JPS635023A - Agent for suppressing growth of malignant tumor cell of animal - Google Patents
Agent for suppressing growth of malignant tumor cell of animalInfo
- Publication number
- JPS635023A JPS635023A JP61148127A JP14812786A JPS635023A JP S635023 A JPS635023 A JP S635023A JP 61148127 A JP61148127 A JP 61148127A JP 14812786 A JP14812786 A JP 14812786A JP S635023 A JPS635023 A JP S635023A
- Authority
- JP
- Japan
- Prior art keywords
- malignant tumor
- tumor cell
- medium
- cultured
- liquid
- 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
- 201000011510 cancer Diseases 0.000 title claims abstract description 21
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 19
- 241001465754 Metazoa Species 0.000 title claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 230000002062 proliferating effect Effects 0.000 claims abstract 2
- 230000005526 G1 to G0 transition Effects 0.000 claims description 2
- 239000003966 growth inhibitor Substances 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims 1
- 230000004565 tumor cell growth Effects 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 21
- 210000004027 cell Anatomy 0.000 abstract description 11
- 239000002609 medium Substances 0.000 abstract description 10
- 239000007788 liquid Substances 0.000 abstract description 6
- 239000006228 supernatant Substances 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 3
- 239000012043 crude product Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 239000011324 bead Substances 0.000 abstract description 2
- 230000035755 proliferation Effects 0.000 abstract description 2
- 239000012679 serum free medium Substances 0.000 abstract description 2
- 229920005654 Sephadex Polymers 0.000 abstract 1
- 210000003360 nephrocyte Anatomy 0.000 abstract 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 208000006265 Renal cell carcinoma Diseases 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 3
- 239000003760 tallow Substances 0.000 description 3
- 241000282412 Homo Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
Abstract
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。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a malignant tumor cell inhibitor for 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１ｈｍ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。BACKGROUND OF THE INVENTION A growth inhibitor for animal malignant 1hm tumor cells, which is obtained by removing the malignant tumor cells from the culture medium of animal malignant tumor cells, 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１ｑ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
肝癌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: An object of the present invention is to provide an animal malignant liver cancer cell proliferation inhibitor that is purer and has a higher inhibitory effect than the malignant tumor cell proliferation inhibitor produced by the prior art. It is.
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Ｌ’ＬＣ等の動物の悪性腫瘍細胞を１０％牛脂児
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１／２５ｆｆｉ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ヶミカル社’ＩＬＨ−２０
）を直径１Ｑｃｍ、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、５．６２５〜７゜２５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 the Problems The agent for suppressing the growth of malignant tumor cells in animals including humans according to the present invention can be applied to animals such as human renal cell carcinoma-derived established cell line H-RC, mouse-derived established cell line L'LC, etc. Malignant tumor cells are cultured in a medium such as E-MEM (Eagles Minimum Essential Medium) supplemented with 10% tallow serum, and then transferred to an extraction medium such as a serum-free medium for 4 to 7 days at 35 to 37°C. An extracted culture solution is obtained by culturing and then removing the malignant tumor cells, and this crude product is dissolved by adding 1/25 ffi of methanol to the original solution, and the supernatant is mixed with methanol-swollen beads of hydroxypropylated dextran. Gel (Pharmacia Fine Chemicals, USA) ILH-20
) packed in a column with a diameter of 1Qcm and a length of 120cm.
It is characterized by being eluted with methanol through a flow rate of 800 ml per hour, and fractionated from a section that flows out between 5.625 and 7.25 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、Ｎ
ＭＲ（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。As a detector for the fractionation, an ultraviolet detector that measures absorbance at a wavelength of 254 nm is used. This fraction contains several types of amino acids as its main components, does not contain carbohydrates, and is
The content of substances that show lactic acid-like signals in MR (nuclear magnetic resonance absorption) and 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。It has a molecular weight of 1000 or less, has an extremely high affinity with water, and easily binds to inorganic salts.
メタノールに可溶であって、−般に有機溶剤に溶けにく
い性状を有している。It is soluble in methanol and generally has a property of being difficult to dissolve in organic solvents.
また、■種水浴中に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 left in a water bath for 1 hour, when left overnight at room temperature at a pH of 2 to 10, and when treated with pronase or glycosidase.
実廠例
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。Practical example 1) Cells used HRC, an established cell line derived from human renal cell carcinoma, was used as the malignant tumor cell of the animal.
２）培 養
成長用培地には１０％牛脂児血清を添加したＥ−ＭＥＭ
に４ａ ／１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２〜５（１／１添加したものを使用した。2) Culture The growth medium is E-MEM supplemented with 10% tallow serum.
The extraction medium used was serum-free E-MEM to which 2 to 5 (1/1) glucose was 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３
５℃〜３７℃で培養し、抽出培養液を得る。First, malignant tumor cells are grown in an incubator until saturated using a growth medium, and then washed with serum-free E-MEM medium to remove serum. Next, transfer this to extraction medium and
Culture at 5°C to 37°C to obtain an extracted culture solution.
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５Ｑｍｍ、内容積２５０ｍｕ、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て毎分
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 solution collected to remove and separate malignant tumor cells was placed in a polycarbonate lidded container with a diameter of 5 Q mm and an internal volume of 250 mu, set in a centrifuge, and rotated at 10,000 revolutions per minute for 10 minutes. Centrifuge for minutes, collect the supernatant,
Further, this is evaporated to dryness under reduced pressure to obtain a crude product.
