【発明の詳細な説明】
本発明はヒト腫瘍細胞変性因子の製造方法に関
する。
本発明者等は広範囲に各種動物細胞を培養中、
ヒト胎児線維芽細胞の培養液にヒトガン細胞を縮
退させる作用のあることを発見し、本培養液中の
有効成分について鋭意研究した結果新規なヒト腫
瘍細胞変性因子〔以下TDF（Tumor
Degeneration Factor）と略称〕の抽出精製に成
功した。TDFはヒト正常細胞また異種動物のガ
ン細胞には何ら作用せず、ヒトのガン細胞にのみ
特異的に縮退作用を発揮し、しかもその効果はヒ
ト白血球インターフエロンにより増強されること
を発見した。さらに本発明者等はヒト胎児線維芽
細胞に効率良くTDFを産出させるためTDF誘導
剤について検討を行なつた結果、ヒトガン細胞と
ヒト線維芽細胞を混合培養することによりTDF
が高濃度に培養液中に産出することを見出した。
本発明はこれらの知見に基づくもので、ヒト胎
児線維芽細胞培養液を水に透析し、透析内液を酸
性条件下に吸着剤と接触させて有効成分を吸着さ
せ、次いで吸着剤から弱アルカリ性水溶液で有効
成分を抽出し、抽出液を熱処理後ゲル過して有
効成分を分取することを特徴とするヒト腫瘍細胞
障碍因子の製造方法、ヒト胎児線維芽細胞とヒト
上皮性ガン細胞との混合培養液を水に対して透析
し、透析内液を酸性条件下に吸着剤と接触させて
有効成分を吸着させ、次いで吸着剤から弱アルカ
リ性水溶液で有効成分を抽出し、抽出液を熱処理
後ゲル過して有効成分を分取することを特徴と
するヒト腫瘍細胞変性因子の製造法、およびヒト
線維芽細胞培養液またはヒト胎児線維芽細胞とヒ
ト上皮性ガン細胞との混合培養液を水に対して透
析し、透析内液を酸性条件下に吸着剤と接触させ
て有効成分を吸着させ、次いで吸着剤から弱アル
カリ性水溶液で有効成分を抽出し、抽出液を熱処
理後ゲル過して有効成分を分取し、これにヒト
白血球インターフエロンを添加することを特徴と
する作用の増強されたヒト腫瘍細胞変性因子含有
剤の製造方法である。
本発明におけるTDFの抽出精製法の骨子は次
のとおりである。ヒト胎児線維芽細胞培養液また
はヒト胎児線維芽細胞とヒトガン細胞の混合培養
液を水に対して十分透析して低分子物質を除去し
た後酸性条件下で吸着剤と接触、吸着させこの吸
着剤を弱アルカリ性水溶液で処理して有効成分る
抽出し、TDFが失活しない程度で熱処理し生じ
た不純物の沈澱を除いた後ゲル過法により
TDFを分離溶出する。
本発明に用いるヒト胎児線維芽細胞は次のよう
にして得ることができる。
ヒト妊娠初期の流産胎児を等張塩類溶液中で細
切し、次いでトリプシンで消化した後、細胞過
器を通して単細胞浮遊液を得、これを遠心分離し
て細胞を集める。この細胞を細胞培養液中で培養
し、一週間後に増殖してくる初代ヒト胎児線維芽
細胞を得る。これに用いる培養液は10％（Ｗ／
Ｖ）牛胎児血清（米国、メリーランド州、エム・
エイ・バイオプロダクツ製）を含むイーグルー
MEM培地〔東京、日水製薬(株)製〕、ダルベツコ
変法イーグルーMEM培地および199培地（同上
製）が適している。
TDFを含むヒト胎児線維芽細胞培養液は次の
ようにして得ることができる。初代ヒト胎児線維
芽細胞をトリプシン処理により培養器から細胞を
はがし、細胞濃度を１〜５×105個／mlに調整し
新たに培養器に移殖し37℃で培養する。こゝで用
いる培養器は動物組織培養用シヤーレ、フラスコ
及びローラーボトルまたはサイドデツクス（フア
ルマシヤ製）の様なビーズに細胞を付着させたス
ピンナーフラスコが適している。ヒトガン細胞と
混合培養する場合は培養液中のヒト胎児線維芽細
胞は５×105個／mlとし、ガン細胞の濃度は１〜
５×104個／mlとするのがよい。ここで用いるこ
とのできるヒトガン細胞は継代可能な樹立株であ
る。例へばHela細胞（Hela、CCD2）、KB細胞
（KBCCL17）、FL細胞（ヒトの正常前膜細胞由
来）等が用いられる。ヒト胎児線維芽細胞のみを
培養した場合約一週間で細胞は培養壁全面に広が
つた時にTDFを含む培養液を採取する。培養液
を採取した後のヒト胎児線維芽細胞はくり返し継
代培養をつづけてこの目的に使用可能である。ま
ずトリプシン処理により細胞をはがし前記の細胞
濃度で新しい培養器に移植することにより細胞が
増殖能力を失うまで再使用できる。このようにし
て得られるTDFを含む培養液を過して不溶性
異物を除去し、水に対して十分透析し、透析性の
低分子物質を除いた後、たとえば希塩酸で酸性に
し適当な吸着剤に接触させる。吸着剤としてはカ
オリン、ベントナイト、活性白土等を用いること
ができる。吸着操作は上記溶液に吸着剤を添加、
撹拌、接触させ、有効成分を吸着させることによ
つて行われる。
つぎに吸着剤を傾しや法、過法、遠心分離法
等の適当な方法で溶液から分離した後、吸着剤か
ら弱アルカリ性水溶、例えばアンモニア水等で有
効成分を抽出し、抽出液を希塩酸などで中性にし
熱処理により不純物を沈澱させる。熱処理の操作
は30〜60分間60゜〜80℃に保持する方法が好まし
い。沈澱物を除いた後、限外過、減圧蒸留もし
くは凍結乾燥などにより抽出液の濃縮を行ない、
これをセフアデツクスＧ−100のゲルを充填した
カラムにかけ、アルブミン溶出位の前部位に流出
されてくる活性画分を集めTDFを得る。
本発明によつて得られるTDFの性状は淡黄色
粉末、水に可溶、メタノール、エタノールに難
溶、エーテル、アセトン、ベンゼンに不溶、分子
量はゲル過法で145000紫外部最大吸収は275〜
280nｍに存在する。元素分析による窒素含量は
10.1〜15.8％、ニンヒドリン、フエノール硫酸、
ビユレツト反応などいずれも陽性、非透析性で糖
蛋白質と推定される。TDFは以下に示すような
