JPS63129993A - Production of human-originated physiologically active substance - Google Patents
Production of human-originated physiologically active substanceInfo
- Publication number
- JPS63129993A JPS63129993A JP27647986A JP27647986A JPS63129993A JP S63129993 A JPS63129993 A JP S63129993A JP 27647986 A JP27647986 A JP 27647986A JP 27647986 A JP27647986 A JP 27647986A JP S63129993 A JPS63129993 A JP S63129993A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- cell
- human
- inducing
- substance
- 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.)
- Granted
Links
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- 238000004519 manufacturing process Methods 0.000 title claims description 7
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- 239000000126 substance Substances 0.000 claims abstract description 39
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- 238000000034 method Methods 0.000 claims description 20
- 238000001155 isoelectric focusing Methods 0.000 claims description 6
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- 239000000243 solution Substances 0.000 description 19
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 238000011282 treatment Methods 0.000 description 10
- 239000001569 carbon dioxide Substances 0.000 description 9
- 229910002092 carbon dioxide Inorganic materials 0.000 description 9
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- 239000000203 mixture Substances 0.000 description 9
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 5
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- 201000006894 monocytic leukemia Diseases 0.000 description 2
- 208000025113 myeloid leukemia Diseases 0.000 description 2
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野]
本発明は新規な生理活性物質の製造方法に間するもので
ある。更に詳しくは、ヒト由来の白血病*mを培養する
ことによって産生される、細胞分化誘導作用を有する蛋
白性のヒト由来生理活性物質(以下、細胞分化誘導物質
と略記)の製造方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel method for producing a physiologically active substance. More specifically, it relates to a method for producing a proteinaceous human-derived physiologically active substance (hereinafter abbreviated as cell differentiation-inducing substance) that has a cell differentiation-inducing effect and is produced by culturing human-derived leukemia*m. .
[従来の技術]
免疫反応をつかさどっている細胞群の中で、最近マクロ
ファージ(以下Mφと略す)が注目されるようになって
きた。Mφが、どん食作用による抗原の処理を始めとし
て、生体の防御機構の中で、・I返し、成熟して機能細
胞へ到達するのである。こ)・
°、の分化成熟の過程で増殖能をもち、腫よう化してし
まったものが白血病細胞である。このような腫よう細胞
を正常な機能をもった細胞へと分化を誘導するのが、分
化誘導能をもった物質であり、これら分化誘導能をもっ
た物質を用いることにより、新しい癌の治療方法を開発
できるものとして、近年、注目されている。[Prior Art] Among the cell groups that control immune reactions, macrophages (hereinafter abbreviated as Mφ) have recently attracted attention. Mφ undergoes .I return, matures, and reaches functional cells during the body's defense mechanisms, including the processing of antigens through phagocytosis. Leukemia cells are cells that have the ability to proliferate and become tumors during the differentiation and maturation process. Substances with differentiation-inducing ability induce the differentiation of such tumor cells into cells with normal functions, and by using these substances with differentiation-inducing ability, new cancer treatments are possible. In recent years, it has attracted attention as a method for developing methods.
分化誘導能をもった物質としては、安全性の高い蛋白性
の物質、特にヒト由来の蛋白性の物質が1IIN待を集
め、近年活発に研究がなされている。現在までに、ヒト
末梢血リンパ球をレクチンで刺激することにより、分化
誘導活性が生成されることが報告されている(ジャーナ
ル ナショナル カンサー インステチュート(,1,
National CancerInstitute)
67巻、1225頁(1981年)、カンサーリサー
チ(Cancer Re5earch) 42巻、 3
!328頁(1982年))。しかしながらこれらの報
告において、ヒ4o、oooの蛋白性物質であることが
報告されているのみであったが、その後の研究において
、公開特許公報、昭60 28934号に示される分子
量45.000〜60 、000または100,000
、等電点5〜7の物質てあり、トリプシンに感受性を
示し、熱に不安定な物質であるとされた。一方、ヒ)−
T−リンパ球性白血病細胞の培養上清中に見いだされた
分化誘導活性は、アクリルアミドゲル電気泳動法によっ
て、分子!50.000〜60,000の蛋白性の物質
に由来するものとされたが、単離工程中で、その活性の
約60〜90%が失われた(日本MJ織培百学会要旨、
43頁、(1983年))。As substances capable of inducing differentiation, highly safe proteinaceous substances, particularly human-derived proteinaceous substances, are attracting attention and have been actively researched in recent years. To date, it has been reported that stimulation of human peripheral blood lymphocytes with lectins produces differentiation-inducing activity (Journal National Cancer Institute, 1999).
National Cancer Institute)
Volume 67, page 1225 (1981), Cancer Research Volume 42, 3
! 328 pages (1982)). However, in these reports, it was only reported that it was a proteinaceous substance of Hi4o, ooo, but in subsequent research, it was found that the molecular weight was 45.000 to 60 as shown in Japanese Patent Publication No. 1982-28934. , 000 or 100,000
It is a substance with an isoelectric point of 5 to 7, is sensitive to trypsin, and is unstable to heat. On the other hand,
The differentiation-inducing activity found in the culture supernatant of T-lymphocytic leukemia cells was determined by acrylamide gel electrophoresis. It was thought to be derived from a proteinaceous substance of 50,000 to 60,000, but approximately 60 to 90% of its activity was lost during the isolation process (Japan MJ Oribai Hyakukai Abstracts,
p. 43 (1983)).
[発明が解決しようとする問題点]
このように、ヒト末梢血リンパ球およびヒト−丁−リン
パ球性白血病細胞から生成される分化誘導活性は、その
活性をもたらす物質の性質はほとんどわかっていないか
、熱や蛋白質分解酵素に強い感受性を示す不安定な物質
であると共に、これらの物質の活性は弱く、ビタミンA
誘導体などが共存しないと、白血病細胞に充分な分化を
誘導することができなかった。[Problems to be Solved by the Invention] As described above, the nature of the substance that causes the differentiation-inducing activity produced from human peripheral blood lymphocytes and human lymphocytic leukemia cells is largely unknown. In addition to being unstable substances that are highly sensitive to heat and proteolytic enzymes, the activity of these substances is weak, and vitamin A
Without the coexistence of derivatives, leukemia cells could not be induced to differentiate sufficiently.
また、ヒト末梢血リンパ球はヒトの血液から採取される
血球を分画して調製されるため、大量に取得することは
困難であり、工業的生産は容易ではなかった。またヒト
−ニーリンパ性白血病細胞は、ヒトのT細胞白血病ウィ
ルスに維持感染することによって、増殖性を獲得したも
のであり、大量の細胞培養は安全性の点て問題が多かっ
た。Furthermore, since human peripheral blood lymphocytes are prepared by fractionating blood cells collected from human blood, it is difficult to obtain them in large quantities, and industrial production has not been easy. Furthermore, human knee lymphocytic leukemia cells have acquired proliferative properties through maintenance infection with human T-cell leukemia virus, and large-scale cell culture has been problematic in terms of safety.
