JPH03236772A - New cell proliferation promoter and additive containing the same as active ingredient for culture medium used in cell culture - Google Patents
New cell proliferation promoter and additive containing the same as active ingredient for culture medium used in cell cultureInfo
- Publication number
- JPH03236772A JPH03236772A JP2031750A JP3175090A JPH03236772A JP H03236772 A JPH03236772 A JP H03236772A JP 2031750 A JP2031750 A JP 2031750A JP 3175090 A JP3175090 A JP 3175090A JP H03236772 A JPH03236772 A JP H03236772A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- culture
- bovine colostrum
- cells
- proliferation promoter
- 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.)
- Pending
Links
- 239000000654 additive Substances 0.000 title claims abstract description 11
- 230000000996 additive effect Effects 0.000 title claims abstract description 9
- 239000004480 active ingredient Substances 0.000 title claims description 5
- 239000001963 growth medium Substances 0.000 title abstract description 8
- 238000004113 cell culture Methods 0.000 title abstract description 7
- 230000004663 cell proliferation Effects 0.000 title abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 31
- 210000003022 colostrum Anatomy 0.000 claims abstract description 29
- 235000021277 colostrum Nutrition 0.000 claims abstract description 29
- 241000283690 Bos taurus Species 0.000 claims abstract description 22
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 5
- 230000001737 promoting effect Effects 0.000 claims description 20
- 230000010261 cell growth Effects 0.000 claims description 19
- 239000006143 cell culture medium Substances 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 abstract description 30
- 210000004102 animal cell Anatomy 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 5
- 239000003102 growth factor Substances 0.000 abstract description 4
- 235000013336 milk Nutrition 0.000 abstract description 3
- 239000008267 milk Substances 0.000 abstract description 3
- 210000004080 milk Anatomy 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 241000030939 Bubalus bubalis Species 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract description 2
- 235000013365 dairy product Nutrition 0.000 abstract description 2
- 230000002062 proliferating effect Effects 0.000 abstract description 2
- 210000002966 serum Anatomy 0.000 description 14
- 239000005862 Whey Substances 0.000 description 11
- 102000007544 Whey Proteins Human genes 0.000 description 11
- 108010046377 Whey Proteins Proteins 0.000 description 11
- 244000309466 calf Species 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000012010 growth Effects 0.000 description 5
- 238000001155 isoelectric focusing Methods 0.000 description 5
- 239000012141 concentrate Substances 0.000 description 4
- 238000002523 gelfiltration Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000005277 cation exchange chromatography Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241001325209 Nama Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (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、牛初乳から得ることができる新規な細胞増殖
促進物質およびこれを有効成分とする細胞培養用培地添
加物に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a novel cell growth promoting substance obtainable from bovine colostrum and a cell culture medium additive containing this substance as an active ingredient.
（従来の技術）
近年、成長因子に関する研究が進められており、一部は
乳汁からも単離され、その構造および機能が解析されて
いる。(Prior Art) Research on growth factors has been progressing in recent years, and some have been isolated from milk, and their structures and functions have been analyzed.
乳汁に由来する成長因子の１つには牛初乳威長因子（Ｂ
ＣＧＦ）があり、これは分子量１４，０００〜１７，８
００、等電点４．４〜４．８であり、各種細胞に対して
増殖効果を有することが報告されている（Ｙ、 Ｈ，Ｓ
ｈｉｎｇｅｔ ａｔ、、 Ｅｎｄｏｃｒｉｎｏｌｏｇｙ
１１５．２７３（１９８４））。One of the growth factors derived from milk is bovine colostrum prestige factor (B
CGF), which has a molecular weight of 14,000 to 17,8
00, has an isoelectric point of 4.4 to 4.8, and is reported to have a proliferative effect on various cells (Y, H, S
Hinget at,, Endocrinology
115.273 (1984)).