生理的性質を示す。TDFを１〜100μｇを水溶液
として培養ヒトガン細胞培養液に加えるとたとえ
ば第１図に示すようにガン細胞縮退作用を示す。
またこの時にTDFと同時にヒトインターフエロ
ン１〜1000IUを加えるとガン細胞はさらに縮退
する（第２Ａ，２Ｂ，３Ａ，３Ｂ図参照）。上記
作用はKB、Hela細胞のようなヒト上皮性ガン細
胞に特異的で、マウスＬ細胞には以上のような操
作を行なつても縮退作用は認められない。以下実
施例によつてさらに詳しく説明する。
実施例 １
ヒト胎児線維芽細胞６日目の培養液を集めザイ
ツ過器にて過して細胞残渣などの不溶性異物
を除いた後セルロースチユーブに入れ、純水に対
して一夜透析を行つた後1N−塩酸で培養液のPH
を4.3に調整し液量の２％のカオリン〔大板、和
光純薬(株)製〕を添加し２時間撹拌接触させた後２
時間静置し、大部分の上清を傾しやして除きカオ
リンをヌツチエ上に減圧過して採取する。カオ
リンの含湿重量の1.5倍量の１％アンモニア水で
３回抽出を行ない、抽出液を合して1N−塩酸で
PH7.0とし、80゜で30分間加温する。加温後生じた
沈澱を遠心分離して除き上清液を純水に対して透
析した後減圧蒸留により水を留去、残留物を少量
の水に溶解し、セフアデツクスＧ−100〔東京、フ
アルマシア・ジヤパン(株)製〕のカラムにかけ溶出
液を分画しガン細胞縮退活性を指標としてアルブ
ミン溶出位の前溶出位の活性ピークを集め、凍結
乾燥してTDFを得る。収量は培溶液１当り約
1.2mgである。
実施例 ２
ヒト脂児線維芽細胞とKB細胞の混合培養液を
集め無菌過器にかけ不溶物を除き、セルローズ
チユーブに入れ純水で一夜透析した後PHを4.3と
して液量の２％のカオリンを加えてTDFを吸着、
カオリンを１％アンモニア水で抽出し、抽出液を
PH7.0に調整し、70℃で40分間加温し、生じた沈
澱を除去し、純水で透析後減圧下に水を留去して
残留物をセフアデツクスＧ−100のカラムにかけ
ガン細胞縮退活性を示すピークを分取し凍結乾燥
してTDFを得る。収量は培養液1.0当り約2.3mg
である。
TDFは淡黄色無定形の粉末で、水に可溶、メ
タノール、エタノールには殆んど溶解しない。エ
ーテル、アセトン、ベンゼンには全く不溶。紫外
部吸収：λmax275〜280nｍ、ニンヒドリン反応、
フエノール硫酸反応、ビユレツト反応何れも陽性
である。また水に対して非透析性である。
実施例 ３
インターフエロンのTDFガン細胞縮退作用に
対する増強効果を次のような方法で調べた。
内径３cmのプラスチツクペトリー皿に1.2〜1.4
×106／mlのガン細胞培養液２mlを加え一夜37℃、
５％炭酸ガスふらん器で培養し、これにTDF10μ
ｇを加えて培養し、その上にヒト白血球インター
フエロン〔京都赤十字血液センターから入手、
Dye−Uptake法（今西ら：Biken Joural 34(3)95
（1981））で力価を測定〕液を加え２−３日間培養
した後、ギムザ液で染色し、このシヤーレを写真
に撮り、マヌユアル・オプテイカル・ピクチユ
ア・アナライジング・システム（西独、ミユヘ
ン、コントロン社MOPAM03）にかけて細胞破
壊領域の比率（％）を計測する。各試験は２〜４
回くり返し行なつた。その結果を第１表にまとめ
た、ｔ−検定の結果Ｐ−値0.05以下を有義とす
る。
上記表で明らかなようにヒト胎児線維芽細胞の
ガン細胞変性因子の活性はヒト白血球インターフ
エロンにより増強された、また異種動物ガン細胞
であるネズミガン細胞L929には上記障碍因子は
活性を示さず、またヒト白血球インターフエロン
も増強作用を示さなかつた。
【表】DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing human tumor cell degeneration factor. The present inventors are currently culturing various animal cells over a wide range of areas.
It was discovered that the culture solution of human fetal fibroblasts has the effect of degenerating human cancer cells, and as a result of intensive research on the active ingredients in this culture solution, a novel human tumor cell degeneration factor [hereinafter referred to as TDF (Tumor)] was discovered.
Degeneration Factor) was successfully extracted and purified. They discovered that TDF has no effect on normal human cells or cancer cells of xenogeneic animals, but specifically degenerates only human cancer cells, and that this effect is enhanced by human leukocyte interferon. Furthermore, the present inventors investigated TDF inducers in order to efficiently produce TDF in human fetal fibroblasts.
was found to be produced in high concentrations in the culture solution. The present invention is based on these findings. Human fetal fibroblast culture fluid is dialyzed against water, the dialyzed fluid is brought into contact with an adsorbent under acidic conditions to adsorb the active ingredient, and then the adsorbent is used to remove weakly alkaline A method for producing a human tumor cell-damaging factor, which comprises extracting the active ingredient with an aqueous solution, heat-treating the extract and then gel-filtering it to separate the active ingredient; The mixed culture solution is dialyzed against water, the dialyzed solution is brought into contact with an adsorbent under acidic conditions to adsorb the active ingredients, the active ingredients are then extracted from the adsorbent with a weakly alkaline aqueous solution, and the extract is heat-treated. A method for producing a human tumor cell degenerative factor, which is characterized by separating the active ingredient by gel filtration, and a method for producing a human tumor cell degeneration factor, which is characterized by separating a human tumor cell degeneration factor by gel filtration, and a method for producing a human fibroblast culture solution or a mixed culture solution of human fetal fibroblasts and human epithelial cancer cells in water. The dialyzed fluid is brought into contact with an adsorbent under acidic conditions to adsorb the active ingredient, then the active ingredient is extracted from the adsorbent with a weakly alkaline aqueous solution, and the extract is heat-treated and then gel-filtered to remove the active ingredient. This is a method for producing a human tumor cell degeneration factor-containing agent with enhanced action, which comprises separating the components and adding human leukocyte interferon thereto. The outline of the TDF extraction and purification method in the present invention is as follows. A human fetal fibroblast culture solution or a mixed culture solution of human fetal fibroblasts and human cancer cells is sufficiently dialyzed against water to remove low-molecular substances, and then brought into contact with an adsorbent under acidic conditions to be adsorbed. The active ingredients are extracted by treating with a weakly alkaline aqueous solution, followed by heat treatment to an extent that does not deactivate the TDF, removing the precipitated impurities, and then using a gel filtration method.