[問題点を解決するための手段]
木発明者らは、」二記先行知見を認識し、マクロファー
ジ(N・1φ)系の細胞が、より安定性に優れた、強い
細胞分化誘導作用を有する蛋白性の生理活性物質を産生
ずるとの作業仮説に基いて、鋭意検討を続け、ヒトのN
1φ前駆細胞に作用して、単独で、Mφへと分化を誘導
しろる活性を有するヒト由来の蛋白性の細胞分化誘導物
質を見いだし、発明を完成し、特許出願を行なった。し
かしながら、先願てはMφ前駆細胞である白血病細胞を
使用する場合においても、Mφ前駆細胞を分化誘導能を
有する物質(以下、分化誘導剤と略す)Mφ様細胞へ変
化させた後、分化誘導剤を洗浄、除去するために、大変
手間取り、大量の細胞を処理することがむずかしかった
。そこで、細胞分化誘導物質を天竜かつ容易に取得する
ための手段について、検討をかさね、Mφ様細胞に分化
しうるヒト白血病細胞を用い、分化誘導能を有する物質
およびMφ活性化物質の存在下に培養することにより、
安全に大量の細胞を取り扱うことができ、かつ細胞分化
誘導物質を大量に取得できることを見いだし本発明を完
成した。[Means for Solving the Problems] The inventors of the present invention recognized the prior knowledge described in Section 2 and concluded that macrophage (N・1φ) cells have superior stability and a strong cell differentiation-inducing effect. Based on the working hypothesis that human N
We have discovered a human-derived proteinaceous cell differentiation inducer that acts on 1φ progenitor cells and has the activity of inducing differentiation into Mφ by itself, completed the invention, and filed a patent application. However, even when using leukemia cells, which are Mφ progenitor cells, the previous application proposed that after changing the Mφ progenitor cells into Mφ-like cells with a substance capable of inducing differentiation (hereinafter abbreviated as a differentiation inducing agent), differentiation is induced. It was very time-consuming to wash and remove the agent, and it was difficult to process a large amount of cells. Therefore, we have repeatedly investigated ways to easily obtain cell differentiation-inducing substances. By culturing,
The present invention was completed by discovering that it is possible to safely handle large amounts of cells and to obtain large amounts of cell differentiation-inducing substances.
[発明の内容]
すなわち、本発明はマクロファージ様細胞に分化しろる
ヒト白血病細胞を分化誘導能を有する物質およびマクロ
ファージ活性化物質の存在下に培養することによる、下
記の特性を有し、a)分子j!l 50.000±5
,000(ゲルろ過性)50.000±5.000 (
S D S−ポリアクリルアミド電気泳動法)
b)等電点 6.5±1.0(等電点電気泳動法)C)
熱安定性 70’Cにて失活しない白血病細胞に対して
細胞分化誘導活性を有するヒト由来生理活性物質の生産
方法に関する。[Contents of the invention] That is, the present invention has the following characteristics by culturing human leukemia cells that differentiate into macrophage-like cells in the presence of a substance capable of inducing differentiation and a macrophage activating substance, and a) Molecule j! l 50.000±5
,000 (gel filtration) 50.000±5.000 (
S D S-polyacrylamide electrophoresis method) b) Isoelectric point 6.5 ± 1.0 (isoelectric focusing method) C)
The present invention relates to a method for producing a human-derived physiologically active substance that does not become inactivated at 70'C and has cell differentiation-inducing activity against leukemic cells.
本発明において、細胞分化誘導物質とはヒ!・由来のM
φ様細胞が産生ずる物質であってin vitr。In the present invention, the cell differentiation inducer is Hi!・Origin M
A substance produced by φ-like cells in vitro.
で少なくともマウスM−1m胞を分化させ、どん本発明
で用いられるMφ様細胞に分化しうるヒト白血病細胞と
は、Mφ前駆細胞に相当し、分化誘導剤の作用により始
めてMφ様細胞に変化する細胞又は本来Mφの性質の一
部を有しているが、分化誘導剤の作用により更にMφの
性質を有するように変化する細胞を意味する。Mφ前駆
細胞としては、白血病患者から分離した初代細胞及び株
化細胞などから得られるが、株化細胞が大量に得やすく
好ましい。本発明で用いられる、白血病細胞の株(ヒ細
胞の例としては、HL−60細胞(ネイチャー (N
ature)、270巻、347頁(1977年))、
T HP−1胞(インターナショナル ジャーナルカン
サー(Int、 、1. Cancer) 26巻、1
7!頁(1980年) ) 、Mono−1−207*
胞(ウィルヒョーズ アルヒーフ ァー バソロジカル
アナトミー ヒスドパソロジー(Virchows
Arch、 A Path、 Anat。The human leukemia cells capable of differentiating into Mφ-like cells used in the present invention correspond to Mφ precursor cells, and only change into Mφ-like cells by the action of a differentiation-inducing agent. Cells or cells that originally have some of the properties of Mφ, but which change to have the properties of Mφ by the action of a differentiation-inducing agent. Mφ progenitor cells can be obtained from primary cells isolated from leukemia patients, established cell lines, etc., but established cell lines are preferred because they are easy to obtain in large quantities. Leukemia cell lines (an example of human cells used in the present invention include HL-60 cells (Nature (N
ature), vol. 270, p. 347 (1977)),
T HP-1 cells (International Journal Cancer (Int, 1. Cancer) vol. 26, 1
7! (1980), Mono-1-207*
Virchows Virchows Bathological Anatomy Hisdopathology
Arch, A Path, Anat.
anrl Ili個、ol、 371巻、15頁(1
976年))などが挙げられる。ここで用いられる分化
誘導剤としてはN1φ様細胞へ変化し得る白血病細胞の
Mφ様細胞への変化を誘導する物質を意味し、例えば、
ホ好ましく、中でも、12−0−テトラデカノイルホル
ボール−13−アセテート(以下、TPAと略記する)
が特に好ましい。anrl Ili pieces, OL, vol. 371, p. 15 (1
976)). The differentiation-inducing agent used here refers to a substance that induces the transformation of leukemic cells that can transform into N1φ-like cells into Mφ-like cells, such as:
Among these, 12-0-tetradecanoylphorbol-13-acetate (hereinafter abbreviated as TPA) is preferable.
is particularly preferred.
本発明において用いられるMφ活性化物71としては、
ビタミンA誘導体(例えば、ビタミンA酸、ビタミンA
アルコール、ビタミンAアセテート、ビタミンAパルミ
テート)、ジメチルスルホキシド、酪酸ナトリウム塩、
ハイドロコーチシン、ダラム陰性菌由来のりボボリサッ
力ライト(以下LPSと略す)、リビッドA、BCGW
なとの菌体壁、ムラミルジペプチドなどが挙げられ、そ
れぞれ単独、あるいは適宜組み合わせて用いることによ
り、Mφを活性化させ、細胞分化誘導物質の産生を促す
ことができる。これらMφ活性化物質の中でも、約1〜
5000ng/ml 、好ましくは約100〜3000
ng/ml 、より好ましくは約500〜2000ng
/m IのビタミンA誘導体、中でもビタミンA酸の使
用が特に好ましい。As the Mφ activated substance 71 used in the present invention,
Vitamin A derivatives (e.g., vitamin A acid, vitamin A
alcohol, vitamin A acetate, vitamin A palmitate), dimethyl sulfoxide, butyric acid sodium salt,
Hydrocortiscin, Durum-negative bacterium-derived Noriboborisatsuryite (hereinafter abbreviated as LPS), Livid A, BCGW
Examples include the bacterial cell wall of Nato, muramyl dipeptide, etc., and by using each alone or in an appropriate combination, Mφ can be activated and production of cell differentiation-inducing substances can be promoted. Among these Mφ activators, about 1 to
5000ng/ml, preferably about 100-3000
ng/ml, more preferably about 500-2000 ng
Particular preference is given to using vitamin A derivatives of /m I, especially vitamin A acids.