さらに、乳汁または乳製品から人手でき、５ＤＳＰＡＧ
ｆ！による測定で分子量約２５，０００、等電点ｐＩ９
．０〜９．５の塩基性ポリペプチドである乳成長因子（
ＭＧＦ） も知られている（特開平１−１２１３００号
公報）。In addition, 5DSPAG
f! Molecular weight approximately 25,000, isoelectric point pI9 as measured by
．． Milk growth factor (
MGF) is also known (Japanese Unexamined Patent Publication No. 1-121300).
本発明者らは、畝上の状況に鑑みて、牛初乳中の新規な
生理活性物質を検索すべく研究を重ねたところ、従来知
られている成長因子とはその理化学的性質において明ら
かに相違する新たな細胞増殖促進物質を見出して本発明
を完成するに至った。In view of the situation on ridges, the present inventors conducted repeated research to search for new physiologically active substances in bovine colostrum, and found that they were clearly different from conventionally known growth factors in terms of their physicochemical properties. The present invention was completed by discovering a new and different cell growth promoting substance.
すなわち、本発明は、牛初乳から得られた新規な細胞増
殖促進物質及びこれを有効成分とする細胞培養用培地添
加物を提供することを課題とする。That is, an object of the present invention is to provide a novel cell growth promoting substance obtained from bovine colostrum and a cell culture medium additive containing the same as an active ingredient.
（課題を解決するための手段）
本発明は、下記の理化学的性質を有する新規な細胞増殖
促進物質に関する。(Means for Solving the Problems) The present invention relates to a novel cell proliferation-promoting substance having the following physical and chemical properties.
牛初乳から抽出でき、５０５−ＰＡＧＥによる測定で、
３３．０００〜３６．０００の分子量を有しており、か
つｐＩ８．４〜１０の等電点を有することを特徴とする
細胞増殖促進物質。It can be extracted from bovine colostrum and measured by 505-PAGE.
A cell growth promoting substance having a molecular weight of 33,000 to 36,000 and an isoelectric point of pI 8.4 to 10.
さらに、本発明は、上記新規細胞増殖促進物質を有効成
分とする細胞、特に動物細胞の培養用培地添加物に関す
る。Furthermore, the present invention relates to a medium additive for culturing cells, particularly animal cells, containing the above novel cell growth promoting substance as an active ingredient.
従来、イン・ビトロで正常Ｂａ１ｂ／ｃ ３Ｔ３繊維芽
細胞やＮａｍａ １ｗａ リンパ芽球細胞を増殖する際
に、標準培地に血清を添加しているが、初乳そのものを
含む培地中では良好な増殖ができないことが報告されて
いる。Conventionally, when normal Ba1b/c 3T3 fibroblasts and Nama 1wa lymphoblastoid cells are grown in vitro, serum is added to the standard medium, but good growth is achieved in a medium containing colostrum itself. It has been reported that it is not possible.
本発明者らは、牛初乳中に上記細胞の増殖と抑制に作用
する物質が存在するとの考えに立ち、牛初乳の分画と精
製に取り組んだ。The present inventors worked on the fractionation and purification of bovine colostrum based on the idea that there is a substance in bovine colostrum that acts on the proliferation and suppression of the above-mentioned cells.
その結果、増殖促進物質を以下の手順に従って初乳から
得ることができた。すなわち、分娩後５日以内に搾乳し
た牛初乳を集め、脱脂処理して脱脂初乳を調製した後、
不活性タンパク質を除去し、ホエーを得た。この初乳ホ
エーに対して、等電点電気泳動、ゲル濾過、陽イオン交
換クロマトグラフィー処理を順次行い、本発明の細胞増
殖促進物質を得た。また、この物質の分子量を測定する
ため、Ｌａｅｍｍｌｉ の方法に従い、ＳＤＳ−ＰＡＧ
Ｅ！を行なった結果、分子量は３３，０００〜３６，０
００であった。As a result, growth-promoting substances could be obtained from colostrum according to the following procedure. That is, after collecting cow colostrum milked within 5 days after calving and preparing defatted colostrum by defatting treatment,
Inactive proteins were removed to obtain whey. This colostrum whey was sequentially subjected to isoelectric focusing, gel filtration, and cation exchange chromatography to obtain the cell growth promoting substance of the present invention. In addition, in order to measure the molecular weight of this substance, we used SDS-PAG according to the method of Laemmli.