Separate and elute TDF. Human fetal fibroblasts used in the present invention can be obtained as follows. Aborted human fetuses in early pregnancy are cut into small pieces in an isotonic salt solution, then digested with trypsin, passed through a cell sieve to obtain a single cell suspension, and centrifuged to collect the cells. These cells are cultured in a cell culture solution, and primary human fetal fibroblasts that proliferate one week later are obtained. The culture solution used for this is 10% (W/
V) Fetal bovine serum (M, Maryland, USA)
Eagle-based products (manufactured by A. Bioproducts)
MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd., Tokyo), Dulbecco's modified Eagle-MEM medium and 199 medium (manufactured by the same company) are suitable. A human fetal fibroblast culture medium containing TDF can be obtained as follows. Primary human fetal fibroblasts are stripped from the culture vessel by trypsin treatment, the cell concentration is adjusted to 1 to 5 x 10 5 cells/ml, and the cells are transferred to a new culture vessel and cultured at 37°C. Suitable culture vessels to be used here include a shear dish for animal tissue culture, a flask, a roller bottle, or a spinner flask in which cells are attached to beads such as a side deck (manufactured by Pharmacia). When performing mixed culture with human cancer cells, the number of human fetal fibroblasts in the culture solution should be 5 x 10 5 cells/ml, and the concentration of cancer cells should be 1 to 1.
It is preferable to set it at 5×10 4 pieces/ml. The human cancer cells that can be used here are established strains that can be passaged. For example, Hela cells (Hela, CCD2), KB cells (KBCCL17), FL cells (derived from normal human anterior membrane cells), etc. are used. When only human fetal fibroblasts are cultured, the culture medium containing TDF is collected when the cells have spread over the entire culture wall in about one week. Human fetal fibroblast cells after harvesting the culture medium can be subcultured repeatedly and used for this purpose. First, the cells are peeled off by trypsin treatment and transplanted into a new culture vessel at the above cell concentration, allowing reuse until the cells lose their ability to proliferate. The TDF-containing culture solution obtained in this way is filtered to remove insoluble foreign substances, thoroughly dialyzed against water to remove dialyzable low-molecular substances, and then acidified with dilute hydrochloric acid and applied to an appropriate adsorbent. bring into contact. Kaolin, bentonite, activated clay, etc. can be used as the adsorbent. The adsorption operation involves adding an adsorbent to the above solution.
This is done by stirring, contacting and adsorbing the active ingredient. Next, after separating the adsorbent from the solution by an appropriate method such as decanting, filtration, or centrifugation, the active ingredients are extracted from the adsorbent with a weak alkaline aqueous solution, such as aqueous ammonia, and the extract is diluted with dilute hydrochloric acid. Neutralize the material using a method such as a heat treatment, and precipitate impurities by heat treatment. The heat treatment is preferably carried out at 60° to 80°C for 30 to 60 minutes. After removing the precipitate, the extract is concentrated by ultrafiltration, vacuum distillation, or freeze-drying.
This is applied to a column packed with Sephadex G-100 gel, and the active fraction flowing out before the albumin elution site is collected to obtain TDF. The properties of TDF obtained by the present invention are pale yellow powder, soluble in water, poorly soluble in methanol and ethanol, insoluble in ether, acetone, and benzene, molecular weight determined by gel filtration method is 145,000, and maximum ultraviolet absorption is 275 ~
Exists at 280nm. Nitrogen content by elemental analysis is
10.1-15.8%, ninhydrin, phenol sulfate,
It is non-dialyzable and is presumed to be a glycoprotein. TDF exhibits the following physiological properties. When 1 to 100 μg of TDF is added as an aqueous solution to a culture medium of cultured human cancer cells, it exhibits a cancer cell degeneration effect as shown in FIG. 1, for example.
Furthermore, if 1 to 1000 IU of human interferon is added at the same time as TDF, the cancer cells will further degenerate (see Figures 2A, 2B, 3A, and 3B). The above action is specific to human epithelial cancer cells such as KB and Hela cells, and no degenerative action is observed in mouse L cells even after the above manipulations. The present invention will be explained in more detail below using examples. Example 1 A culture solution of human fetal fibroblasts on the 6th day was collected and passed through a Seitz filter to remove insoluble foreign substances such as cell debris, and then placed in a cellulose tube and dialyzed against pure water overnight. PH of culture solution with 1N-hydrochloric acid
was adjusted to 4.3, and 2% of the liquid volume of kaolin (Oita, manufactured by Wako Pure Chemical Industries, Ltd.) was added and left in contact with stirring for 2 hours.