細胞分化誘導物質産生に充分な時間、ヒト由来の白面病
細胞を培養した後、培養上清を収集し、遠心分離により
細胞層を除去すれば、細胞分化誘導物質動等を適宜組み
合わせて精製することにより、高純度の細胞分化誘導物
質を得ることができろ。After culturing human-derived white-faced disease cells for a sufficient period of time to produce a cell differentiation inducer, the culture supernatant is collected and the cell layer is removed by centrifugation, followed by purification by appropriately combining the activities of the cell differentiation inducer. By doing so, it is possible to obtain a highly pure cell differentiation inducer.
細胞分化誘導物質の活性の測定は、in vitroで
マウス骨髄性白血病細胞M−1細胞に、どん食能を誘起
する効果を測定することにより行なった。本発明者らが
用いている方法は、林の方法(トキシコロジーフォーラ
ム、7巻、50頁、(1984年))を改良したもので
ある。即ち、増M門にあるN1−1細胞5XIO5ce
lls/ml (培地:イーグルMEM+2培量ビタミ
ン・アミノ酸+10%牛脂児血清)浮遊液に細胞分化誘
導物質溶液(試験液)を混し、その1mlを10mlガ
ラス管にとり、横に倒して、炭酸ガス培養器中で、37
℃で2日間培養後、遠心処理(+000 rpm、 1
0分間)を施し、上澄ミ液ヲ捨て、血清を含まない培養
液1mlを加えて再び細胞を懸濁し、2μm/m1の濃
度のポリスチレン・ラテックス粒子(1,004μm:
積水化学社製)を加λ攪はんした後、さらに4時間培養
する。この細、スをのせ、顕微鏡で観察する。赤く染色
されろ死、!s鞄を除き、生細胞のみについて、ラテッ
クス粒1子をとん食した細胞と非どん食細胞とを計数し
、どん食細胞の比率を求めろ。試験液を適宜希釈して、
上記の測定を行ない、どん食細胞の比率が10%になる
のに必要な、細胞分化誘導物質の試料の希釈尤の逆数を
もって、本発明における細胞分化誘導物質の活性量を
1単侍(U)/it と定義する。The activity of the cell differentiation inducer was measured in vitro by measuring the effect of inducing phagocytosis in mouse myeloid leukemia M-1 cells. The method used by the present inventors is an improvement on Hayashi's method (Toxicology Forum, Vol. 7, p. 50, (1984)). That is, N1-1 cells 5XIO5ce in the phylum
lls/ml (Medium: Eagle MEM + 2 medium vitamins and amino acids + 10% tallow serum) Mix the cell differentiation inducer solution (test solution) with the suspension, put 1 ml of it into a 10 ml glass tube, turn it sideways, and add carbon dioxide gas. In the incubator, 37
After culturing at ℃ for 2 days, centrifugation treatment (+000 rpm, 1
0 minutes), discard the supernatant, add 1 ml of serum-free culture medium, suspend the cells again, and add polystyrene latex particles at a concentration of 2 μm/ml (1,004 μm:
(manufactured by Sekisui Chemical Co., Ltd.) and stirred for a further 4 hours. Place this thin strip on top and observe it under a microscope. Dyed red, death! Count the cells that have phagocytosed one latex particle and the non-phagocytic cells for only living cells, excluding the bag, and find the ratio of phagocytic cells. Dilute the test solution appropriately,
Perform the above measurements, and calculate the amount of activity of the cell differentiation inducer in the present invention using the reciprocal of the dilution of the sample of the cell differentiation inducer necessary for the ratio of phagocytic cells to be 10%.
Defined as 1 single samurai (U)/it.
以下本発明における細胞分化誘導物質の活性量は、この
とん食能測定法によって測定した単位で示されている。Hereinafter, the activity amount of the cell differentiation-inducing substance in the present invention is shown in units measured by this phagocytosis measurement method.
上記の弧胞の培養により産生きれる、本発明の細胞分化
誘導物質の性質を詳しく述べる。The properties of the cell differentiation-inducing substance of the present invention, which can be produced by culturing the above-mentioned arcuate, will be described in detail.
A)分子量 : 50.000±5.000(ゲルろ違
法)ダルヘラコリン酸緩衡液(塩化カリウム0.2g/
l、塩化ナトリウム8g/1.リン酸第1カリウム0.
2y、/1.リン酸第2ナトリウム1.15g/1.
p H7,4)に0.01χポリエチレングリコールを
添加した溶液にて平衡化した5uperose 6 +
5uperose 12 (ファル5e2restら
の方法[メソッド イン エンザイモロジ−(Meth
od in Enzvmology) 28−8巻、5
4頁(1972年)コに従い、トリス/グリシン/5D
s(pH8,3)で、電気泳動を行なった。標Lt分子
量キット(ファルマシア社製)を用いて分子量検量線を
作成し、分画したゲルからの抽出物のM−1細胞のとん
食能誘起活性評価により分子量を決定する。A) Molecular weight: 50.000±5.000 (gel filtration illegal) Dalhera cholic acid buffer solution (potassium chloride 0.2g/
l, sodium chloride 8g/1. Potassium phosphate 0.
2y, /1. Sodium phosphate 1.15g/1.
5uperose 6 + equilibrated with a solution containing 0.01χ polyethylene glycol at pH 7.4)
5uperose 12 (Method of Fal5e2rest et al. [Method in Enzymology (Meth
od in Enzvmology) Volume 28-8, 5
Tris/glycine/5D according to page 4 (1972)
Electrophoresis was performed at pH 8.3. A molecular weight calibration curve is created using a standard Lt molecular weight kit (manufactured by Pharmacia), and the molecular weight is determined by evaluating the phagocytosis-inducing activity of M-1 cells of the extract from the fractionated gel.
以上の結果より、本発明の細胞分化誘導物質はサブ・ユ
ニット構造をとっていない物質であることが分かる。From the above results, it can be seen that the cell differentiation-inducing substance of the present invention does not have a subunit structure.
C)等電点:6.5±1.0(等電点電気泳動法)アト
−株式会社製の等電点電気泳動装置(SJ107IEc
型)を用い、ファルマライト(ファルマシア社製、pH
4〜8)とグリセロールを含む5%ポリアクリルアミド
平板ゲルを作成する。陽極側に0.04 M DL−
グルタミン酸、陰極側に0.2M L−ヒスチジンを使
用して、700vで50分間の前泳動を行なう。続いて
試料を付与し、700vで1時間、500■で16時間
泳動を行なう。泳動終了後ゲルを2.5 mm巾で切り
出し、次いて各ゲル片を0.15 M塩化ナトリウムを
含む0.02 M )リス−塩11を衝in (pH8
,2) 0.2 mlで抽出し、各抽出液について、M
−1細胞を用いた細胞分化誘導活性の評価を行なう。C) Isoelectric point: 6.5 ± 1.0 (isoelectric focusing method) Isoelectric focusing device (SJ107IEc manufactured by At-Co., Ltd.)
Pharmalite (manufactured by Pharmacia, pH
A 5% polyacrylamide plate gel containing 4 to 8) and glycerol is prepared. 0.04 M DL- on the anode side
Perform pre-phoresis at 700v for 50 minutes using glutamic acid and 0.2M L-histidine on the cathode side. Subsequently, a sample is applied and electrophoresis is performed at 700V for 1 hour and at 500V for 16 hours. After the electrophoresis was completed, the gel was cut into a 2.5 mm width, and each gel piece was soaked in 0.02 M Lithium salt 11 (pH 8) containing 0.15 M sodium chloride.
, 2) Extract with 0.2 ml, and for each extract, M
The cell differentiation inducing activity will be evaluated using -1 cells.