E! As a result, the molecular weight was 33,000 to 36,0
It was 00.
本発明の細胞増殖促進物質は、従来孔に由来するＢＣＧ
ＦやＭＧＦと比較すると分子量、等電点等において下記
のように相違し、この点において新規な細胞増殖促進物
質である。The cell growth promoting substance of the present invention is BCG derived from conventional pores.
Compared with F and MGF, it differs in molecular weight, isoelectric point, etc. as shown below, and in this respect it is a novel cell growth promoting substance.
さらに、該細胞増殖促進物質の増殖活性を調査するため
、基本培地となるＤＭＥＭ （Ｄｕｌｂｅｃｃｏ’ｓｍ
ｏｄｉｆｉｅｄ Ｍｉｎｉｍｕｍ Ｅｓ５ｅｎｔｉａｌ
Ｍｅｄｉｕｓ＋）に仔牛血清、初乳ホエーおよび本発
明の該細胞増殖促進物質をそれぞれ０−１ｏ％の範囲内
で加え、Ｂａ１ｂ／ｃ ３Ｔ３細胞を各培地にそれぞれ
３Ｘ１０’個／ｄになるように接種して８日間培養した
。Furthermore, in order to investigate the proliferation activity of the cell proliferation-promoting substance, we used DMEM (Dulbecco's
odified Minimum Es5nential
Add calf serum, colostrum whey, and the cell growth promoting substance of the present invention to Medius+) within the range of 0-10%, and inoculate Ba1b/c 3T3 cells into each medium at 3 x 10' cells/d. and cultured for 8 days.
なお、Ｂａ１ｂ／ｃ ３Ｔ３細胞の増殖試験に当たって
は、０．２５％クリスタルバイオレット７２０％メタノ
ール溶液で生細胞を固定染色して、その吸光度から細胞
数を測定した。In addition, in the proliferation test of Ba1b/c 3T3 cells, living cells were fixed and stained with a 0.25% crystal violet 720% methanol solution, and the number of cells was determined from the absorbance.
初乳ホエーを添加した培地中では、いずれも生育が良く
なかったが、基本培地に仔牛血清および該細胞増殖促進
物質を組合せて添加し、た培地中では、いずれも仔牛血
清単独で加えた場合より良好な増殖曲線を示し、培養５
日後には３〜８Ｘ１０’個／＋ｄとなった。Growth was not good in any medium supplemented with colostrum whey, but in both cases, a combination of calf serum and the cell growth promoting substance was added to the basal medium, and calf serum alone was added to the basal medium. Showing a better growth curve, culture 5
After a few days, the number was 3 to 8×10′ pieces/+d.
また、Ｎａｍａ１ｗａ細胞では、ＲＰＭＩ−１６４０を
基本培地として、？ｌＴＴアッセイにより細胞数を測定
した結果、同様な増殖促進効果が認められた。In addition, for Nama1wa cells, RPMI-1640 was used as the basic medium. As a result of measuring the cell number by lTT assay, a similar proliferation promoting effect was observed.
以上のことから、得られた抽出物は、細胞、特に動物細
胞培養の増殖促進物質および培地添加物として有用であ
る。From the above, the obtained extract is useful as a growth promoter and medium additive for cell, especially animal cell culture.
