminate.5For example, Killer T
The patient's normal cells and surgery are required for cell induction;
There are many problems that need to be resolved, such as the fact that killer T cells can only be induced in some cancer patients and not all cases, and that the operation is extremely complicated.
（発明が解決しようとする問題点）
本発明者らは、従来の方法よりも飛躍的に実用性を向上
させｆｃｌｉ規な腫膓障吾性細胞誘導活性化法、すなわ
ち、容易に入手できる物ｉｔ刺激剤として用い、はとん
どすべての癌患者で強力な＠瘍障害性細胞を短時間で、
かつ操作性よく誘導活性比できる方法を見い出す之め瑣
々のリンパ球活性化材を慣策し、′＄！ｉ々の悪性＠膓
治僚用白血球訪導材を見出してきた（特願昭５８−２２
８４９６号。(Problems to be Solved by the Invention) The present inventors have developed a method for inducing and activating tumescent cells that is dramatically more practical than conventional methods, that is, an easily available method. Used as an IT stimulant, it has a strong effect on tumor-causing cells in almost all cancer patients in a short period of time.
In addition, we have found a method that allows us to increase the induced activity ratio with ease of use, and have developed various lymphocyte activation materials. We have discovered a leukocyte-conducting material for malignant @Eiji staff (Patent application 1986-22)
No. 8496.
同５９−２０５５５９号、同６０−１６８５７２号）。No. 59-205559, No. 60-168572).
［Ｌ悪性１練膓治僚用白血球誘導方法についても鋭意恢
討し九結果、種々の発明を成してきた（特願昭５８−２
２８４９６号）。[We have also conducted intensive research on leukocyte induction methods for use in L-malignant 1 training, and have made various inventions as a result.
No. 28496).
本発明は、悪性嘘勃治僚用白血球誘導方法および誘４装
置に関し詳細に検討され九結果成されたものであり、抗
凝固剤に工らず効率よく白血球を刺激できるようにし次
刺激方法および装置に関するものである。The present invention is the result of a detailed study on a leukocyte induction method and an induction device for treating malignant erectile dysfunction. It is related to the device.
（問題点を解決するための手段）
本発明者らは、前記目的に旧って悪性株膓治療用白血球
誘導方法について鋭意研究してき友が。(Means for Solving the Problems) For the above purpose, the present inventors have been conducting intensive research on leukocyte induction methods for treating malignant strains.
驚くへきことに、多価カチオンのキレート化剤で抗血液
凝固し之血液からは、悪性肺膓治僚用の白血球がほとん
ど誘導されないの九対し、多価カチオンのキレート化剤
以外の抗凝固剤で抗血液凝固し几血液からは、非常に高
い効率、活性で悪性腫瘍治療用の白血球が誘導されるこ
とを見出し、さらＶｃは、白血球の刺激時にカルシウム
イオン全存在させれは、悪性腫瘍治療用の白血球が誘導
できること、すなわち、多価カチオンのキレート化剤で
抗凝固した血液でも、悪性核鳴治僚用白血球刺激材と白
血球とを接触させる際に、カルシウムイオンと多価カチ
オンのキレート化剤以外の抗凝固剤を加えるなどして、
カルシウムイオンが存在できるようにしてやれば、悪性
Ｍ瘍治僚用の白血球が高ぶかつ尚活性に４４されること
を見出し１本発明′！ｉ−′７ｒＳ成するに至った。Surprisingly, blood anticoagulated with polyvalent cation chelating agents hardly induces leukocytes for malignant lung disease, whereas anticoagulants other than polyvalent cation chelating agents It was discovered that white blood cells for the treatment of malignant tumors can be induced from anti-coagulant blood with very high efficiency and activity. In other words, even in blood that has been anticoagulated with a polyvalent cation chelating agent, calcium ions and polyvalent cations can be chelated when the leukocyte stimulating material for malignant nuclei is brought into contact with the leukocytes. By adding an anticoagulant other than the anticoagulant,
We have discovered that by allowing calcium ions to exist, white blood cells used to treat malignant tumors can be increased and become more active. 1. The present invention'! i-'7rS was completed.
すなわち１本発明は、悪性粗錫治療用白血球刺激材と白
血球とをカルシウムイオン存在下で衾融させることｙｋ
！徴とする紐１−僚用白血球刺激方法であり、さらに、
白血球導入口、悪性筒易治療用白血球刺激材を充填して
成る悪性腫膓治僚用白血琢刺倣器、白血球導出口の順に
配置されてｂる装置であって、白血球導入口から悪性１
虚易治僚用白血球刺激器までの間に、多価カチオンのキ
レート化剤以外の抗凝固剤ま几は多価カチオンのキレー
ト化剤以外の抗凝固剤とカルシウム塩を導入するための
注入口を少なくとも一つ設けてなることを特徴とする悪
性）庫瘍治僚用白血球刺激装置である。That is, 1 the present invention is to fuse a leukocyte stimulating material for the treatment of malignant protease and leukocytes in the presence of calcium ions.
! Characteristic String 1 - A method for stimulating white blood cells, and further,
A leukocyte inlet, a leukocyte stimulation device for treating malignant tumors filled with a leukocyte stimulating material for treating malignant tumors, and a leukocyte outlet are arranged in this order, and the device is arranged in this order:
Between the white blood cell stimulator and the white blood cell stimulator, there is an inlet for introducing anticoagulants other than polyvalent cation chelators and calcium salts. This is a leukocyte stimulator for use in treating malignant tumors, characterized in that it is provided with at least one of the following.