その後、これに抽出培養液の１ ／２５ｍのメタノール
を加え、溶解して直径６０＋１１１１１、深さ１００ｍ
ｍ、内容積２５０ｍ１でポリカーボネート製の蓋付分離
容器に入れ遠心分類機にセットし遠心分離（１万回転、
１０分間）し、その上澄み液３ｉ分をメタノールで膨潤
した米国ファルマシアフ？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１Ｑｃｌｎ、長さ１
２０ｃｍ）に通し、メタノールで８００ｍ１／ｈの流迅
速度で溶出する。Then, add 1/25 m of methanol to the extracted culture solution and dissolve it to a diameter of 60+11111 and a depth of 100 m.
Pour into a polycarbonate separation container with an internal volume of 250 m1, set it in a centrifugal sorter, and centrifuge it (10,000 rotations,
(for 10 minutes) and swollen 3 hours of the supernatant liquid with methanol. LH-20 manufactured by In-Chemical Co., Ltd.
A column packed with a stationary phase consisting of (diameter 1Qcln, length 1
20 cm) and elute with methanol at a flow rate of 800 ml/h.
検出には２５４ ｎｍ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、５．６２５〜
７．２５時間に流出する区分より分画を１ｑだ。なお、
チャートスピードは４ ａｍ７ ｈｏｕｒである。For detection, an ultraviolet detector that measures absorbance at a wavelength of 254 nm is used.
7. The fraction that flows out in 25 hours is 1q. In addition,
Chart speed is 4 am 7 hours.
次に、このようにして採取した分画を１０ｍ１ｌｌＨ９
，４０℃で減圧蒸発乾固し、ざらにこれを１００倍濃度
の１縮水溶液とする。Next, the fraction collected in this way was added to 10 ml H9
, evaporate to dryness under reduced pressure at 40°C, and roughly make a 100-fold concentrated aqueous solution.
４）検 定
（イ）２４穴培養皿に２Ｘ１０５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％牛脂
児血清をＥ−ＭＥＭに混合したものを培地として金穴に
１．５ｍｕ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 x 10 cells/well of human renal cell carcinoma-derived established cell lines HRC were implanted in a 24-well culture dish, and the experimental group was cultured using a mixture of 10% beef tallow serum in E-MEM as a medium. 1.5 mu was added to the culture medium and cultured at 37°C, 5% CO2, and 100% humidity. The medium was replaced every other day, and on the 7th day, the number of cells was counted to examine proliferation.
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
立株柵胞ＨＲＣの数を１００％とし、分画を原液に換算
してｉ ｏ＠ａ度としたものをΔとし、５倍１度とした
ものをＢ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。As a control standard, the number of the established strain HRC derived from human renal cell carcinoma cultured in fresh medium is taken as 100%, and the fraction is converted to the stock solution and io@a degree is Δ, and 5 times 1 degree. Table 1 shows the results of administering the sample B to a fresh medium and examining its effect.
第１表
（ロ）マウスに０５７ＢＬを用い、このマウスにマウス
ルイス肺癌細胞１０８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０．２５ｍ１注射によって
投与し、投与８日目の腫瘍のｗｉ×横の大きさ及び投与
１２日目の解剖によるＩｌ！ｉ瘍の重量を計潤した。対
照標準に対する計測値を第２表及び第４表に示す。Table 1 (b) Using 057BL in mice, 108 mouse Lewis lung cancer cells were subcutaneously transplanted into the dorsal side of the mice, and a 50-fold concentrated aqueous solution of the fraction was administered by injection of 0.25 ml twice a day. Wi x horizontal size of the tumor on the 8th day and Il according to the dissection on the 12th day of administration! The weight of the i.c. tumor was weighed. Measured values for the control standards are shown in Tables 2 and 4.
第２表 第３表Table 2 Table 3
図は、本発明の動物の悪性腫ｇ、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 animal malignant tumor G and the cytostatic agent of the present invention.
Claims (1)
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
８００ミリリットルの流量で溶出し、５．６２５〜７．
２５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
。After culturing and proliferating malignant tumor cells of animals including humans, they were transferred to an extraction medium and cultured at 35 to 37°C, and the malignant tumor cells were removed. Elution was carried out at a flow rate of 800 ml for 1 hour using a column packed with a stationary phase of 5.625 to 7.0 cm in diameter and 120 cm in length.
1. A malignant tumor cell growth inhibitor for animals including humans, characterized in that the agent is obtained by collecting fractions that flow out over a period of 25 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61148127A JPH0720871B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61148127A JPH0720871B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS635023A true JPS635023A (en) | 1988-01-11 |
| JPH0720871B2 JPH0720871B2 (en) | 1995-03-08 |
Family
ID=15445862
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61148127A Expired - Fee Related JPH0720871B2 (en) | 1986-06-26 | 1986-06-26 | Animal malignant tumor cell growth inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0720871B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7741426B2 (en) | 2003-06-23 | 2010-06-22 | Nippon Shokubai Co., Ltd | Method for production of fluorinated phenylenediamine |
-
1986
- 1986-06-26 JP JP61148127A patent/JPH0720871B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7741426B2 (en) | 2003-06-23 | 2010-06-22 | Nippon Shokubai Co., Ltd | Method for production of fluorinated phenylenediamine |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0720871B2 (en) | 1995-03-08 |
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