本発明における牛初乳は、乳牛、水牛等の牛の分娩直後
〜約１週間のうちに搾乳されたものを意味する。また細
胞培養用培地添加物は、本発明の物質を単独で用いても
よいが、また、その他の細胞培養用培地添加物と併用し
て組成物として用いてもよい、また、本発明の物質を単
離して用いることは勿論、これを含有する濃縮物等も増
殖促進効果がある限り使用することができる。さらに、
本発明の物質は、動物細胞培地にもまた植物細胞培地に
も添加することができるが、特に動物細胞培地に添加す
ると、血清の使用量を軽減することができる点で有利で
ある。添加量は、培地１２当り、１０〜１００■程度が
望ましい。In the present invention, bovine colostrum refers to colostrum obtained from cows such as dairy cows and water buffaloes that are milked from immediately after calving to within about one week. In addition, the substance of the present invention may be used alone as a cell culture medium additive, but it may also be used as a composition in combination with other cell culture medium additives. Not only can it be isolated and used, but also concentrates containing it can be used as long as they have a proliferation-promoting effect. moreover,
The substance of the present invention can be added to both animal cell culture medium and plant cell culture medium, but it is particularly advantageous when added to animal cell culture medium in that the amount of serum used can be reduced. The amount added is desirably about 10 to 100 ml per 12 of the culture medium.
（発明の効果）
本発明により、新規な細胞増殖促進物質を得ることがで
きた。また、該物質を、細胞、特に動物細胞培養の際に
培地に添加することによって、細胞の増殖を促進するこ
とができ、高価な血清の添加量を軽減でき、生産コスト
を安価にすることが可能となった。(Effects of the Invention) According to the present invention, a novel cell growth promoting substance could be obtained. Furthermore, by adding this substance to the culture medium during cell, especially animal cell culture, cell proliferation can be promoted, the amount of expensive serum added can be reduced, and production costs can be reduced. It has become possible.
以下、実施例により本発明を具体的に説明する。Hereinafter, the present invention will be specifically explained with reference to Examples.
実施例１
分娩後５日以内に搾乳した牛初乳３００−から、常法に
従い脱脂乳さらにホエーを調製した。次に、初乳ホエー
を透析あるいは、５ｅｐｈａｄｅｘ Ｇ−２５（ファル
スシア社製）をサイズφ５０ｍａ＋ Ｘ ３００ｍｍ０
カラムへ充填し、水で平衡化したカラムを用いたゲル濾
過により脱塩し、凍結乾燥により濃縮物２０ｇを得た。Example 1 Skim milk and whey were prepared according to a conventional method from 300 g of cow colostrum milked within 5 days after calving. Next, the colostrum whey was dialyzed or 5ephadex G-25 (manufactured by Farussia) was used with a size of φ50 mm + x 300 mm0.
The mixture was packed into a column, desalted by gel filtration using a column equilibrated with water, and lyophilized to obtain 20 g of a concentrate.
この濃縮物を０．５〜１．０％（Ｗ／Ｖ）の濃度で水に
溶解し、さらに１〜２％（Ｖ／Ｖ）の濃度となるように
静ρｈｏｌｉｔｅ等の両性電解質を加え、調製用等電点
電気泳動装置（Ｂｉｏ−Ｒａｄ社製）を用い、等電点電
気泳動を行った。この結果を第１図に示す。This concentrate is dissolved in water at a concentration of 0.5 to 1.0% (W/V), and an amphoteric electrolyte such as static ρholite is further added to a concentration of 1 to 2% (V/V). Isoelectric focusing was performed using a preparative isoelectric focusing device (manufactured by Bio-Rad). The results are shown in FIG.
ｐ＋ ８．４〜１０．０の活性画分（画分数１６〜１９
）を回収し、ＵＰ−ＰＦＩＯＰＳ （東ソー社製）を用
いたＵＦ処理による濃度を行った後、等張リン酸緩衝液
で平衡化したＴＯＹＯＰＥＡＬ−ＨＷ５５Ｆ （東ソー
社製）を充填したカラムを用い、ゲル濾過を実施した。Active fraction with p+ 8.4-10.0 (number of fractions 16-19
) was collected and concentrated by UF treatment using UP-PFIOPS (manufactured by Tosoh Corporation), using a column packed with TOYOPEAL-HW55F (manufactured by Tosoh Corporation) equilibrated with isotonic phosphate buffer. Gel filtration was performed.