本発明における白血球とは、血液細胞の内赤血球および
血小板を除いた。いわゆる白血球を指すが、この白血球
ニジ顆粒球あるいはＢ細胞を除去し７ｃ細胞分画も１本
発明における白血球の概念に含まれる。本発明において
活性化を行なう白血球は、公知の連続逼心分離法にて末
梢血より採取した白血球分画を用いてもよ＜、’！７ｔ
−公知のフィコールバーク重層遠心分離法にて分離した
単核細胞分画でもよく、あるいは末梢血単核細胞より公
知のノイラミニダーゼ処理羊赤血球とのロゼツト形既で
分離＆縮し九Ｔ細胞分画を使用しても、強力な腫瘍障害
性細胞の誘導が可能である。In the present invention, white blood cells include blood cells excluding red blood cells and platelets. This term refers to so-called white blood cells, but the concept of white blood cells in the present invention also includes white blood cells, granulocytes, and 7c cell fractions obtained by removing B cells. The leukocytes to be activated in the present invention may be a leukocyte fraction collected from peripheral blood by a known continuous separation method. 7t
- The mononuclear cell fraction separated by the known Ficoll-Birk multilayer centrifugation method may be used, or the 9T cell fraction may be obtained by separating and shrinking peripheral blood mononuclear cells in a rosette shape with known neuraminidase-treated sheep red blood cells. Even when used, induction of potent tumor-toxic cells is possible.
本発明において誘導活性化する穫＠障害性細胞は、白血
球の中で顆粒球、単球、マクロファージを除くリンパ球
分画に属し、とりわけで細胞の性質を有している。The harmful cells to be induced and activated in the present invention belong to the lymphocyte fraction of white blood cells, excluding granulocytes, monocytes, and macrophages, and have particularly cell characteristics.
本発明で言う悪性腫膓治僚用白血球刺激材とは。What is the leukocyte stimulating material for treating malignant tumors in the present invention?
白血球と接触することにより肺ＩＪ！障害性細胞を誘導
できる水に不溶な物質であるが１例えば、不溶性の担体
に、白血球と接触することによｐ腫傷障害性細胞を誘導
可能な刺激材を共有結合させて得られる。Lung IJ due to contact with white blood cells! A water-insoluble substance capable of inducing tumor-damaging cells can be obtained, for example, by covalently bonding to an insoluble carrier a stimulating material capable of inducing tumor-damaging cells by contacting leukocytes.
本発明において用いる刺激剤としては、リンパ球を活性
化する作用を持った物’Ｊ［を使用するが。The stimulant used in the present invention is a substance that has the effect of activating lymphocytes.
符にＴ Ｕンバ球を活性化する物質が好ましＬｎ、 Ｔ
リンパ球を活性化する物質としてよく知られてｂるもの
にレクチンがあるが１本発明の目的全達成するためには
、＠ｌ！障害性細胞誘導能を有するレクチンを使用する
。Preferably a substance that activates the T Unba sphere, Ln, T
Lectin is a well-known substance that activates lymphocytes, but in order to achieve all the objectives of the present invention, @l! A lectin that has the ability to induce harmful cells is used.
すなわち、ファセオラス・ブルガリス
（Ｐｈａｓｅｏｌｕｓ ｖｕｌｇａｒｉｓ ）由来のア
カインゲンマメレクチン（ＰＨＡ ）、コンカナバリア
・エンシフオルミス（Ｃｏｎｃａｎａｖａｌｉａ ｅｎ
ｓｉｆｏｒｍｉｓ ）由来のコンカナバリンＡ （Ｃｏ
ｎ Ａ ） 、ライステリア・アオリバンダ（Ｗｉｓｔ
ｅｒｉａ ａｏｒｉｂａｎｄａ ）由来のノダクジマメ
レクテン（ＷＦＡ ）、レンズ・キュリナリ、ｘ、 （
Ｌｅｎｓ ｃｕ！１ｎａｒｉｓ ）由来のレンズマメレ
クチン（ＬＣＨ）、フィトラッカ・アメリカーナ（Ｐｈ
ｙｔｏｌａｃｃａ ａｍｅｒｉｃａｎａ ）由来のアメ
リカヤマゴボウレクチン（ＰＷＭ）、グリシン・マック
ス（Ｇｌｙｃｉｎｅ ｍａｘ ）由来のダイズレクチ７
（ＳＢＡ）。Namely, red bean lectin (PHA) derived from Phaseolus vulgaris, Concanavalia ensiformis
concanavalin A (Co
nA), Riceteria aoribanda (Wist
eria aoribanda), Lens culinari, x, (
Lens cu! 1naris), lentil lectin (LCH) from Phytolacca americana (Ph.
ytolacca americana) and soybean lectin 7 derived from Glycine max.
(SBA).