D)熱安定性
本発明の細胞分化誘導物質を0.01χポリエチレング
リコールを添加したpH7,4リン酸緩衝液にて3倍に
希釈し、所定の時間、所定の温度にて加熱した後、M−
1細胞を用いた細胞分化誘導活性の評111iを行なう
。本発明の細胞分化誘導物質は、70℃、1時間の熱処
理において、その活性を失わない、熱的に安定な生理活
性物質である。D) Thermostability The cell differentiation inducer of the present invention is diluted 3 times with a pH 7.4 phosphate buffer containing 0.01x polyethylene glycol, heated at a predetermined temperature for a predetermined time, and then −
Evaluation 111i of cell differentiation inducing activity using 1 cell is performed. The cell differentiation-inducing substance of the present invention is a thermally stable physiologically active substance that does not lose its activity even after heat treatment at 70°C for 1 hour.
E)レクチンカラムへの吸着性
市販の各種レクチン固定化樹脂を市販セパコールミニカ
ラム(バイオラット社製)に充填し150mMの塩化ナ
トリウムを含む50 mMリン酸緩衝液(pH7,5)
で充分に洗浄後、本発明の細胞分化誘導物質試料を添加
し、同緩衝液にて洗浄し、次いて、各種糖類を含む溶離
lαで溶出を行なう。E) Adsorption to lectin columns Various commercially available lectin immobilization resins were packed into a commercially available Sepacol mini column (manufactured by Bio-Rat), and 50 mM phosphate buffer (pH 7.5) containing 150 mM sodium chloride was added.
After thorough washing with water, the sample of the cell differentiation inducer of the present invention is added, washed with the same buffer, and then eluted with elution lα containing various saccharides.
コンカナバリン−A、および、レンチルレクチンのカラ
ムを用いた場合に、レクチンカラムへの吸着が認められ
、いずれのカラムにおいても0.2Mα−メチル−d−
マンノシド溶液により、細胞分化誘導活性が溶出する。When concanavalin-A and lentil lectin columns were used, adsorption to the lectin column was observed, and in both columns 0.2M α-methyl-d-
Cell differentiation inducing activity is eluted by the mannoside solution.
F)ジスルフィド結合の還元剤による影響ジスルフィド
結合の還元剤としてジチオスレイトール(DTT) 、
又は、2−メルカプトエタノール(2−M E )を、
本発明になる細胞分化誘導物質の溶液中に加え、37℃
で、4時間反応させる。F) Effect of reducing agents on disulfide bonds Dithiothreitol (DTT) as a reducing agent on disulfide bonds,
Or 2-mercaptoethanol (2-ME),
Added to the solution of the cell differentiation inducing substance of the present invention, at 37°C.
Let it react for 4 hours.
反応後、M−1細胞に対するとん食能誘起活性を測定す
る。50 mMのジチオスレイトールを添加した場合に
、その活性の低下が認められる。After the reaction, the phagocytosis-inducing activity on M-1 cells is measured. A decrease in the activity is observed when 50 mM dithiothreitol is added.
G)pH安定性
本発明になる細胞分化誘導物質を含む溶液に、4倍量の
各種pHの纏衝液を添加し、24時間、37℃に加温し
た後、pHを中性にもどし、M−1細胞に対する細胞分
化誘導活性を測定する。pH2〜10の範囲において、
活性の低下は認められない。G) pH stability To the solution containing the cell differentiation inducing substance of the present invention, four times the amount of buffer solution of various pH was added, and after heating at 37°C for 24 hours, the pH was returned to neutral, and the pH was adjusted to neutral. Measure the cell differentiation inducing activity for -1 cells. In the pH range of 2 to 10,
No decrease in activity was observed.
■)蛋白分解酵素安定性
゛る細胞分化誘導活性の低下は認められない。上記実験
に0.1%SDSを添加したこと以外は、同一の条件に
て蛋白分解酵素の効果を検討したところ、0.1%SD
Sの添加時のプロナーゼ−Eにより、細胞分化誘導活性
が消失ずろことが認められろ。■) No decrease in cell differentiation inducing activity due to protease stability was observed. The effect of protease was investigated under the same conditions as in the above experiment except that 0.1% SDS was added.
It was observed that the cell differentiation inducing activity of pronase-E when S was added disappeared.
1)ヒト白血病細胞に対する生理作用
牛胎児血清を10%含むRP M l−1640培地に
ヒト単球性白血病細胞(THP−1)、ヒト前骨髄性白
血病細胞(HL−60)を37°C5炭酸ガス培養器中
でそれぞれ培養し、増殖間にある細胞をリン酸緩衝液で
よく洗浄した後5%生胎児血清および 10 nMヒタ
ミンAmを添加したR P M I −11340培地
くヒタミン、アミノ酸強化)に、それぞれ2X105個
/ ml培地になるように懸濁する。細胞懸濁?αlO
Oμmと細胞分化誘導物質溶液(試験液)100111
との混合液を96穴プレートに入れ、37℃、5%炭炭
酸ガス台空気の下で、3日間培養する。1) Physiological effect on human leukemia cells Human monocytic leukemia cells (THP-1) and human promyelocytic leukemia cells (HL-60) were incubated at 37°C with carbon dioxide in RP M l-1640 medium containing 10% fetal bovine serum. After each cell was cultured in a gas incubator and the cells between the growth stages were thoroughly washed with phosphate buffer, the cells were transferred to RPM I-11340 medium supplemented with 5% live fetal serum and 10 nM hitamine (enriched with hitamine and amino acids). Then, suspend each cell at 2 x 105 cells/ml medium. Cell suspension? αlO
Oμm and cell differentiation inducer solution (test solution) 100111
The mixture with the above was placed in a 96-well plate and cultured for 3 days at 37°C under 5% charcoal and carbon dioxide atmosphere.
0.2%ニトロブルーテトラゾリウム(NET、シグマ
社)の溶液(0,2μg/m1TPA含有培地に溶かし
たme)IO07zlを加えて、さらに45分間、−、
とにより、T HP−1細胞、HL−60細胞にNBT
゛還元能が誘導されるのみならず、ラテックス粒子どん
食油、培養容器壁への付着能、酵母菌殺菌能、i性ホス
ファターゼ活性、β−グルクロニダーセ活性など、Mφ
の特徴として、細胞鑑定に常用される(「マニュアル
オブ マクロファージ メソドロジー(Manual
of Macropha、ge Me↑、hodolo
gy)、マーセル デツカ−社、米国、1981年」
「図解白血球、金芳堂、1982年」)各種指標の活性
の増強が認められる。A solution of 0.2% nitroblue tetrazolium (NET, Sigma) (0.2 μg/ml me dissolved in TPA-containing medium) IO07zl was added for an additional 45 min.
NBT was applied to T HP-1 cells and HL-60 cells by
Not only is the reducing ability induced, but the latex particles also have the ability to adhere to food oil, culture vessel walls, yeast bactericidal ability, i-phosphatase activity, β-glucuronidase activity, etc.
As a characteristic, it is commonly used for cell identification (“Manual
of macrophage methodology (Manual
of Macropha, ge Me↑, hodolo
gy), Marcel Detzker, USA, 1981.
"Illustrated White Blood Cells, Kinpodo, 1982") enhancement of the activity of various indicators was observed.