その結果を第２図に示す。図に見られる活性画分を再び
回収し、陽イオンクロマトグラフィーに供した。陽イオ
ン交換体として、５Ｐ−ＴＯＹＯＰＥＡＬ６５０ （東
ソー社製）を用い、これをゲル濾過と同様に等張リン酸
緩衝液で平衡化し、サイズφ２６＋ｕ＋ Ｘ ２５０ｍ
ｍのカラムに充填してクロマトグラフィーを行った。溶
出は、平衡化に用いた緩衝液とこれに、ＩＭとなるよう
にＮａＣ１を加えた緩衝液との塩濃度勾配溶出とした。The results are shown in FIG. The active fraction seen in the figure was collected again and subjected to cation chromatography. As a cation exchanger, 5P-TOYOPEAL650 (manufactured by Tosoh Corporation) was used, and as in gel filtration, it was equilibrated with isotonic phosphate buffer, and the size was φ26+u+×250m.
Chromatography was performed by packing the sample into a column of m. Elution was carried out by salt concentration gradient elution using the buffer used for equilibration and a buffer to which NaCl was added to give IM.
その結果、活性画分はシャープなピークとなり、この画
分を回収、脱塩処理した後、凍結乾燥して１２０ｍｇの
粉末を得た。これを細胞増殖促進物質とした（第３図参
照）。As a result, the active fraction had a sharp peak, and this fraction was collected, desalted, and lyophilized to obtain 120 mg of powder. This was used as a cell growth promoting substance (see Figure 3).
また、ＳＤＳ−ＰＡＧＥ （Ｌａｅｍｍｌｉ法）により
分子量を測定した結果、３３．０００〜３６，０００で
あった。Further, the molecular weight was measured by SDS-PAGE (Laemmli method) and was 33,000 to 36,000.
実施例２
ＤＭＥＨに仔牛血清９％および実施例１によって得られ
た細胞増殖促進物質１％を加えた培地１００μｌを０．
３２ｃｊの９６穴培養用プレートに入れ、Ｂａ１ｂ／ｃ
３Ｔ３細胞数が３Ｘ１０’個／ｄになるように接種した
。Example 2 100 μl of a medium prepared by adding 9% calf serum and 1% of the cell growth promoting substance obtained in Example 1 to DMEH was added to 0.0 μl of DMEH.
Place Ba1b/c in a 32cj 96-well culture plate.
The cells were inoculated so that the number of 3T3 cells was 3×10′ cells/d.
別に初乳から調製した酸ホエーおよび仔牛血清をＤＭＥ
Ｈに１０％加えて、同様に細胞数が３Ｘ１０’個／ｄに
なるように接種した。Acid whey and calf serum prepared separately from colostrum were added to DME.
10% was added to H and inoculated in the same manner so that the number of cells was 3 x 10' cells/d.
これらを５％ＣＯ，、湿度９８％の細胞培養インキュベ
ーター中で３７゛Ｃで培養した。These were cultured at 37°C in a cell culture incubator with 5% CO and 98% humidity.
経時的に試料を取り出し、培養液を除いた後ＰＢＳで洗
浄し、０．２５％クリスタルバイオレット７２０％メタ
ノール溶液１００μｌを添加し、生細胞を固定染色した
後、５９５ｎｍで吸光度を測定した。培養結果を第４図
に示す、初乳ホエーのみを添加した培地では細胞の増殖
はまったく認められなかったが、細胞増殖促進物質を含
む培地では、仔牛血清と同等あるいはそれ以上の増殖活
性を示した。Samples were taken out over time, the culture medium was removed, and then washed with PBS. 100 μl of 0.25% crystal violet 720% methanol solution was added to fix and stain the living cells, and then the absorbance was measured at 595 nm. The culture results are shown in Figure 4. In the medium containing only colostrum whey, no cell proliferation was observed, but in the medium containing cell growth-promoting substances, the cell growth activity was equal to or greater than that of calf serum. Ta.
実施例３
実施例１と同様にして、牛初乳ホエーを調製用等電点電
気泳動に供し、ｐＩ７以上の画分を回収して細胞増殖促
進物質を含む分画物を得た。Example 3 In the same manner as in Example 1, bovine colostrum whey was subjected to preparative isoelectric focusing, and fractions with a pI of 7 or more were collected to obtain a fraction containing a cell growth promoting substance.