フォセオラス会すメンシス（Ｐｈａｓｅｏｌｕｓ ｌｉ
ｍｅｎｓｉｓ）由来のりママメレクチン（ＬＢＡ）、ロ
ビナ・プソイドアカシア（Ｒｏｂｉｎａ ｐｓｅｕｄｏ
ａｃａｃｉａ ）由来のニセアカシアレクテン（ＲＰＡ
Ｊ、ノホラ・ジャポニカ（５ｏｐｈｏｒａ ｊａｐｏｎ
ｉｃａ ）由来のイヌエンジュマメレクチン（ＳＪＡ）
、 ビサム・サチバム（Ｐｉｓｕｍ ｓａｔｉｖｕｍ
）由来のｘ７ドウマメレクチン（ＰＳＡ）、 ビシア−
７アパ（Ｖｉｃｉａ ｆａｂａ ）由来のソラマメレク
テン（ＶＦＡ）等を使用する。中でも特にアカインゲン
マメレクチン（ＰＨＡ）。Phaseolus mensis (Phaseolus li)
Glue mamame lectin (LBA) from Robina mensis, Robina pseudoacacia (Robina pseudoacacia)
acacia) derived from locust acacia lectene (RPA)
J, Nophora japonica (5ophora japonica)
dog bean lectin (SJA) derived from ica
, Pisum sativum
), x7 horse bean lectin (PSA), derived from Vicia
Vicia faba (VFA) derived from Vicia faba is used. Among them, red bean lectin (PHA).
コンカナバリンＡ （Ｃｏｎ Ａ ） 、ノダクジマメ
レクテン（ＷＦＡ）、レンズマメレクチン（ＬＣＨ）。Concanavalin A (Con A), black bean lectin (WFA), lentil lectin (LCH).
アメリカヤマゴボウレクチン（ＰＷＭ）は０強力に腫膓
陣害性細胞を誘導する。また、スタフィロコッカス・ア
ウレウス（５ｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕ
ｓ）由来のプロティンＡも強力にＭ腸障害性細胞を誘導
する。また、ヒト末梢血白血球全レクチンで刺激した培
養上清中に含まれるリンフ才力インも刺激剤として使用
でき、特にゲルクロマトグラフィー等公知の方法によっ
てｄ’Ａしたインターリューキン２（ＩＬ２）分画が強
力な刺激剤として使用できる。Pokeweed lectin (PWM) strongly induces tumorigenic cells. Also, Staphylococcus aureus (5taphylococcus aureus)
Protein A derived from s) also strongly induces M enteropathic cells. Lymphin, which is contained in the culture supernatant stimulated with human peripheral blood leukocyte whole lectin, can also be used as a stimulant, especially interleukin 2 (IL2) fractions that have been d'A'd by known methods such as gel chromatography. can be used as a powerful stimulant.
この他、核酸塩基、負電荷を持つ物質等も刺激剤として
使用できる。In addition, nucleic acid bases, negatively charged substances, etc. can also be used as stimulants.
本発明で用いられる不溶性担体は、親水性担体。The insoluble carrier used in the present invention is a hydrophilic carrier.
疎水性担体いずれも使用できるが、疎水性担体を用いる
場合には、符に担体への血清成分の非特異的吸着が生じ
る沈め、親水性担体の方が好ましｈ結果を与える。不溶
性担体の形状は１粒子状、繊維状、中空糸状、膜状等い
ずれの公知の形状も用いることができる。粒状もしくは
球状不溶性担体としては１粒径１〜３０００ミクロンの
ものカ使用できる。粒径１ミクロン以下では活性化白血
球との分離が困難である。とくに粒径５０ミクロン以上
であれば、容易に活性化白血球との濾過分離が可能であ
シ１粒径３０００ミクロン以上では。Any hydrophobic carrier can be used, but when a hydrophobic carrier is used, a hydrophilic carrier is preferable, since non-specific adsorption of serum components to the carrier may occur. The shape of the insoluble carrier may be any known shape such as a single particle, a fiber, a hollow fiber, or a membrane. As the granular or spherical insoluble carrier, one having a particle size of 1 to 3000 microns can be used. If the particle size is 1 micron or less, it is difficult to separate it from activated leukocytes. In particular, if the particle size is 50 microns or more, it can be easily separated from activated leukocytes by filtration, whereas if the particle size is 3000 microns or more.
白血球との担体単位重量あたりの接触面積が低下するた
め好ましくない。さらに好ましくは１粒径８０〜２００
０ミクロンのものが本発明において良好である。また１
粒状もしくは球状不溶性担体の比重が１．０７以上であ
れば、容易に活性化白血球との遠心もしくは静置による
分離が可能である。This is not preferable because the contact area per unit weight of the carrier with leukocytes decreases. More preferably, one particle size is 80 to 200.
0 micron is good in the present invention. Also 1
If the specific gravity of the granular or spherical insoluble carrier is 1.07 or more, it can be easily separated from activated leukocytes by centrifugation or standing still.
また、平膜状あるいは中空糸状多孔性担体を使用する場
合、その孔径が、細胞は通過できなりが培地成分は自由
にｍ過できる０、０５〜１０ミクロンのもの全使用すれ
ば、膜内面に結合した白血球に膜外面より栄９！全補給
でき、高濃度の白血球を刺激活性化することが可能であ
る。さらに好ましくは、０．１〜５ミクロンの孔径の多
孔性担体が良好に使用できる。In addition, when using a flat membrane-like or hollow fiber-like porous carrier, if the pore size is 0.05 to 10 microns, which cells cannot pass through but medium components can freely pass through, it is recommended that the inner surface of the membrane be Sakae 9 from the outer surface of the membrane to the bound white blood cells! It can fully replenish and stimulate and activate a high concentration of white blood cells. More preferably, a porous carrier having a pore size of 0.1 to 5 microns can be used successfully.