なお、これまでの説明で明らかなように、本発明になる
新規生理活性物質は、Mφ前駆細胞であるヒトおよびマ
ウスの骨髄性白血病細胞に作用して、Mφ様細胞へと分
化を誘導し、Mφに特有な各種機能の赤道をもたらし、
溶液中での分子量、すなわちゲルろ適時の分子量が50
.000±5,000てあり、5DS−ポリアクリルア
ミドゲル電気泳動ての分子量も50.000±5,00
0であること、コンカナバリン−へ、レンチルレチン等
のレクチンに対して結合性を有することから、本発明の
細胞本発明で使用される細胞の培養には、高等動物細胞
の培養に適した各種合成培地が用いられる。As is clear from the above explanation, the novel physiologically active substance of the present invention acts on human and mouse myeloid leukemia cells, which are Mφ precursor cells, and induces differentiation into Mφ-like cells. Bringing the equator of various functions unique to Mφ,
The molecular weight in solution, that is, the molecular weight at the time of gel filtration is 50
.. 000±5,000, and the molecular weight by 5DS-polyacrylamide gel electrophoresis is 50.000±5,00.
0 and has binding properties to concanavalin and lectin such as lentilretin. Therefore, the cells of the present invention can be cultured in various synthetic media suitable for culturing higher animal cells. is used.
代表的な培地としては、例えばRP M l−1640
培地、イーグルのMEM培地、ダルベツコ変法のMEM
培地、a −M E M培地、)Jamの培地、199
培地、 McCoy 5A培地、I 5coveの培地
などを単独もしくは適宜混合した培地が用いられる。こ
れらの培地の組成は「細胞、I織培養マニュアル、講談
社、1982年」に記載されている。これらの培地には
、アルブミン、インシュリン、トランスフェリンなとの
血清由来の蛋白質、ヒト血清、牛胎児血清、牛血清、馬
血清などの動物血清を単独で、あるいは適宜組み合わせ
て添加してもよい。また必要に応じて、微生物による汚
染を防止するために、例えばペニシリン10−100単
位/m11g1t!!、硫酸ストレプトマイシン10〜
100μg/ml培地、硫酸カナマイシン40〜60I
1g/ml培地などの抗生物質を添加することができろ
。培養液のpHの制i、TIIや、炭酸イオン濃度の:
A節のために、例えは10〜60mMのヘペス[4−(
2−ハドロキシエチル)−1−ビベラ優れたものが好ま
しい。As a typical medium, for example, RP M l-1640
Medium, Eagle's MEM medium, Dulbecco's modified MEM
Medium, a-MEM medium,) Jam's medium, 199
A medium such as McCoy 5A medium, I5cove medium, etc. may be used alone or in an appropriate mixture. The compositions of these media are described in "Cell, Iori Culture Manual," Kodansha, 1982. Serum-derived proteins such as albumin, insulin, and transferrin, and animal serum such as human serum, fetal bovine serum, bovine serum, and horse serum may be added to these media alone or in appropriate combinations. In addition, if necessary, in order to prevent contamination by microorganisms, for example, 10-100 units of penicillin/m11g1t! ! , streptomycin sulfate 10~
100μg/ml medium, kanamycin sulfate 40-60I
Antibiotics such as 1 g/ml medium may be added. Control of the pH of the culture solution, TII, and carbonate ion concentration:
For the A node, e.g. 10-60 mM Hepes[4-(
2-Hadroxyethyl)-1-Vivera is preferred.
細胞分化誘導物質を産生さ仕るためには、適当な培地を
用いて、ヒト白血病細胞を、培地1 ml当たりに約l
XIO3〜4 X l 06個、好ましくは、約4×1
05〜2XI06個となるように懸濁し、培養容器へ1
+Wえ込む。次いて、分化誘導能を有する物質(分化誘
導剤)およびMφ活性1ヒ物質を添加する。細胞、分化
誘導剤及びMφ活性化vIJ質を含む培養容器を約35
〜38°(:、好ましくは約37℃、約5〜lO%炭酸
ガス含有空気中、温度約90〜100%の条件の下で、
40〜100時間培養することにより、細胞分化誘導物
質が産生され、培養上清中に放出される。In order to produce cell differentiation-inducing substances, human leukemia cells are grown in an appropriate medium at a concentration of about 1 liter per ml of medium.
XIO3-4 X l 06 pieces, preferably about 4 x 1
Suspend to make 05-2X06 cells and transfer to culture container 1
+W Equipped. Next, a substance capable of inducing differentiation (differentiation inducing agent) and a substance with Mφ activity are added. A culture vessel containing cells, a differentiation inducer, and Mφ activated vIJ substance was prepared for approximately 35 minutes.
~38° (:, preferably at about 37°C, in air containing about 5-10% carbon dioxide, at a temperature of about 90-100%,
By culturing for 40 to 100 hours, a cell differentiation inducer is produced and released into the culture supernatant.
培地のpHは、培養間間中約6.0〜7.5に維持ずろ
ことが好ましい。The pH of the medium is preferably maintained at about 6.0 to 7.5 throughout the culture.
次に実施例を挙げて、本発明を更に具体的に説明するか
、本発明はこれらに限定されるものでないことは言うま
でもない。なお以下の記載において、「%」は特に記載
しない限り容量パーセント(V/V%)を表わす。また
特に記載がない限り、培養は37°C,湿度90〜10
0%、5%炭酸ガス含有空気中で1〒なった。Next, the present invention will be explained in more detail with reference to Examples, and it goes without saying that the present invention is not limited thereto. In the following description, "%" represents capacity percentage (V/V%) unless otherwise specified. Unless otherwise specified, cultivation was carried out at 37°C and humidity 90-10°C.
1 in air containing 0% and 5% carbon dioxide.
実施例1
ヒト急性単球性白血病細胞T HP−1細胞を1.50
単位/mlのペニシリンおよび5071g/mlのスト
レプトマイシンを含有し、血清を含まないRP M 1
1640培地にて、細胞密度6xlo5個/mlとなる
ように、細胞浮遊液を調製し、その10 mlを100
枚の鞘織培青用プラスチック製ペトリディッシュに植え
込み、分化誘導剤として、12−0−テトラデカノイル
ホルボール−13−ア七チー)(TPA)、Mφ活性化
物質としてビタミンA酸(RA)をそれぞれ!7zg/
mlとなるように添加し、37℃、5%炭酸ガス含有空
気中で72時間培養した。72時間後に各ディツシュの
tqi上清を収集し、300Orpmで10分間遠心し
、細胞層を除去した後、上清中の細胞分化誘導物質の活
性を測定した。即ち、iv殖期にあるM−1胞5X10
”ce!Is/ml (培地:イーグルMEM+2培量
ビタミン・アミノ酸+10%牛脂児血清)浮遊液に得ら
れた培養液を混し・、その1m1を10m1ガラス管に
とり、横に倒して、炭酸ガス培養器中で、37℃で2日
間培養後、遠心処理(1000rpm、 10分間)を
施し、上澄み液を捨て、血清を含まない培養液1mlを
加えて再び細胞を懸濁し、2μI/mlの濃度のポリス
チレン・ラテックス粒子(1,o04zzm :積木化
学社製)を加え攪はんした後、さらに4時間培養する。Example 1 Human acute monocytic leukemia T HP-1 cells at 1.50
Serum-free RP M 1 containing units/ml of penicillin and 5071 g/ml of streptomycin
Prepare a cell suspension in 1640 medium at a cell density of 6xlo5 cells/ml, and add 10 ml of the suspension to 100
12-0-tetradecanoylphorbol-13-acyl (TPA) was used as a differentiation inducer, and vitamin A acid (RA) was used as an Mφ activator. Each! 7zg/
ml and cultured for 72 hours at 37°C in air containing 5% carbon dioxide. After 72 hours, the tqi supernatant of each dish was collected and centrifuged at 300 rpm for 10 minutes to remove the cell layer, and then the activity of the cell differentiation inducer in the supernatant was measured. That is, 5 x 10 M-1 cells in the iv propagation stage.
ce!Is/ml (Medium: Eagle MEM + 2 volumes of vitamins and amino acids + 10% tallow baby serum) Mix the obtained culture solution with the suspension, take 1 ml of it into a 10 ml glass tube, turn it sideways, and add carbon dioxide gas. After culturing at 37°C for 2 days in an incubator, centrifuge the cells (1000 rpm, 10 minutes), discard the supernatant, add 1 ml of serum-free culture medium, resuspend the cells, and bring the cells to a concentration of 2 μI/ml. After adding polystyrene latex particles (1,o04zzm, manufactured by Block Chemical Co., Ltd.) and stirring, the mixture was further cultured for 4 hours.