ＤＭＥＭに６％の仔牛血清および４％の該分画物を加え
た培地１００μｌを０．３２ｃｄの９６穴培養用プレー
トに入れ、Ｂａ１ｂ／ｃ ３７３の細胞数が３Ｘ１０’
個／ｍ１になるように接種した。100 μl of a medium containing 6% calf serum and 4% of the fraction in DMEM was placed in a 0.32 cd 96-well culture plate, and the number of Ba1b/c 373 cells was 3X10'.
The cells were inoculated at a concentration of 5 cells/m1.
別に仔牛血清をＤＭＥＭに１０％を加えて、同様に細胞
数が３Ｘ１０’個／Ｉｉになるように接種した。これら
を５％ＣＯｔ、湿度９８％の細胞培養インキュベーター
中で３７°Ｃで培養した。Separately, 10% calf serum was added to DMEM and inoculated in the same manner so that the number of cells was 3×10′ cells/Ii. These were cultured at 37°C in a cell culture incubator with 5% COt and 98% humidity.
実施例２と同様にして、経時的に試料を取り出し、生細
胞を固定染色した後、５９５開で吸光度を測定した。培
養結果を第５図に示す、細胞増殖促進物質を含む分画物
は、仔牛血清とほぼ同等増殖活性を示した。In the same manner as in Example 2, samples were taken out over time, living cells were fixed and stained, and then the absorbance was measured at 595 mm. The culture results are shown in FIG. 5, and the fraction containing the cell growth promoting substance showed approximately the same growth activity as calf serum.
第１図は、実施例１における牛初乳ホエー濃縮物の等電
点電気泳動に供した結果のｐＨ（−〇−）光度（棒グラ
フ）を、第２図は、そのｐｌ ８．４〜１Ｏ１０の両分
をゲル濾過に供した結果を、第３図は、その活性画分を
陽イオンクロマトグラフィーに供した結果をそれぞれ示
す。
第４図は、実施例２における本発明の増殖促進物質（−
〇−）と仔牛血清（−・−）、初乳ホエー（−Δ−）と
の細胞増殖活性の比較を、第５図は、実施例３における
本発明の増殖促進物質（−〇−）と仔牛血清（−・−）
との細胞増殖活性の比較をそれぞれ示す。Figure 1 shows the pH (-〇-) luminous intensity (bar graph) of the results of subjecting bovine colostrum whey concentrate to isoelectric focusing in Example 1, and Figure 2 shows its pl 8.4 to 1O10. Figure 3 shows the results of subjecting both fractions to gel filtration, and Figure 3 shows the results of subjecting the active fraction to cation chromatography. FIG. 4 shows the growth promoting substance (-) of the present invention in Example 2.
Figure 5 shows a comparison of the cell proliferation activity between the cell growth promoting substance (-〇-) of the present invention in Example 3, calf serum (-・-), and colostrum whey (-Δ-). Calf serum (-・-)
Comparison of cell proliferation activity with
Claims (2)
定で分子量３３，０００〜３６，０００を有しており、
かつ等電点ｐＩ８．４〜１０を有することを特徴とする
細胞増殖促進物質(1) It can be extracted from bovine colostrum and has a molecular weight of 33,000 to 36,000 as measured by SDS-PAGE.
A cell growth promoting substance characterized by having an isoelectric point pI of 8.4 to 10.
定で分子量３３，０００〜３６，０００を有しており、
かつ等電点ｐＩ８．４〜１０を有する細胞増殖促進物質
を有効成分とする細胞培養培地添加物(2) It can be extracted from bovine colostrum and has a molecular weight of 33,000 to 36,000 as measured by SDS-PAGE.