不溶性担体の材質としては、アガロース系、デキストラ
ン系、セルロース系、ポリアクリルアミド系、ガラス系
、活性炭系、ポリアミド系、ポリエステル系、ポリウレ
タン系等の物質あるいは再生セルロース系、ナイロン、
アクリル、ポリエステル等公知の繊維性物ＩＸを一般に
用いることができる。Materials for the insoluble carrier include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, activated carbon-based, polyamide-based, polyester-based, polyurethane-based materials, regenerated cellulose-based, nylon, etc.
Known fibrous materials IX such as acrylic and polyester can generally be used.
刺激剤を不溶性担体の表面に固定する方法としては、共
有結合、イオン結合、物理吸着等あらゆる公知の方法を
用めることができるが、刺激剤の溶出性から考えると、
共有結合で固定して用いることが望ましい。そのために
は通常固定化酵素。All known methods such as covalent bonding, ionic bonding, and physical adsorption can be used to immobilize the stimulant on the surface of the insoluble carrier, but considering the leaching properties of the stimulant,
It is preferable to use it by fixing it with a covalent bond. For this purpose usually immobilized enzymes are used.
アフイニテイクロマトグラフィで用いられる公知の方法
を用いることができる。例えば、臭化水素（ＣＮＢｒ
）でアガロース、セファロース等全活性化し、あるｂは
シリカガラスピーズ全γ−アミノプロピルトリエトキシ
シランと反応させてアルキルアミノガラスを得、これを
グルタルアルデヒドで活性化し、刺激剤と結合させる等
の方法を用することができる。ま友必要に応じて、不溶
性担体と刺激剤の間に任意の長さの分子（スペーサー）
を導入して使用することもできる。例えは、アガロース
のヒドロキシル基とへキサメチレンジイソシアナートの
片側のインシアナート基を反１６結合さぜ、残つ九イソ
シアナート基と刺激剤のアミン基を反応結合させるごと
〈実施することができる。Known methods used in affinity chromatography can be used. For example, hydrogen bromide (CNBr
) to activate all agarose, Sepharose, etc., and (b) react with silica glass beads with all γ-aminopropyltriethoxysilane to obtain alkylamino glass, which is activated with glutaraldehyde and combined with a stimulant. can be used. If necessary, any length molecule (spacer) between the insoluble carrier and the stimulant
It is also possible to introduce and use it. For example, it can be carried out by combining the hydroxyl group of agarose and the incyanate group on one side of hexamethylene diisocyanate through an anti-16 bond, and then reacting and bonding the remaining 9 isocyanate groups with the amine group of the stimulant.
悪性腫瘍治療用白血球刺激材による白血球の活性化は、
ａ胞の栄養成分と酸素、二酸化炭素等の存在下で行なわ
れるが、この時カルシウムイオンが存在しないと、悪性
棟湯治療用の白血球が誘導されないので、カルシウムイ
オンを存在させる必要がある。Activation of leukocytes by leukocyte stimulating materials for malignant tumor treatment is
It is carried out in the presence of nutritional components of a-cells, oxygen, carbon dioxide, etc., but if calcium ions are not present at this time, white blood cells for the treatment of malignant ridges will not be induced, so calcium ions must be present.
血液を体外にとり出す際には、抗血液凝固剤が使用され
るが、一般に用いられているのは、クエン酸ナトリウム
のように２価のイオンをキレート固定してしまうキレー
ト化剤、ヘパリンのようにプロトロンビンがトロンビン
に変化するのを防ぐ薬剤等である。このうち、キレート
化剤は、連続遠心分離装置で白血球を採取する場合には
好んで用いられているが、この場合、得られる白血球浮
遊液にはカルシウムイオンがほとんど存在せず。Anticoagulants are used when blood is taken out of the body, but the commonly used agents are chelating agents such as sodium citrate, which chelate and fix divalent ions, and heparin, which fixes divalent ions. These include drugs that prevent prothrombin from converting into thrombin. Among these, chelating agents are preferably used when leukocytes are collected using a continuous centrifuge, but in this case, the resulting leukocyte suspension contains almost no calcium ions.
これを白血球刺激剤と接触させても、悪性１産湯治療用
の白血球はほとんど誘導されなり０そのｔめ。Even if this is brought into contact with a white blood cell stimulant, most of the white blood cells for the treatment of malignant disease are induced, and the result is 0.
連続遠心分離装置で白血球′ｔ−採取する際にも、抗ａ
ｔ 固剤トして、ヘパリンのようにカルシウムイオンを
キレートしない薬剤を用いるか、またはキレート化剤を
用いて白血球を採取した後、カルシウム塩とキレート化
剤以外の抗凝固剤を加えて、カルシウムイオンが存在す
るようにしてやる必要がある。When collecting white blood cells using a continuous centrifuge, anti-a
After collecting white blood cells using a solid agent and using a drug that does not chelate calcium ions, such as heparin, or using a chelating agent, calcium salts and an anticoagulant other than the chelating agent are added. It is necessary to make sure that ions exist.