この細胞をPBSlmlでよく洗浄、遠心し、細胞外の
ラテッ・クス粒子を除去する。この操作を2回繰り返し
たのち、遠心管の底に沈殿した細胞をピペットで吸い」
二げ、スライドガラス上に1部落とす。これに 10.
5%エオシン液1 ’Iltを加え、カバーガラスをの
せ、顕微鏡で観察する。赤く染色される死細胞を 1
除き、生細胞のみについて、ラテックス粒子をど ′ん
食した細胞と非どん食細胞とを計数し、どん食細胞の比
率を求める。培Rmを適宜希釈して、上記の測定を行な
い、どん食細胞の比率す月0%になるのに必要な、培養
液の希釈率の逆数をもって、1単位(U)/ml と定
義される本発明の細胞分化誘導物質の活性型は、得られ
た培養液において、138単位/mlであった。The cells are thoroughly washed with 1ml of PBS and centrifuged to remove extracellular latex particles. After repeating this procedure twice, pipette the cells that have settled at the bottom of the centrifuge tube.
Drop one portion onto a glass slide. 10.
Add 5% eosin solution 1'Ilt, place a cover glass, and observe with a microscope. Dead cells stained red 1
For living cells only, count the cells that have phagocytosed latex particles and non-phagocytic cells, and determine the ratio of phagocytic cells. Dilute the culture medium Rm appropriately and perform the above measurements.The reciprocal of the dilution rate of the culture medium required to make the ratio of phagocytic cells 0% is defined as 1 unit (U)/ml. The active form of the cell differentiation inducer of the present invention was 138 units/ml in the obtained culture solution.
得られた培養液のレクチンにたいする吸着性を検討した
。市販の各種レクチン固定化樹脂を市販セパコールミニ
カラム(バイオラット社製)に充填し 150 TOM
の塩化ナトリウムを含む50 mMリンλンのカラムを
用いた場合に、レクチンカラムへの吸着が認められ、い
ずれのカラムにおいても0.2M誘導活性が溶出した。The adsorption of the obtained culture solution to lectin was investigated. Various commercially available lectin-immobilized resins were packed into a commercially available Sepacol mini column (manufactured by Biorat) and 150 TOM
When a column containing 50 mM phosphorus λ containing sodium chloride of
Con−Aカラムに吸着、溶出した細胞分化誘導活性に
ついて、分子量、等電点の測定を行なった。The molecular weight and isoelectric point of the cell differentiation-inducing activity adsorbed and eluted on the Con-A column were measured.
分子量測定のため、該細胞分化誘導物質の活性画分を、
ダルベツコ リン酸緩衝液(塩化カリウム0.2g/I
、塩化ナトリウム8g/ l 、リン酸第1カリウム0
.2g/1.リン酸第2ナトリウム1.15g/1.
pH7,4)にo、otxポリエチレングリコールを
添加した溶液にて平衡化した5uperose 6 +
5uperose 12(ファルマシア社製)を用い
るゲルろ過法により分画し、M−1細胞でのどん食油の
誘起による細胞分化誘導活性を測定したところ、分子:
150,000±5,000の両分に細胞分化誘導物質
の活性が認められた。For molecular weight measurement, the active fraction of the cell differentiation inducer was
Dulbecco's phosphate buffer (potassium chloride 0.2g/I
, sodium chloride 8 g/l, potassium monophosphate 0
.. 2g/1. Sodium phosphate 1.15g/1.
5uperose 6 + equilibrated with a solution containing o, otx polyethylene glycol at pH 7.4)
Fractionation was performed by a gel filtration method using 5uperose 12 (manufactured by Pharmacia), and the cell differentiation-inducing activity induced by Don edible oil in M-1 cells was measured, and the following molecules were found:
The activity of the cell differentiation inducer was observed in both cells of 150,000±5,000.
5DS−ポリアクリルアミド電気泳動法による分子型測
定のため、トリス/グリシン/5DS(p)I 8.3
)で、電気泳動を行なった。標準分子量−分化誘導物質
の活性が認められた。Tris/glycine/5DS(p)I 8.3 for molecular type determination by 5DS-polyacrylamide electrophoresis
), electrophoresis was performed. Standard molecular weight - activity of differentiation inducer was observed.
等電点測定のために、等電点電気泳動法を以下の方法に
よって行なった。即ち、アト−株式会社製の等電点電気
泳動装置(S 、J I0?IEC型)を用い、ファル
マライト(ファルマシア社製、pH4〜8)とグリセロ
ールを含む5%ポリアクリルアミド平板ゲルを作成した
。陽極側に0.04M DL−グルタミン酸、陰極側に
0.2M L−ヒスチジンを使用して、700■て50
分間の前泳動を行なった。続いて試!4を付与し、70
0vて1時間、500 V 116時間泳動を行なった
。泳動終了後ゲルを2.5 mm巾で切り出し、次いて
各ゲル片を0.15 M塩化ナトリウムを含む0.02
M +−リス−塩酸緩衝1夜(pH8,2’)0.2
mlで抽出し、各抽出液について、M−1細胞を用い
た細胞分化誘導活性の評価を行なったところ、等電点は
pH:6.5±1.0であった。For isoelectric point measurement, isoelectric focusing was performed by the following method. That is, a 5% polyacrylamide plate gel containing Pharmalite (manufactured by Pharmacia, pH 4 to 8) and glycerol was prepared using an isoelectric focusing device (S, J I0? IEC type) manufactured by At-Co., Ltd. . Using 0.04M DL-glutamic acid on the anode side and 0.2M L-histidine on the cathode side, 700μ = 50
A pre-run for 1 minute was performed. Next, try it! 4 and 70
Electrophoresis was performed at 0V for 1 hour and at 500V for 116 hours. After the electrophoresis was completed, the gel was cut out into 2.5 mm width, and each gel piece was soaked in 0.02 mm solution containing 0.15 M sodium chloride.
M+-Lis-HCl buffer overnight (pH 8,2') 0.2
When the cell differentiation inducing activity of each extract was evaluated using M-1 cells, the isoelectric point was found to be pH: 6.5±1.0.
