JPH0729933B2 - Animal malignant tumor cell growth inhibitor - Google Patents
Animal malignant tumor cell growth inhibitorInfo
- Publication number
- JPH0729933B2 JPH0729933B2 JP61148130A JP14813086A JPH0729933B2 JP H0729933 B2 JPH0729933 B2 JP H0729933B2 JP 61148130 A JP61148130 A JP 61148130A JP 14813086 A JP14813086 A JP 14813086A JP H0729933 B2 JPH0729933 B2 JP H0729933B2
- Authority
- JP
- Japan
- Prior art keywords
- malignant tumor
- tumor cell
- hours
- cell growth
- growth inhibitor
- 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.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、人を含む動物の悪性腫瘍細胞抑制剤に関する
ものである。TECHNICAL FIELD The present invention relates to an agent for suppressing malignant tumor cells in animals including humans.
従来の技術 動物の悪性腫瘍細胞の培養後培地より前記悪性腫瘍細胞
を除いて抽出したものからなる動物の悪性腫瘍細胞増殖
抑制剤は特開昭59−33223号として提案されている。2. Description of the Related Art An animal malignant tumor cell growth inhibitor comprising an animal malignant tumor cell after culturing and excluding the above malignant tumor cells and extracted has been proposed in JP-A-59-33223.
発明が解決すべき問題点 本発明は前記従来の技術によって得られた悪性腫瘍細胞
増殖抑制剤よりも純粋で抑制効果の高い動物の悪性腫瘍
細胞増殖抑制剤を提供することを目的とするものであ
る。Problems to be Solved by the Invention It is an object of the present invention to provide a malignant tumor cell growth inhibitor for animals that is purer and has a higher inhibitory effect than the malignant tumor cell growth inhibitor obtained by the above-mentioned conventional technique. is there.
問題点を解決するための手段 本発明の人を含む動物の悪性腫瘍細胞増殖抑制剤は、ヒ
ト腎細胞癌由来樹立株細胞Ｈ・Ｒ・Ｃ、マウス由来の株
化細胞LLC等の動物の悪性腫瘍細胞を10％牛胎児血清添
加のＥ−MEM（イーグルスミニマムエッセンシャルメデ
ィアム）等の培地で培養し、その後無血清培地等の抽出
用培地に移して35〜37℃で４〜７日培養し、それから前
記悪性腫瘍細胞を除くことにより、抽出培養液を得、こ
の粗製品に原液の1/25量のメタノールを加えて溶解し、
その上澄みをメタノールで膨潤したビーズ状のハイドロ
キシプロピル化デキストランゲル（米国ファルマシアフ
ァインケミカル社製LH−20）を直径10cm,長さ120cm充填
したカラムで１時間800ミリリットルの流量を通しメタ
ノールで溶出し、7.25〜9.0時間及び11.875〜13.094時
間の間に流出する区分を分画したものを総合したものよ
りなることを特徴とする。Means for Solving Problems The malignant tumor cell growth inhibitor for animals including humans of the present invention is a malignant animal such as human renal cell carcinoma-derived established cell line HRC and mouse-derived cell line LLC. Tumor cells were cultured in a medium such as E-MEM (Eagles minimum essential medium) supplemented with 10% fetal bovine serum, and then transferred to an extraction medium such as a serum-free medium and cultured at 35 to 37 ° C for 4 to 7 days. , And then by removing the malignant tumor cells, to obtain an extraction culture solution, to this crude product is dissolved by adding 1/25 amount of methanol of the stock solution,
The supernatant was swelled with methanol and beaded hydroxypropylated dextran gel (LH-20 manufactured by Pharmacia Fine Chemicals, USA) was packed in a column packed with a diameter of 10 cm and a length of 120 cm at a flow rate of 800 ml for 1 hour and eluted with methanol to give 7.25. It is characterized by being composed of a total of fractionated fractions flowing out during ~ 9.0 hours and 11.875-13.094 hours.
前記分取に際しての検出器としては254nmの波長に対す
る吸光度を計測する紫外線検出器を用いる。この分画
は、数種のアミノ酸を主成分とし、糖質を含まず、NMR
（核磁気共鳴吸収），ガスクロマトグラフィーで乳酸様
シグナルを示す物質の含量は微小である。An ultraviolet detector that measures absorbance at a wavelength of 254 nm is used as a detector for the fractionation. This fraction is mainly composed of several amino acids, contains no sugar, and
(Nuclear magnetic resonance absorption), the content of substances showing a lactate-like signal by gas chromatography is minute.
そして、分子量は1000以下であり、水との親和性が極め
て高く、無機塩と結合し易く、水，メタノールに可溶で
あって、一般に有機溶剤に溶けにくい性状を有してい
る。It has a molecular weight of 1,000 or less, has a very high affinity with water, easily binds to an inorganic salt, is soluble in water and methanol, and generally has a property of being hardly soluble in an organic solvent.