カルシウムイオンの存在量は、白息ｆｆ 刺激材テ白血
球を刺激する場合には、０．１から２００ ｍＥｑ／ｌ
Ｃ）＠囲で抗鑵場性の白血球を誘導することができる。The amount of calcium ions present is 0.1 to 200 mEq/l when stimulating white blood cells.
C) Anti-inflammatory leukocytes can be induced by @surroundings.
より好ましめ範囲は０．２から１００ ｍＥｑ／１．さ
らに好ましいのは０．５から５０ ｍＥｑ／ｌの範囲で
あり、最も好ましいのは０．５から１０ｍＥｑ ／ ｌ
の範囲である。A more preferable range is 0.2 to 100 mEq/1. More preferred is a range of 0.5 to 50 mEq/l, most preferred is 0.5 to 10 mEq/l
is within the range of
＠＠障害性細胞誘導能を持つ悪性腫瘍治療用白血球刺激
材を実際の臨床に用いる場合は、友とえば、第１図に示
し次悪性１ｉ膓治療用白血球刺激器を用いる。このｉ置
は容器１内に白血球刺激材２を収容し１両瑞に白血球流
入口５および流出口４を有し、それぞれフィルター５で
区分されている。＠＠When using a leukocyte stimulator for treating malignant tumors that has the ability to induce harmful cells in actual clinical practice, for example, a leukocyte stimulator for treating malignant tumors shown in FIG. 1 is used. The container 1 contains a leukocyte stimulating material 2, and has a leukocyte inlet 5 and an outlet 4 in both chambers, each separated by a filter 5.
このフィルター５は担体２が容器１外に流出するのを防
ぐものである。This filter 5 prevents the carrier 2 from flowing out of the container 1.
上述し友悪性値膓治療用白血球刺激器は、例えばＳ第２
図に示すような装置で使用される。The above-mentioned leukocyte stimulator for treating leukemia is, for example, S-2.
Used in a device like the one shown in the figure.
７は全血より分取されｔ白血球浮遊液または白血球を含
む全血の導入口、すなわち、白血球導入口であり、８は
これらの血球の導出口、すなわち。7 is an inlet for introducing a leukocyte suspension or whole blood containing leukocytes separated from whole blood, ie, a leukocyte inlet, and 8 is an outlet for these blood cells, ie.
白血球導出口である。６は悪性＠易治療用白血球刺激器
であり、９は多価カチオンキレート化剤以外の抗凝固剤
または多価カチオンキレート化剤とカルシウム塩を導入
するための注入口である。This is the leukocyte outlet. 6 is a leukocyte stimulator for malignant treatment; 9 is an injection port for introducing an anticoagulant other than the polyvalent cation chelating agent or a polyvalent cation chelating agent and calcium salt.
白血球導入ロアから全血が導入される場合で説明すると
、全血は白血球導入ロアから導入され。To explain the case where whole blood is introduced from the leukocyte introduction lower, whole blood is introduced from the leukocyte introduction lower.
注入口９から注入され几多価カチオンキレート化剤以外
の抗凝固剤（５１えは、ヘパリン）と混合され、血液が
固まらないようにされ九後、悪性稙鳴治療用白血球刺激
器６に送られ、ここで全血中の白血球が刺激され、抗植
務性の白血球が誘導される。この後、刺激され九白血球
と共に全血が白血球導出口８より導出され、悪性１重湯
治療用の白血球を含む血液が得られる。The blood is injected from the injection port 9 and mixed with an anticoagulant (51, heparin) other than the polyvalent cation chelating agent to prevent blood from clotting, and then sent to the leukocyte stimulator 6 for treating malignant chorion. Here, leukocytes in whole blood are stimulated, and anti-implantation leukocytes are induced. Thereafter, the stimulated whole blood together with the leukocytes is drawn out from the leukocyte outlet 8 to obtain blood containing leukocytes for treatment of malignant ichijuto.
次に、遠心分離器等で分離され、全血より分取されたキ
レート化剤を含む白血球浮遊液が白血球導入ロアより導
入されるケースについて説明する。Next, a case will be described in which a leukocyte suspension containing a chelating agent separated from whole blood by a centrifuge or the like is introduced from the leukocyte introduction lower.
キレート化剤を含む白血球浮遊液は、白血球導入ロアか
ら導入された後、注入口９から圧入され九カルシウム塩
によりキレート化剤が中和され。A leukocyte suspension containing a chelating agent is introduced from the leukocyte introduction lower, and then press-injected through the injection port 9, and the chelating agent is neutralized with a nine-calcium salt.
同時に注入された多価カチオンキレート化剤以外の抗凝
固剤と混合され、血液が固まらないようにされる。この
後、白血球浮遊液は悪性腫瘍治療用白血球刺激器６に送
られ、ここで抗＠感性の白血球が誘導される。この後、
刺激され之白血球は、白血球導出口８より導出される。It is mixed with an anticoagulant other than the polyvalent cation chelator, which is injected at the same time, to prevent blood from clotting. Thereafter, the leukocyte suspension is sent to a leukocyte stimulator 6 for malignant tumor treatment, where anti-@ sensitive leukocytes are induced. After this,
The stimulated leukocytes are led out from the leukocyte outlet 8.
ま友、この装置は連続遠心分離器１０と組合わせて、嶌
３図のようにして便うこともできる。Friend, this device can also be used in combination with a continuous centrifugal separator 10 as shown in Figure 3.