次に示す方法により、細胞分化誘導物質の熱安定性を検
討した。培養液に、0,01χポリエチレングリコール
を添加したpH7,4リン酸緩衝液にて3倍に希釈し、
所定の時間、所定の温度にて加熱表 1
実験番号 条件 M−1細胞とん含率 %(相対値
%)
1 (無処置)58%(100%)256°C91
5分 57%(98%)3 60分
59% (102%)4 70℃ 15分
54% (93%)5 60分
53% (91%)61006C15分 22%
(38%)ジスルフィド結合の還元剤による影響を検
討するために、ジチオスレイトール(DTT) 、又は
、2−メルカプトエタノール(2−ME)を、培養)α
中に加え、37℃で、4時間反応させた。反応後、M−
1細胞に対するとん食油誘起活性を測定した。The thermostability of cell differentiation-inducing substances was investigated using the following method. Dilute the culture solution 3 times with pH 7.4 phosphate buffer containing 0.01χ polyethylene glycol,
Heating at a predetermined temperature for a predetermined time Table 1 Experiment number Conditions M-1 cell content % (relative value %) 1 (No treatment) 58% (100%) 256°C 91
5 minutes 57% (98%) 3 60 minutes
59% (102%) 4 70℃ 15 minutes
54% (93%) 5 60 minutes
53% (91%) 61006C15 minutes 22%
(38%) To examine the influence of reducing agents on disulfide bonds, dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) was cultured) α
and reacted at 37°C for 4 hours. After reaction, M-
The dietary oil-induced activity against 1 cell was measured.
表 2
実験番号 条件 M−1細胞どん含率 %(相対値
%)
1 (無処理)54%<100%)2 2MEO
,1% 49%(91%)3 1%
46%(85%)4 DT75mM 43%
(80%)5 50 mM 13%(2
4%)p H安定性を次に示す。細胞分化誘導物質を含
む培養;夜に、4倍量のpH2,4,7,3,9,10
の各種緩衝液を添加し、24時間、37℃に加温した後
、pHを中性にもどし、M−1細胞に対する細胞分化誘
導活性を測定した。pH2〜10の範囲のいずれにおい
ても活性の低下は認められなかった。Table 2 Experiment number Conditions M-1 cell content % (relative value %) 1 (No treatment) 54% < 100%) 2 2MEO
,1% 49% (91%)3 1%
46% (85%)4 DT75mM 43%
(80%) 5 50 mM 13% (2
4%) The pH stability is shown below. Culture containing cell differentiation inducer; at night, 4 times the amount of pH 2, 4, 7, 3, 9, 10
Various buffer solutions were added thereto, and the mixture was heated at 37°C for 24 hours, the pH was returned to neutral, and the cell differentiation-inducing activity for M-1 cells was measured. No decrease in activity was observed in any pH range from 2 to 10.
ついて、蛋白分解酵素に対する安定性を検討した。Therefore, the stability against proteolytic enzymes was investigated.
細胞分化誘導物質を含む培養液に蛋白分解酵素トリプシ
ン、または、プロナーゼ−E (200単位)を添加
し、37°Cにて、3時間反応させた。反応後、M−1
細胞に対する細胞分化誘導活性を測定したが、いずれの
条件においても、活性の低下は認められなかった。−上
記実験に0.1%SDSを添加したこと以外は、同一の
条件にて、蛋白分解酵素の効果を検討したところ、0.
1%SDSの1.后加時のプロナーゼ−Eにより、細胞
分化誘導活性が完全に消失した。Proteinase trypsin or pronase-E (200 units) was added to the culture medium containing the cell differentiation inducer, and the mixture was reacted at 37°C for 3 hours. After reaction, M-1
Cell differentiation inducing activity for cells was measured, but no decrease in activity was observed under any conditions. - The effect of protease was examined under the same conditions as in the above experiment except that 0.1% SDS was added.
1. of 1% SDS. Cell differentiation inducing activity was completely abolished by pronase-E during post-infusion.
さらに、ヒト白血病′s@に対する生理作用を確認すへ
く、牛胎児血清を10%含むRP M I −1640
培地にヒト単球性白血病細胞(THP−1)、ヒト−ガ
ス培養器中でそれぞれ培養し、増Mltllにある細胞
をリン酸緩衝液でよく洗浄した後、lO%牛脂児血清お
よび10 nMビタミンAMを添加したR P M、、
r−1640培地(ビタミン、アミノ酸強化)に、そ1
゛れぞれ2XIO5個/ml培地になるように!?3濁
した。Furthermore, to confirm the physiological effect on human leukemia's@, RPMI-1640 containing 10% fetal bovine serum
Human monocytic leukemia cells (THP-1) were cultured in a human gas incubator, and after thoroughly washing the cells in the expanded Mltll with phosphate buffer, 10% tallow serum and 10 nM vitamin R P M with AM added,
R-1640 medium (fortified with vitamins and amino acids),
2XIO5 cells/ml medium for each! ? 3 It became cloudy.
細胞懸濁液100alと培養#ff100μmとの混合
液を96穴プレートに入れ、37℃、5%炭酸ガス混合
空気の下で、3日間培養した。0.2%ニトロブルーテ
トラゾリウム(NBT、シグマ社)の溶液(0,2μg
/ml T P A含有培地) locμlを加えて
、更に45分間培養下後2顕微鏡下で観察した。青く沈
着した色素を有するNBT還元能陽性細胞が、T HP
−1細胞において33%、)iL−60細胞において2
7%に観察された。A mixture of 100 al of cell suspension and 100 μm of culture #ff was placed in a 96-well plate and cultured for 3 days at 37° C. under air mixed with 5% carbon dioxide gas. A solution of 0.2% nitro blue tetrazolium (NBT, Sigma) (0.2 μg
locμl of TPA-containing medium) was added thereto, and after culturing for an additional 45 minutes, observation was made under a microscope. NBT-reducing ability-positive cells with blue pigment deposited are T HP
-33% in iL-60 cells, 2 in iL-60 cells
It was observed in 7%.
実施例2
ヒト急性単球性白血病細胞T HP−1細胞を用いて、
実施例1と同様にして細胞1度6XIO5個/mlの細
胞浮遊液を調製し、その10 mlを10枚の絹織培養
用プラスチック製ベトリディッシュに植え込み、分化誘
導剤として、12−0−テトラデカノイルホルボール−
13−アセテート(TPA)、ホルボユの培養1.清を
収集し、3000 rpmで10分間遠心し、細胞層を
除去したE連中の細胞分化誘導物質の活性を測定した。Example 2 Using human acute monocytic leukemia T HP-1 cells,
A cell suspension containing 6XIO5 cells/ml was prepared in the same manner as in Example 1, 10 ml of the suspension was implanted into 10 plastic vetri dishes for silk culture, and 12-0- was used as a differentiation inducer. Tetradecanoylphorbol
13-acetate (TPA), culture of horuboyu 1. The supernatant was collected, centrifuged at 3000 rpm for 10 minutes, the cell layer was removed, and the activity of the cell differentiation inducer was measured.
それぞれの培養上清について、Con−Aカラムへの吸
着処理およびα−メチル−d−マンノシド溶)αによる
脱着処理によって得られる活性画分は、分子量約50.
000 (S D S−ポリアクリルアミ)・電気泳動
法)で、pH2〜10での処理にて失活せずに、706
Cの加熱処理においCも、その活性を保持していた。For each culture supernatant, the active fraction obtained by adsorption treatment on a Con-A column and desorption treatment with α-methyl-d-mannoside solution α has a molecular weight of approximately 50.
000 (S D S-polyacrylamide electrophoresis method), it was not deactivated by treatment at pH 2 to 10, and 706
In the heat treatment of C, C also retained its activity.