A cell culture medium additive containing a cell growth promoting substance as an active ingredient and having an isoelectric point pI of 8.4 to 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2031750A JPH03236772A (en) | 1990-02-13 | 1990-02-13 | New cell proliferation promoter and additive containing the same as active ingredient for culture medium used in cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2031750A JPH03236772A (en) | 1990-02-13 | 1990-02-13 | New cell proliferation promoter and additive containing the same as active ingredient for culture medium used in cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03236772A true JPH03236772A (en) | 1991-10-22 |
Family
ID=12339699
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2031750A Pending JPH03236772A (en) | 1990-02-13 | 1990-02-13 | New cell proliferation promoter and additive containing the same as active ingredient for culture medium used in cell culture |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03236772A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6194208B1 (en) * | 1994-04-28 | 2001-02-27 | Gropep Limited | Modified milk growth factor |
-
1990
- 1990-02-13 JP JP2031750A patent/JPH03236772A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6194208B1 (en) * | 1994-04-28 | 2001-02-27 | Gropep Limited | Modified milk growth factor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Schmidt et al. | Silica-stimulated monocytes release fibroblast proliferation factors identical to interleukin 1. A potential role for interleukin 1 in the pathogenesis of silicosis. | |
| DE3280462T2 (en) | Antibodies to a composition with mediator activity and its use in a pharmaceutical composition. | |
| Mjaatvedt et al. | Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin | |
| Moses et al. | Transforming growth factor production by chemically transformed cells | |
| Beutler et al. | Identity of tumour necrosis factor and the macrophage-secreted factor cachectin | |
| Harmon et al. | Changes in lactoferrin, immunoglobulin G, bovine serum albumin, and alpha-lactalbumin during acute experimental and natural coliform mastitis in cows | |
| US4822776A (en) | Lipoprotein lipase suppression by endotoxin-induced mediator (shock assay) | |
| AU649019B2 (en) | Anabolic activity modulator and uses thereof | |
| US4440860A (en) | Stimulating cell growth | |
| CA1229789A (en) | Polypeptides exhibiting skeletal growth factor activity | |
| FI91166B (en) | Crude milk fraction, process for its preparation and its use as a supplement in cell culture media | |
| JP3022566B2 (en) | Macrophage-induced inflammatory neurons | |
| JPH0637520B2 (en) | Polypeptide | |
| EP0061138A2 (en) | Angiotropins of leukocytes and inflamed tissue: a new class of natural chemotropic protein mitogens for specific induction of directional growth of blood vessels, neovascularization of tissues and morphogenesis of blood vessel patterns, process for their biotechnical preparation and pharmaceutical compositions | |
| FI97298C (en) | Method for isolating a tissue factor inhibitor | |
| Nilausen | Role of fatty acids in growth‐promoting effect of serum albumin on hamster cells in vitro | |
| EP0061141A2 (en) | Chemokinesins and chemotaxins of leukocytes and inflamed tissues: natural mediator proteins for reversible promotion of random and directional locomotion (chemokinesis and chemotaxis) for accumulation of specific leukocyte types, process of their biotechnical preparation and pharmaceutical compositions | |
| Moore et al. | Substance P augmentation of CSF-1-stimulated in vitro myelopoiesis. A two-signal progenitor restricted, tuftsin-like effect. | |
| O'Hare et al. | Expression and precursor processing of neuropeptide Y in human and murine neuroblastoma and pheochromocytoma cell lines | |
| Li et al. | Effects of the processing methods of corn grain and soybean meal on milk protein expression profiles in dairy cows | |
| Morgan et al. | The Nutrition of Animal Tissues Cultivated in vitro: IV. Amino Acid Requirements of Chick Embryonic Heart Fibroblasts | |
| JPH03236772A (en) | New cell proliferation promoter and additive containing the same as active ingredient for culture medium used in cell culture | |
| Satoshi et al. | Analysis of non-heme iron in arachidonate 12-lipoxygenase of porcine leukocytes | |
| Yoshida et al. | The binding ability of α-lactalbumin and β-lactoglobulin to mutagenic heterocyclic amines | |
| Ashton et al. | Milk components inhibitory to Bacillus stearothermophilus |