全血を導入をする例で説明すると、全血は白血球導入ロ
アから導入され、圧入口９から注入され九多価カチオン
キレート比剤以外の抗凝固剤と混合され、抗縦固され九
後、遅続遠心分離器１０に送られ、白血球成分とそれ以
外の取分に分離され。To explain the example of introducing whole blood, whole blood is introduced from the leukocyte introduction lower, injected from the injection port 9, mixed with an anticoagulant other than the polyvalent cation chelate ratio agent, and subjected to anti-vertical solidification. It is sent to a slow centrifuge 10 and separated into white blood cell components and other fractions.
白血球成分は悪性腫瘍治療用白血球刺激器６に送られ、
それ以外の成分は、白血球導出口８よシ導出される。白
血球は刺激器６内で刺激され、抗植瘍性の白血球が誘導
される。この後、刺激され几白血球は、白血球導出口８
より導出される。The white blood cell component is sent to a white blood cell stimulator 6 for malignant tumor treatment.
Other components are led out through the white blood cell outlet 8. The leukocytes are stimulated within the stimulator 6, and anti-inplant leukocytes are induced. After this, the stimulated white blood cells are transferred to the white blood cell outlet 8.
It is derived from
このようにして回収した活性化白血球は１強力な＠ｖＪ
障害細胞を含有する。すなわち、活性化白血球を各種ヒ
ト龜膓細胞に作用させ次ところ。The activated leukocytes collected in this way have a strong @vJ
Contains damaged cells. That is, activated leukocytes were allowed to act on various types of human pharyngeal cells.
ＺＲ７７５−５０Ｋ細胞、 ＭＫＮ−１１Ａｌｉ細胞、
ＰＣ−９肺癌細胞、Ｃ−１結腸癌細胞、ＮＥＴ−２膀胱
癌細胞、ＮＲＣ−１２’ｌｉ＋ｉ癌細胞等を強く障害し
友。ZR775-50K cells, MKN-11Ali cells,
It strongly damages PC-9 lung cancer cells, C-1 colon cancer cells, NET-2 bladder cancer cells, NRC-12'li+i cancer cells, etc.
（発明の効果）
以上述べてきたように１本発明の悪性腫傷冶療用白血球
刺激方法を用いることによ、す、抗凝固剤の種類によら
ず、確実に、高い効率で、高い活性の悪性腫瘍治療用白
血球を誘導できるようになつ几。(Effects of the Invention) As described above, by using the leukocyte stimulation method for malignant tumor treatment of the present invention, it is possible to reliably, efficiently, and highly activate the leukocyte, regardless of the type of anticoagulant. It is now possible to induce leukocytes for the treatment of malignant tumors.
また１本発明の悪性肺瘍治療用刺激装置を用いることに
より、全血でも、連続遠心分離装置で分離した白血球分
画でも、悪性＠膓治療用刺激器内で白血球が刺激材と接
触する際に、カルシウムイオンを存在させることができ
るので、確実に、蘭匣に悪性帷易治療用の白血球が誘導
できるようになつ友。In addition, by using the stimulator for malignant lung tumor treatment of the present invention, whether it is whole blood or leukocyte fraction separated with a continuous centrifugation device, when leukocytes come into contact with the stimulant in the stimulator for malignant lung tumor treatment, Since calcium ions can be present in the blood, leukocytes for the treatment of malignant diseases can be definitely induced in the orchid.
さらに、患者末梢血白血球全効率よく活性化し。Furthermore, the patient's peripheral blood leukocytes were all efficiently activated.
安全Ｋかつ操作性よく１強力な１厘易陣害性細胞を誘導
することができ、胃癌、肺癌、乳癌、肝癌等の癌治療に
有用である。It is safe and easy to operate, and is capable of inducing strong and easily invasive cells, and is useful for cancer treatment such as gastric cancer, lung cancer, breast cancer, and liver cancer.
（実施例）
以下、実施例により１本発明の実施の題様をより詳細に
説明する。(Example) Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples.
実施例１
悪性腫瘍治療用白血球刺激材の調製は１次のようにして
行なった。すなわち、 ＣＮＢｒＮＢｒ活性化セファロ
−２６スＢ−デン、ファルマシ７社裂。Example 1 A leukocyte stimulating material for treating malignant tumors was prepared as follows. Namely, CNBrNBr-activated Sepharose-26S B-den, Pharmacy 7-sha.
粒径２５０〜３５０ミクロン）に、刺激剤として。(particle size 250-350 microns) as an irritant.
通常の方法によってＰＨＡを結合せしめ、過剰の活性基
金グリシンでブロッキングした後、ｐＨ４−０，Ｉ Ｍ
酢酸バッファー、ｐＨ８，５炭酸ナトリウムバツフアー
でくり返し洗浄後、生理食塩水で洗浄５水切りして実験
に供した。不溶性担体の刺激剤保持量は、初めに添加；
−之刺激剤量より、結合反応後の上清刺激剤量および洗
＃液中の刺激剤量をさし引いて結合量を求め計算したと
ころ、不溶性担体１ゴあｆｃり１．５ηであった。なお
、刺激剤量は２ Ｂ Ｏｎｍの吸光度で測定し友。After binding PHA by conventional methods and blocking with excess active fund glycine, pH 4-0, IM
After repeated washing with acetic acid buffer, pH 8.5 sodium carbonate buffer, washing with physiological saline and draining, the sample was used for experiments. The stimulant holding amount of the insoluble carrier is added at the beginning;
The amount of binding was calculated by subtracting the amount of stimulant in the supernatant after the binding reaction and the amount of stimulant in the washing solution from the amount of stimulant. Ta. The amount of stimulant is measured by absorbance at 2 B Onm.
