JPH0729931B2 - Animal malignant tumor cell growth inhibitor - Google Patents
Animal malignant tumor cell growth inhibitorInfo
- Publication number
- JPH0729931B2 JPH0729931B2 JP61148128A JP14812886A JPH0729931B2 JP H0729931 B2 JPH0729931 B2 JP H0729931B2 JP 61148128 A JP61148128 A JP 61148128A JP 14812886 A JP14812886 A JP 14812886A JP H0729931 B2 JPH0729931 B2 JP H0729931B2
- Authority
- JP
- Japan
- Prior art keywords
- malignant tumor
- tumor cell
- cell growth
- growth inhibitor
- medium
- 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、10.75〜11.875時間の間に流出する区
分を分画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 swollen 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 to elute with methanol. It is characterized in that it consists of a fraction of the section flowing out within 11.875 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して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 extracted culture broth obtained by removing malignant tumor cells is placed in a container with a polycarbonate lid with a diameter of 60 mm and an internal volume of 250 ml, set in a centrifuge, and centrifuged at 10,000 rpm for 10 minutes. Separate, 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、10.75〜11.875時間
に流出する区分より分画を得た。なお、チャートスピー
ドは4cm/hourである。For detection, an ultraviolet ray detector measuring the absorbance at a wavelength of 254 nm was used, and as shown in the figure, fractions were obtained from the section flowing out at 10.75 to 11.875 hours. 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に混合したものを培地として各穴に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 cell line HRC were implanted in a 24-well culture dish, and the experimental group was prepared by mixing 10% fetal bovine serum with E-MEM as a medium. Add 1.5 ml to each hole,
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、分画を原液に換算して10
倍濃度としたものをＡとし、５倍濃度としたものをＢと
して新鮮倍地に投与してその影響を検べた結果を第１表
に示す。As a reference standard, the number of established HRC cells derived from human renal cell carcinoma cultured in fresh medium was set to 100%, and the fractions were
Table 1 shows the results of examining the effect of a double concentration administered as A and a 5-fold concentration as B, which was administered to 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、分画の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 into the dorsal region of the back of the mouse, and a 50-fold concentrated aqueous solution of the fraction was administered twice a day by 0.25 ml injection to give 8 doses.
The length × width size of the tumor on the day and the weight of the tumor on the 12th day after administration were measured. The measured values for the control are shown in Tables 2 and 4.
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、10.75〜11.875時間の間
に流出する区分を分取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.
An agent for suppressing the growth of malignant tumor cells in animals, including humans, which is characterized in that it is eluted at a flow rate of 0 ml and is collected in a fraction flowing out for 10.75 to 11.875 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61148128A JPH0729931B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61148128A JPH0729931B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS635024A JPS635024A (en) | 1988-01-11 |
| JPH0729931B2 true JPH0729931B2 (en) | 1995-04-05 |
Family
ID=15445883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61148128A Expired - Fee Related JPH0729931B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0729931B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69508328T2 (en) * | 1994-03-31 | 1999-11-04 | Citizen Watch Co., Ltd. | CLOCK WITH A LIGHT-TRANSFERING DISPLAY PLATE |
-
1986
- 1986-06-26 JP JP61148128A patent/JPH0729931B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPS635024A (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 |