JPH08119867A - Fibroblast growth factor derived from milk - Google Patents

Abstract

PURPOSE: To obtain a fibroblast growth factor derived from a mammalian milk. CONSTITUTION: This fibroblast growth factor(FGF) derived from a milk is obtained by using a cream fraction obtained by the centrifugation of the milk of available mammals (e.g. a cow), especially a foremilk until 24hr postpartum. Since a part of cow's foremilk-FGF is present in a whey, this fraction can also be used. In this FGF, isoelectric points exist at the acidic side of 5-6 and at the basic side of 8-10, and further it has a potent affinity to heparin or heparan sulfate. FGF is a factor to enhance growth of mouse fibroblasts, especially demonstrates a potent activity to endotherial cells in blood vessels and widely produced and distributed in tissues and cells of the living body such as hypothalmus, brain, retina, eyes, kidneys, cardiac muscles, hepatic cells, prostate, placenta, cartilage, spleen, endothelial cells, fibroblasts, cerebral gland and macrophages, but its existence in the cells of mammalian gland and in the milk has not been reported.

Description

Detailed Description of the Invention

[0001]

This invention relates to a novel mammalian milk-derived fibroblast growth factor available.

[0002]

2. Description of the Related Art Fibroblast growth factor (FGF) has been isolated from bovine brain and pituitary gland as a factor that promotes the growth of mouse fibroblasts. Initially, it was thought to act specifically on fibroblasts, but now it has been shown to act widely on mesenchymal cells, nerve cells, epithelial cells, etc., and in particular has strong activity on vascular endothelial cells. There is. FGF is an acidic FGF (aFGF) with an isoelectric point (pl) of 5-6 and a pl of 9-10.
It is distinguished by basicity (bFGF).

Both aFGF and bFGF are hypothalamus, brain, retina, eye, kidney, myocardium, hepatocyte (during regeneration), prostate, placenta,
Cartilage, spleen, endothelial cells, fibroblasts, brain lines, macrophages
It is widely produced and distributed in various tissues and cells in vivo such as Ji, but its presence in mammary gland cells and milk has not been reported. However, mammalian milk is the only complete diet for neonates of each species, and among them, in addition to various nutrients essential for development, insulin-like growth factor (Francis,
GL et al, Biochem. J., 251, 95-103, (1988)), epidermal growth factor (Shin. YW et al, Endocrinology, 1
15, 273-282 (1984)), transforming growth factor (Noda, K. et
al, Gann, 75, 109-112 (1984), Tokuyama, H., et al,
Growth factors, such as Growth Factors, 3, 105-114 (1990)), have a large amount of growth factors having various activities, especially in colostrum.

Therefore, the present inventors have developed an FG having various functions.
Considering that F is naturally contained, the present invention was completed by focusing on the affinity of FGF, which is heparin or heparan sulfate (HS). That is, the presence of HS proteoglycans has been clarified in the milk fat globule membrane (MFGM) (Shimizu,
M., et al, Agric. Biol. Chem., 45, 741-745 (198
1)). In general, FGF is produced by each cell in vivo, then bound to HS proteoglycans present in the cell membrane and basement membrane, and stored and stabilized (Folkman, J. et al, Am. J. Path.
ol., 130, 393-400 (1988), Gospodarowicz, D. et al,
J. Cell Physiol., 128, 475-484 (1986)). Therefore, after being produced in mammary gland cells in milk as well, most of it is expected to be present in HS proteoglycan of MFGM.

[0005]

As described above, in searching for growth factors in milk, all researchers must
It was mainly protein. The inventors conducted an immunoassay mainly on MFGM to search for a new growth factor existing in milk, and used BC-aFGF on bovine colostrum (BC).
And found the presence of BC-bFGF.

The present invention has a milk-derived fibroblast having an isoelectric point of 5 to 6 on the acidic side, an isoelectric point of 8 to 10 on the basic side, or having a strong affinity for heparin or heparan sulfate. It proposes a cell growth factor.

[0007]

The function of the present invention is as a raw material for producing BC-FGF,
The use of a cream fraction of mammalian milk (especially colostrum). However, since a part of BC-FGF is also present in whey, this fraction can also be used. The milk may be of any type as long as it is available.

Next, the present invention will be described in detail with reference to examples. Example 1, Preparation of Sample for Immunoassay: A cream fraction obtained by centrifugation (3000 rpm, 20 min) from bovine colostrum up to 24 hours after calving was added with 0.15 M NaCl 0.1 M Tris-hydrochloride buffer ( It was dispersed in pH 7.0) and then centrifuged. This operation was repeated 3 times to wash the cream fraction and then freeze-dried. 5 volumes of acetone was added to 1 volume of dry cream, and the mixture was stirred at room temperature for 1 hour. This operation was repeated 3 times, the acetone insoluble matter was collected by centrifugation, and the residual acetone was removed under reduced pressure to prepare an acetone powder of a cream fraction.