また、沸騰水浴中に１時間置いた場合、PH2〜10の範囲
で常温中に一昼夜放置した場合およびプロナーゼ処理及
びグリコシターゼ処理等に対してその活性は失われない
ものである。In addition, the activity is not lost when it is placed in a boiling water bath for 1 hour, when it is left at room temperature in the range of PH 2 to 10 for a whole day and night, and when it is treated with pronase and glycosidase.
実施例 １）使用細胞 動物の悪性腫瘍細胞としてヒト腎細胞癌由来樹立株細胞
HRCを使用した。Example 1) Cells used Human renal cell carcinoma-derived established cell line as malignant tumor cell of animal
HRC was used.
２）培養 成長用培地には10％牛胎児血清を添加したＥ−MEMに4g/
のグルコースを添加したものを用い、抽出用培地とし
ては、血清無添加のＥ−MEMにグルコースを２〜5g/添
加したものを使用した。2) Culture 4g / E-MEM supplemented with 10% fetal bovine serum was used as the growth medium.
Was used, and the medium for extraction used was E-MEM containing no serum and 2 to 5 g of glucose added.
まず成長用培地を用い、培養器に飽和状態になるまで悪
性腫瘍細胞を増殖し、その後無血清のＥ−MEM培地で洗
って血清を除く。次に、これを抽出用培地に移して35℃
〜37℃で培養し、抽出培養液を得る。First, a growth medium is used to grow malignant tumor cells in a culture vessel until the cells are saturated, and then washed with a serum-free E-MEM medium to remove serum. Next, transfer it to the extraction medium at 35 ° C.
Incubate at ~ 37 ° C to obtain an extraction medium.
３）精製法 まず、悪性腫瘍細胞を除去分離するため採取された抽出
培養液を直径60mm、内容積250ml、ポリカーボネート製
の蓋付分離容器に入れ遠心分離機にセットし毎分１万回
転で10分間遠心分離し、その上澄みを採取し、さらにこ
れを減圧蒸発乾固して粗製品を得る。3) Purification method First, the extraction culture solution collected to remove and separate malignant tumor cells is placed in a separation container with a diameter of 60 mm, an internal volume of 250 ml, a polycarbonate lid and set in a centrifuge at 10,000 rpm for 10 minutes. Centrifuge for minutes, collect the supernatant, and evaporate to dryness under reduced pressure to obtain a crude product.
その後、これに抽出培養液の1/25量のメタノールを加
え、溶解して直径60mm、深さ100mm、内容積250mlでポリ
カーボネート製の蓋付分離容器に入れ遠心分離機にセッ
トし遠心分離（１万回転,10分間）し、その上澄み液３
分をメタノールで膨潤した米国ファルマシアファイン
ケミカル社製LH−20よりなる固定相充填のカラム（直径
10cm,長さ120cm）に通し、メタノールで800ml/hの流量
速度で溶出する。After that, add 1/25 amount of methanol of the extracted culture solution, dissolve and put it into a polycarbonate separation container with a diameter of 60 mm, a depth of 100 mm and an internal volume of 250 ml, set it in a centrifuge and centrifuge (1 10,000 revolutions, 10 minutes), and the supernatant 3
Column packed with stationary phase consisting of LH-20 manufactured by Pharmacia Fine Chemical Co., USA, swollen with methanol (diameter
10 cm, length 120 cm) and elute with methanol at a flow rate of 800 ml / h.
検出には254nmの波長に対する吸光度を計測する紫外線
検出器を使用し、図に示すように、7.25〜9.0時間及び1
1.875〜13.094時間に流出する区分を分取したものを総
合して総合分画を得た。なお、チャートスピードは4cm/
hourである。An ultraviolet detector that measures absorbance at a wavelength of 254 nm was used for detection, and as shown in the figure, 7.25 to 9.0 hours and 1
The fractions collected from 1.875 to 13.094 hours were collected to obtain a total fraction. The chart speed is 4 cm /
hour.
次に、このようにして採取した分画を10mmHg、40℃で減
圧蒸発乾固し、さらにこれを100倍濃度の濃縮水溶液と
する。Next, the fraction thus collected is evaporated to dryness under reduced pressure at 10 mmHg and 40 ° C., and this is made into a concentrated aqueous solution having a concentration of 100 times.
４）検定 （イ）24穴培養皿に２×105個／穴のヒト腎細胞癌由来
樹立株細胞HRCを植え込み、実験群を10％牛胎児血清を
Ｅ−MEMに混合したものを倍地として各穴に1.5ml加え、
37℃,5％CO2,100％湿度で培養する。１日おきに培地交
換し、７日目に細胞数を計り増殖を調べた。4) Assay (a) 2 × 10 5 cells / well of human renal cell carcinoma-derived established HRC cells were implanted in a 24-well culture dish, and the experimental group was mixed with 10% fetal bovine serum in E-MEM. Add 1.5 ml to each hole as
Incubate at 37 ℃, 5% CO 2 and 100% humidity. The medium was replaced every other day, and the number of cells was measured on the 7th day to examine the growth.