ヒト白血球は次のようシで一℃得比。すなわち。Human leukocytes were collected at 1°C as follows: Namely.
採血したヒト末柑血をノ・ンークス液で２倍希釈し。The collected human blood was diluted 2 times with Nox's solution.
フィコールバークｉｇ（ファルマシア社ｆＭ）に重層し
、２０００ｒｐｍで２０分間遠心分離した後。After layering on Ficoll Bark ig (Pharmacia fM) and centrifuging at 2000 rpm for 20 minutes.
中間層の白血球層上分離して、これをリン酸緩賃生理食
塩水（ＰＢＳ）で洗つ几後、ヘパリン加自己血漿に２Ｘ
１０６／−の細胞濃度で浮遊させた。After separating the middle white blood cell layer and washing it with phosphate saline (PBS), it was added to heparinized autologous plasma 2X.
The cells were suspended at a cell concentration of 106/-.
この細胞浮遊液を１ｄずつ、細胞培養用の゛２−ウェル
（ファルコン４５０４７）に分注し、これ疋刺激材を２
０μｔずつ象加し、ＣＯ□インキュベーター中で温度３
７Ｃで培養を行なつ文。培養１時間程度で白血球の刺激
材表面上への付着が観察される。Dispense 1 d of this cell suspension into 2-wells for cell culture (Falcon 45047), and add 2 d of stimulation material to each well.
Add 0μt in incubator at temperature 3 in CO□ incubator.
A statement about culturing at 7C. Adhesion of leukocytes to the surface of the stimulation material is observed after about 1 hour of culturing.
２４時間培養を行なつ友後、培養液をピペッティングし
て活性化白血球を刺激材表面からはがして静置すると、
刺激材は容器の底に沈下するので。After culturing for 24 hours, the activated leukocytes are peeled off from the surface of the stimulation material by pipetting the culture solution and left to stand still.
Because the stimulant will sink to the bottom of the container.
上清細胞液をとり、これをＰＢＳで洗つ之後、自己ヘパ
リン加血漿に５Ｘ１０６／−の細胞濃度で浮遊させた。The supernatant cell solution was collected, washed with PBS, and then suspended in autologous heparinized plasma at a cell concentration of 5×10 6 /−.
この活性化白血球が＠膓細胞障害性を有するかどうかは
１次のようなキラー活性測定法を用いて評価した。培養
プレートに付着して増殖する種々のヒト癌細胞株を標的
細胞として、５Ｘ１０４／ｍ／の細胞濃度で１０チ牛脂
児血清添加ＲＰＭ１１６４！Ｊ培地に浮遊させ、これを
１０μｔずつ１０μを容テラサキプレートに分注し、
ＣＯ，インキュベーター中で温度３７Ｃで培養する。２
４時間培ｇ！を行なうと、癌細胞は培養プレート底面に
強く付着する。Whether or not these activated leukocytes had cytotoxicity was evaluated using the following killer activity measurement method. Targeting various human cancer cell lines that adhere to and proliferate on culture plates, RPM1164 was added with 10 T tallow serum at a cell concentration of 5 x 104/m/m. Float in J medium, dispense 10 μt each into Terasaki plates,
Incubate at a temperature of 37C in a CO, incubator. 2
4 hours cultivation! When this is done, cancer cells strongly adhere to the bottom of the culture plate.
これ全ヘパリン加血漿で洗った後、活性化白血球浮遊液
１０μｔを添加し、３７Ｃで４時間、 Ｃｏ。After washing with whole heparinized plasma, 10 μt of activated leukocyte suspension was added and incubated at 37C for 4 hours with Co.
インキュベーター中で培養し、プレートに付層している
癌細胞を障害させる。障害を受けた癌細胞は、プレート
底面への付着性を喪失し、ハンクス液で洗うと活性化白
血球とともに除去さする。生残してプレート底面に付着
している癌細胞全アセトンで固定し、ギムザ液で染色し
ｔ後、顕微鏡で計数する。キラー活性は次式によシ計算
する。Cultivate in an incubator to damage cancer cells layered on the plate. Damaged cancer cells lose their adhesion to the bottom of the plate and are removed along with activated leukocytes when washed with Hank's solution. All cancer cells that remain alive and adhere to the bottom of the plate are fixed with acetone, stained with Giemsa solution, and then counted using a microscope. Killer activity is calculated using the following formula.
キラー活性＝
このようにして評価したキラー活性は、ＺＲ７５−３ｏ
ヒト乳癌、１ｉｆｆｌｉｌｃ対し、テ８０９に、ＭＫ
Ｎ −１ヒト胃癌細胞に対して８４チであつ次。Killer activity = Killer activity evaluated in this way is ZR75-3o
Human Breast Cancer, 1ifflilc, Te 809, MK
84 against N-1 human gastric cancer cells.
比較例１ 実施例１でヘパリン加血漿を用い友ところに。Comparative example 1 In Example 1, heparinized plasma was used.