Acetone powder was dissolved in 0.1M Tris-HCl buffer (pH 7.0) containing 2M NaCl and 6M urea, and then dissolved in 2M NaCl.
And equilibrated with the same buffer containing 6M urea
A Dex 75 (Pharmacia) column was loaded and gel filtration was performed. The fraction is monitored with BIAcore (Pharmacia) using a sensor chip with FGF antibody immobilized.
The fractions that react with FGF antibody were collected and
It was dialyzed against 0 mM Tris-buffered hydrochloric acid (pH 7.0).
This was loaded on a column in which a retinoic acid-immobilized cellulophane (manufactured by Seikagaku Corporation) column and a Cu-chelate cellulophanein (manufactured by Seikagaku Corporation) column were directly bound to collect non-adsorbed fractions, and concanavalin A cephalo- It was loaded on a column (manufactured by Pharmacia).

The non-adsorbed fraction was directly loaded on heparin-sepharose (manufactured by Pharmacia) and eluted by a linear concentration gradient method up to 2.0 M NaCl. The substances eluted near 1 M NaCl and around 1.5 M NaCl were collected, dialyzed against deionized water, and then freeze-dried. Based on the standard method, SDS
After electrophoresis, Western blotting was performed according to a conventional method. The antibodies used for staining were aFGF antibody and bFGF antibody (both manufactured by Sigma). In addition, the same preparation was subjected to ELISA by a conventional method to measure the contents of aFGF and bFGF. As for aFGF and bFGF, 1 μL of bovine colostrum contained a few μg of aFGF and 20 to 30 μg of bFGF.

Example 2, Preparation of activity measurement sample: Solid urea was added to the wash cream described in Example 1 so that the final urea concentration was 6 M, and the pH was adjusted to 7.0. The mixture was stirred with a polytron under ice cooling (12,000 rpm, 10 min), and the resulting butter lumps and insoluble matter were removed by centrifugation (3,000 rpm, 15 min). NaCl was added to the resulting clarified solution so as to make the concentration 2.0 M, and the supersol described in Example 1
A Dex 75 (Pharmacia) column was loaded. Thereafter, aFGF and bFGF were prepared by the method described in Example 1. In addition, a concanavalin A sepharose column non-adsorbed fraction before loading on a heparin-sepharose column, a heparin-sepharose column 1.0M NaCl elution fraction, and a 1.5M Na
The Cl-eluted fractions were isoelectrically fractionated in the range of pH 3.5 to 10 for isoelectric focusing (manufactured by Bio-Rad), and the activity of each fraction was measured. From the whey
The whey obtained was decaseinized by the acid-ethanol method, and the obtained whey was adjusted to 7.0 and then the method described in Example 1.

The activity was measured by pre-culturing bovine umbilical cord-derived endothelial cells in RPMI 1640 supplemented with 5% fetal bovine serum at 5% CO ₂ and 37 ° C. for 48 hours, and replacing them with the same medium containing no serum. .
0M NaCl eluate (BC-aFGF) and 1.5M NaCl eluate (BC-bFGF)
Was added to 0-100 ng / ml medium, and 72
Cultured for hours. The number of cells after culturing was counted and the activity was measured.

The results are shown in FIG. As shown in FIG. 1, BC-FGF contains growth factors which are roughly classified into two having isoelectric points at pH 5 to 6 and 8 to 10. The isoelectric point of the 1.0 M NaCl elution fraction on the heparin-sepharose column was 5-6, and that of the fraction eluted with 1.5 M NaCl was 8-10. They showed very strong activity on endothelial cells and also acted on mouse 3T3 cells (fibroblasts).

FG is also added to human milk within 1-2 weeks after delivery.
Since F was found, FGF was distributed in the colostrum of both monogastric and ruminant animals. In addition, 16 postpartum
Mammary gland cells separated from bovine or human colostrum within a period of time were stained with bFGF antibody, and each culture medium was partially purified and subjected to Western blotting according to a conventional method. It showed a positive reaction. From this, FG present in bovine and human milk
It was confirmed that F was produced in mammary cells.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, a milk-derived novel fibroblast growth factor can be easily produced by using an available cream fraction of mammalian milk.

[Brief description of drawings]

FIG. 1 shows the isoelectric point of bovine colostrum under various conditions.

Claims (3)

[Claims] 1. A milk-derived fibroblast growth factor, which has an isoelectric point on the acidic side of 5 to 6. 2. A milk-derived fibroblast growth factor, which has an isoelectric point on the basic side of 8 to 10. 3. A milk-derived fibroblast growth factor, which has a strong affinity for heparin or heparan sulfate.