(以下 余白)
表 3
実験番号 分化誘導剤 細胞分化誘導物質Mφ活性化
物質
I TPA long/ml 97単位
/m1RA 17B/m1
2 TPA 100 ng/ml 121R
A 1. u g/m1
3 T P A 1000 ng/ml 1
09RA O,Iμg/ml
L P S lOμg/m1
4 PDD 1000 ng/ml 85
RA Iμg/m1
6 M E Z 1000 ng/ml
91RA 1μg/ml
(比較例) T P A 1000 ng/ml
13S−ConA 20 tt g/ml実施例3
ヒト急性単球性白血病細胞T HP−1細胞を用いて、
細胞濃度を変えたこと以外は、実施例1と同様にして細
胞浮遊液を調製し、その10 mlを 10枚のM1織
培襖mプラスチック製ペトリディッシュに植え込み、T
PAを100 ng/ml、RAをlug/m1となる
ように添加し、37℃、5%炭酸ガス含有空気中で72
時間培養した。72時間後に各ディツシュの培養上清を
収集し、3000 rpmで10分間遠心し、細胞層を
除去した上清中の細胞分化誘導物0肩の活性を測定した
。それぞれの培養上清ζじいりアクリルアミド電気泳動
法)で、pH2〜lOでの処理にて失活せずに、70℃
の加熱処理においても、その活性を失わなかった。(Margin below) Table 3 Experiment number Differentiation inducer Cell differentiation inducer Mφ activator I TPA long/ml 97 units/m1RA 17B/m1 2 TPA 100 ng/ml 121R
A1. u g/ml 3 T P A 1000 ng/ml 1
09RA O, Iμg/ml L P S lOμg/ml 4 PDD 1000 ng/ml 85
RA Iμg/ml 6 M EZ 1000 ng/ml
91RA 1 μg/ml (comparative example) TPA 1000 ng/ml
13S-ConA 20 tt g/ml Example 3 Using human acute monocytic leukemia THP-1 cells,
A cell suspension was prepared in the same manner as in Example 1, except that the cell concentration was changed, and 10 ml of the suspension was implanted into 10 M1 tissue culture fusuma plastic Petri dishes.
Add PA to 100 ng/ml and RA to 1 ug/ml, and incubate at 37°C in air containing 5% carbon dioxide.
Cultured for hours. After 72 hours, the culture supernatant of each dish was collected, centrifuged at 3000 rpm for 10 minutes, the cell layer was removed, and the activity of cell differentiation derivative 0 in the supernatant was measured. By acrylamide electrophoresis using each culture supernatant, it was found that the culture supernatant was not inactivated by treatment at pH 2 to 1O at 70°C.
It did not lose its activity even after heat treatment.
(以下 余白)
表 4
実験番号 細胞濃度 細胞分化誘導物質1
1 X 105/ml 8G単位/m12
4 X 105/ml +043
8 X 105/ml 1294
13 X 105/ml 735
25 X 105/ml 41いること以外は
、実施例2と同様に培養を行ない、培養上清を得た。培
養上清中の細胞分化誘導物質の活性を測定したところ、
119単位/mlの活性が認められた。ConAカラム
への吸着処理およびα−メチル−d−マンノシド溶液に
よる脱着処理にて得られる活性画分は、その分子量約5
0,000 (5DS−ポリアクリルアミド電気泳動法
)であり、70℃の前払処理においても、その活性を保
持していた。(See margin below) Table 4 Experiment number Cell concentration Cell differentiation inducer 1
1 X 105/ml 8G units/m12
4 X 105/ml +043
8 x 105/ml 1294
13 x 105/ml 735
Culture was carried out in the same manner as in Example 2, except that 25 × 10 5 /ml 41 cells were used, and a culture supernatant was obtained. When we measured the activity of cell differentiation inducers in the culture supernatant, we found that
An activity of 119 units/ml was observed. The active fraction obtained by adsorption treatment on a ConA column and desorption treatment with an α-methyl-d-mannoside solution has a molecular weight of approximately 5.
0,000 (5DS-polyacrylamide electrophoresis method), and retained its activity even after pretreatment at 70°C.
以上詳細に説明した、本発明の細胞分化誘導物質を医薬
品製造の常套手段、例えば加熱処理、ろ過滅菌、凍結乾
燥、分注等の処理を適宜族して製剤化することにより、
従来にない新しい医薬品を得ろことができる。By formulating the cell differentiation-inducing substance of the present invention, which has been explained in detail above, by appropriately subjecting it to conventional methods for manufacturing pharmaceuticals, such as heat treatment, sterilization by filtration, freeze-drying, and dispensing,
It is possible to obtain new medicines that have never existed before.
Claims (1)
胞を分化誘導能を有する物質およびマクロファージ活性
化物質の存在下に培養することを特徴とする、下記の特
性を有し、 a)分子量50,000±5,000(ゲルろ過法)5
0,000±5,000(SDS−ポリアクリルアミド
電気泳動法) b)等電点6.5±1.0(等電点電気泳動法) c)熱安定性70℃にて失活しない白血病細胞に対して
細胞分化誘導活性を有するヒト由来生理活性物質の生産
方法(1) Human leukemia cells capable of differentiating into macrophage-like cells are cultured in the presence of a substance capable of inducing differentiation and a macrophage activating substance, and have the following characteristics: a) Molecular weight 50,000 ±5,000 (gel filtration method) 5
0,000±5,000 (SDS-polyacrylamide electrophoresis) b) Isoelectric point 6.5±1.0 (isoelectric focusing) c) Thermostable leukemia cells that do not inactivate at 70°C Method for producing a human-derived physiologically active substance having cell differentiation-inducing activity against
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP27647986A JPS63129993A (en) | 1986-11-21 | 1986-11-21 | Production of human-originated physiologically active substance |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP27647986A JPS63129993A (en) | 1986-11-21 | 1986-11-21 | Production of human-originated physiologically active substance |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS63129993A true JPS63129993A (en) | 1988-06-02 |
JPH0348797B2 JPH0348797B2 (en) | 1991-07-25 |
Family
ID=17570026
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP27647986A Granted JPS63129993A (en) | 1986-11-21 | 1986-11-21 | Production of human-originated physiologically active substance |
Country Status (1)
Country | Link |
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JP (1) | JPS63129993A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63129994A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Production of human-originated physiologically active substance |
JPS63130600A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Physiologically active substance and production thereof |
JP2011033544A (en) * | 2009-08-04 | 2011-02-17 | Hoyu Co Ltd | Isoelectric electrophoretic gel and isoelectric electrophoretic method |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6028934A (en) * | 1983-07-25 | 1985-02-14 | Green Cross Corp:The | Cell differentiation introduction substance |
JPS63130600A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Physiologically active substance and production thereof |
JPS63129994A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Production of human-originated physiologically active substance |
-
1986
- 1986-11-21 JP JP27647986A patent/JPS63129993A/en active Granted
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6028934A (en) * | 1983-07-25 | 1985-02-14 | Green Cross Corp:The | Cell differentiation introduction substance |
JPS63130600A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Physiologically active substance and production thereof |
JPS63129994A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Production of human-originated physiologically active substance |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63129994A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Production of human-originated physiologically active substance |
JPS63130600A (en) * | 1986-11-21 | 1988-06-02 | Agency Of Ind Science & Technol | Physiologically active substance and production thereof |
JPH0348798B2 (en) * | 1986-11-21 | 1991-07-25 | Kogyo Gijutsuin | |
JPH0474360B2 (en) * | 1986-11-21 | 1992-11-26 | ||
JP2011033544A (en) * | 2009-08-04 | 2011-02-17 | Hoyu Co Ltd | Isoelectric electrophoretic gel and isoelectric electrophoretic method |
Also Published As
Publication number | Publication date |
---|---|
JPH0348797B2 (en) | 1991-07-25 |
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