ＡＣＤ（キレート化剤）加血漿を用い几以外は全て実施
例１と同様に実験した。The experiment was conducted in the same manner as in Example 1 except for using ACD (chelating agent)-added plasma.
その結果、キラー活性はＺ Ｒ７５−３０ヒト乳癌−に
対し４チ、ＭＫＮ−１ヒト胃癌細胞に対し７％であつ友
。As a result, the killer activity was 4% against ZR75-30 human breast cancer cells and 7% against MKN-1 human gastric cancer cells.
すなわち、カルシウムイオンをキレート化剤でキレート
した血漿中では、 ＠Ｖｉ＆障害性白血球はほとんど誘
導されなかった。That is, in plasma in which calcium ions were chelated with a chelating agent, @Vi & impaired leukocytes were hardly induced.
実施例２
実施例１のヘパリン加血漿の代りに、ＡＣＤ（キレート
化剤）加血漿にグルコン酸カルシウムを加えてＡＣＤを
中和すると同時にヘパリンを加え九血Ｗｔを用い友外は
、全て実施例１と同様にして実験した。Example 2 Instead of the heparin-added plasma in Example 1, calcium gluconate was added to the ACD (chelating agent)-added plasma to neutralize ACD, and at the same time, heparin was added and Kuketsu Wt was used. The experiment was conducted in the same manner as in 1.
その結果、キラー活性はＺ Ｒ７５−３０ヒト乳癌細胞
に対し７２チ、ＭＫＮ−１ヒト胃癌細胞に対し７４％で
あつ友。As a result, the killer activity was 72% against ZR75-30 human breast cancer cells and 74% against MKN-1 human gastric cancer cells.
すなわち、カルシウムイオンをキレート化剤でキレート
し次血漿でも、キレート化剤以外の抗凝固剤とカルシウ
ムを加えることにより、 啜＠＊″４性白血球が誘導で
きるようになつ九。In other words, by chelating calcium ions with a chelating agent and then adding calcium and an anticoagulant other than the chelating agent to plasma, it becomes possible to induce 4-type leukocytes.
５ｇ１図は悪性嘘易治僚用白血球刺激材を充填してなる
白血球刺激器の一例を示す模式図、忍２図は悪性嘘城治
僚用白血球刺激装置の一例を示す模式図、第３図は悪性
＠易治僚用白血球刺激装置の他の例を示す模式図である
。
１−・・・・・容器、２・・・・・・白血球刺激材、３
・・・・・・白血球流入０．４・・・・・・白血球流出
０．５・・・・・・フィルター。
６・・−・・・悪性値膓治療用白血球刺激器。Figure 5g1 is a schematic diagram showing an example of a leukocyte stimulator filled with a white blood cell stimulating material for malignant lizards, Figure 2 is a schematic diagram showing an example of a leukocyte stimulator for malignant lizards, and Figure 3 is a schematic diagram showing another example of a leukocyte stimulator for malignancy. 1- Container, 2 White blood cell stimulating material, 3
...Leukocyte inflow 0.4...Leukocyte outflow 0.5...Filter. 6.--Leukocyte stimulator for the treatment of malignant blood cells.
Claims (2)
ウムイオン存在下で接触させることを特徴とする悪性腫
瘍治療用白血球刺激方法。(1) A method for stimulating leukocytes for treating malignant tumors, which comprises bringing a leukocyte stimulating material for acute tumor treatment into contact with leukocytes in the presence of calcium ions.
填して成る悪性腫瘍治療用白血球刺激器。 白血球導出口の順に配置されている装置であつて、白血
球導入口から悪性腫瘍治療用白血球刺激器までの間に、
多価カチオンのキレート化剤以外の抗凝固剤または多価
カチオンのキレート化剤以外の抗凝固剤とカルシウム塩
を導入するための注入口を少なくとも一つ設けてなるこ
とを特徴とする悪性腫瘍治療用白血球刺激装置。(2) A leukocyte stimulator for malignant tumor treatment, comprising a leukocyte inlet and a leukocyte stimulating material for malignant tumor treatment. A device arranged in the order of the leukocyte outlet, between the leukocyte inlet and the leukocyte stimulator for malignant tumor treatment,
A treatment for malignant tumor characterized by having at least one injection port for introducing an anticoagulant other than a polyvalent cation chelating agent or an anticoagulant other than a polyvalent cation chelating agent and a calcium salt. Leukocyte stimulator for use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61086084A JPS62243562A (en) | 1986-04-16 | 1986-04-16 | Method and apparatus for stimulating leucocyte for remedy ofmalignant tumor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61086084A JPS62243562A (en) | 1986-04-16 | 1986-04-16 | Method and apparatus for stimulating leucocyte for remedy ofmalignant tumor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62243562A true JPS62243562A (en) | 1987-10-24 |
Family
ID=13876837
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61086084A Pending JPS62243562A (en) | 1986-04-16 | 1986-04-16 | Method and apparatus for stimulating leucocyte for remedy ofmalignant tumor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62243562A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60252423A (en) * | 1984-05-29 | 1985-12-13 | Asahi Chem Ind Co Ltd | Method and apparatus for inducing antitumor immunocyte |
-
1986
- 1986-04-16 JP JP61086084A patent/JPS62243562A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60252423A (en) * | 1984-05-29 | 1985-12-13 | Asahi Chem Ind Co Ltd | Method and apparatus for inducing antitumor immunocyte |
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