Leave to stand for a while, and remove most of the supernatant by decanting and collect the kaolin by passing it through a Nutssie filter under reduced pressure. Extract three times with 1% ammonia water in an amount of 1.5 times the wet weight of kaolin, combine the extracts, and add 1N hydrochloric acid.
Adjust the pH to 7.0 and heat at 80° for 30 minutes. The precipitate formed after heating was removed by centrifugation, the supernatant liquid was dialyzed against pure water, the water was distilled off under reduced pressure, the residue was dissolved in a small amount of water,・The eluate is fractionated using a column manufactured by Japan Co., Ltd., and the activity peak at the pre-eluting position of albumin is collected using the cancer cell degeneration activity as an index, and then freeze-dried to obtain TDF. The yield is approximately per 1 medium solution.
It is 1.2mg. Example 2 A mixed culture solution of human adipose tissue fibroblasts and KB cells was collected, passed through a sterile filter to remove insoluble matter, placed in a cellulose tube, and dialyzed against pure water overnight.The pH was then adjusted to 4.3, and 2% of the solution volume was added with kaolin. In addition, adsorbs TDF,
Extract kaolin with 1% ammonia water and extract the extract.
Adjust the pH to 7.0, heat at 70℃ for 40 minutes, remove the resulting precipitate, dialyze with pure water, evaporate the water under reduced pressure, and apply the residue to a Sephadex G-100 column to degenerate cancer cells. The peak showing activity is collected and lyophilized to obtain TDF. Yield is approximately 2.3 mg per 1.0 culture solution
It is. TDF is a pale yellow amorphous powder that is soluble in water and almost insoluble in methanol and ethanol. Completely insoluble in ether, acetone and benzene. Ultraviolet absorption: λmax275-280nm, ninhydrin reaction,
Both the phenol sulfuric acid reaction and Biuretz reaction were positive. It is also nondialyzable against water. Example 3 The enhancing effect of interferon on TDF cancer cell degeneration was investigated by the following method. 1.2 to 1.4 in a plastic petrie dish with an inner diameter of 3 cm.
Add 2 ml of ×10 6 /ml cancer cell culture solution and incubate at 37°C overnight.
Cultivate in a 5% carbon dioxide gas bubbler and add 10μ of TDF to this.
g and cultured, and then human leukocyte interferon [obtained from Kyoto Red Cross Blood Center,
Dye-Uptake method (Imanishi et al.: Biken Journal 34(3)95
(1981)) was added and cultured for 2-3 days, and then stained with Giemsa solution. (MOPAM03) and measure the ratio (%) of the cell destruction area. Each test is 2-4
I did it over and over again. The results are summarized in Table 1. A t-test result P-value of 0.05 or less is considered significant. As is clear from the table above, the activity of cancer cell degeneration factors in human fetal fibroblasts was enhanced by human leukocyte interferon, and the above-mentioned inhibitory factors did not show activity in murine cancer cells L929, which are xenogeneic cancer cells. Human leukocyte interferon also showed no enhancing effect. 【table】
【図面の簡単な説明】[Brief explanation of drawings]
第１図はヒトガン細胞（KB）培養液にTDFを
加えたときのガン細胞縮退作用を示す写真で、第
２Ｂ図は同細胞の培養液にTDFと同時にヒトイ
ンターフエロンを加えたときのガン細胞縮退作用
を示す写真（100倍）、第２Ａ図は無添加の対照写
真（100倍）である。第３Ｂ図はヒトガン細胞
（Hela）培養液にTDFと同時にヒトインターフ
エロンを加えたときのガン細胞縮退作用を示す写
真（100倍）、第３Ａ図は同無添加の対照写真
（100倍）である。第１図下方左側はヒト線維芽細
胞、下方右側はKB細胞を示し、第１，２Ｂ，３
Ｂ図において空洞部は細胞破壊領域を示す。
Figure 1 is a photograph showing the degeneration of cancer cells when TDF is added to the human cancer cell (KB) culture solution, and Figure 2B is a photograph showing the regression effect of cancer cells when TDF and human interferon are added to the culture solution of the same cells. A photograph showing the degeneracy effect (100x magnification), and Figure 2A is a control photograph (100x magnification) without additives. Figure 3B is a photograph (100x) showing the cancer cell degeneration effect when human interferon was added to human cancer cell (Hela) culture solution at the same time as TDF, and Figure 3A is a control photograph (100x) without the same additive. be. Figure 1 shows human fibroblasts on the lower left and KB cells on the lower right.
In Figure B, the cavity indicates the cell destruction area.