対照標準として新鮮培地で培養したヒト腎細胞癌由来樹
立株細胞HRCの数を100％とし、7.25〜9.0時間に流出す
る分画及び11.875〜13.094時間に流出する分画を原液に
換算して10倍濃度としたものをそれぞれA1及びB1とし、
５倍濃度としたものをそれぞれA2及びB2として新鮮培地
に投与してその影響を検べた結果を第１表に示す。The number of established human renal cell carcinoma-derived HRC cells HRC cultured in a fresh medium as a control is set to 100%, and the fractions flowing out at 7.25 to 9.0 hours and the fractions flowing out at 11.875 to 13.094 hours are converted into undiluted solution to 10%. fold concentration were ones and a 1 and B 1, respectively,
Table 1 shows the results of examining the effect of 5-fold concentrated A 2 and B 2 each administered to a fresh medium.
（ロ）マウスにC57BLを用い、このマウスにマウスルイ
ス肺癌細胞106個を側背部皮下に移植し、7.25〜9.0時間
に流出する分画A3、11.875〜、13.094時間に流出する分
画B3及びこれら分画の総合分画Ｃの50倍濃縮水溶液を１
日２回0.25ml注射によって投与し、投与８日目の腫瘍の
縦×横の大きさ及び投与12日目の解剖による腫瘍の重量
を計測した。対照標準に対する計測値を第２表及び第４
表に示す。 (B) C57BL was used as a mouse, and 10 6 mouse Lewis lung cancer cells were subcutaneously transplanted to the lateral dorsal region of this mouse, and fraction A 3 flowed out at 7.25 to 9.0 hours, and fraction B flowed out at 11.875 to 13.094 hours. 3 and a 50 times concentrated aqueous solution of the total fraction C of these fractions
It was administered by 0.25 ml injection twice a day, and the vertical and horizontal dimensions of the tumor on the 8th day of administration and the weight of the tumor by dissection on the 12th day of administration were measured. The measured values for the control standard are shown in Tables 2 and 4.
Shown in the table.
以上のように、7.25〜9.0時間に流出する分画Ａと11.87
5〜13.094時間に流出する分画Ｂとは悪性腫瘍細胞抑制
効果を異にしており、分画Ｂは増殖の初期抑制率が高
く、分画Ａは増殖途上における抑制力が高い。動物実験
において、この両者を混合した分画を投与することによ
り、より高く、安定した抑制効果が得られる。 As described above, Fraction A and 11.87 flowing out from 7.25 to 9.0 hours
The inhibitory effect on malignant tumor cells is different from that of Fraction B flowing out at 5 to 13.094 hours, Fraction B has a high initial inhibition rate of proliferation, and Fraction A has a high inhibitory effect in the process of proliferation. In animal experiments, a higher and more stable inhibitory effect can be obtained by administering a fraction in which both are mixed.
図は、本発明の動物の悪性腫瘍細胞増殖抑制剤の分画抽
出を示す線図である。The figure is a diagram showing the fractional extraction of the malignant tumor cell growth inhibitor of the animal of the present invention.
Claims (1)
た後、抽出用培地に移して35〜37℃で培養し、前記悪性
腫瘍細胞を除いたものを、メタノールで膨潤したビーズ
状のハイドロキシプロピル化デキストランゲルよりなる
固定相を直径10cm,長さ120cm充填したカラムで１時間80
0ミリリットルの流量で溶出し、7.25〜9.0時間及び11.8
75〜13.094時間の間に流出する区分を分取したものを総
合したものよりなることを特徴とする人を含む動物の悪
性腫瘍細胞増殖抑制剤。1. A malignant tumor cell of an animal including a human being cultured and proliferated, transferred to an extraction medium and cultured at 35 to 37 ° C., and excluding the malignant tumor cell, a bead-shaped product swollen with methanol A stationary phase composed of hydroxypropylated dextran gel was packed in a column packed with a diameter of 10 cm and a length of 120 cm for 80 hours for 1 hour.
Elute at a flow rate of 0 ml, 7.25 to 9.0 hours and 11.8
An agent for suppressing the growth of malignant tumor cells in animals, including humans, which is composed of a total of fractions collected during the period of 75 to 13.094 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61148130A JPH0729933B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61148130A JPH0729933B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS635026A JPS635026A (en) | 1988-01-11 |
| JPH0729933B2 true JPH0729933B2 (en) | 1995-04-05 |
Family
ID=15445928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61148130A Expired - Fee Related JPH0729933B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0729933B2 (en) |
-
1986
- 1986-06-26 JP JP61148130A patent/JPH0729933B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS635026A (en) | 1988-01-11 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |