JPH02279698A - Vascular endothelial cell growth factor and its preparation - Google Patents

Abstract

NEW MATERIAL:A vascular endothelial cell growth factor having the following physico-chemical properties. Mol.wt.: approximately 45000 (measured by SDS- PAGE method); amino acid composition (contents per 100 residues): D: 6.2, T: 3.5, S: 6.1, E: 11.4, P: 8.2, G: 11.7, A: 13.2, C: 2.5, V: 6.9, M: 1.5, I: 2.4, L: 14.5, F: 1.4, H: 1.2, K: 2.5, R: 6.6 and the factor has a partial structure of the amino acid sequence in the formula at the N terminal (or at the near place). USE:A preventing and treating drug for arterial sclerosis and hypertension, a tissue repairing agent for fracture and tissue injuries, or a reagent for clinical examinations. PREPARATION:For example, human placenta is cultured in serum-free medium and the prepared culture supernatant is subjected to ammonium sulfate precipitation, anion exchange chromatography, gel filtration, hydroxy appetite gel chromatography and alkylsuperlose column chromatography for collecting a fraction having a vascular endothelial cell multiplication-promoting activity to provide the vascular endothelial cell growth factor.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel vascular endothelial cell growth factor and a method for producing the same.

[Conventional technology]

Many vascular endothelial cell growth factors have been reported to date. However, one substance whose structure has been clarified and which definitely has a proliferative effect on vascular endothelial cells is basic fibroblast growth factor (Basic Fibroblast Growth Factor).
Last Growth Factor; Basic FG
F) and acidic II fibroblast growth factor (＾cidic
FibrobtasLGrowth Factor;
There are only two types: acidic FGF).

Basic FGF was first isolated from the pituitary gland of bovines, and is also produced in various tissues and cells, such as the brain, nervous tissues such as the pituitary gland, retina, adrenal glands, corpus luteum, kidneys, and macrophages. It has now been found that the protein is isolated from brain tissue. Initially, as the name suggests, it showed proliferative activity against fibroblasts (3T3 cells) and was isolated for the purpose of its use, but later on, it was isolated from mesophyllogenic cells such as vascular endothelial cells and adrenal cells. It was found that it also exhibits growth-promoting activity against many cells, making it clear that it is a growth factor with extremely poor specificity. In addition, basic PGF is known to have an effect of promoting proliferation of vascular endothelial cells as well as regulating the function of vascular endothelial cells in an in vivo test. More tumor cells contain basic FG
It has been reported that F is produced, and it has been suggested that it may be a so-called tumor angiogenic factor.

The cDN^ of basic FGP has already been cloned, and it is known that basic PGP has a structure in which a leader peptide consisting of 9 amino acids is followed by a peptide consisting of 146 amino acids. ing. On the other hand, basic FGF isolated from tissues has an N-terminus of 1
There are two types of structures starting from the th or 6th amino acid.

Furthermore, basic PGF has a strong affinity for heparin, shows 55% homology with heparin at the amino acid level, and can be separated using heparin affinity column chromatography. It also shows weak homology with interleukin-1.

Acidic FGF was initially developed by Machining into endothelial cell growth factor (Bndothelial Growth Factor).
Factor; BCGF) was isolated from bovine nerve tissue. After that, acidic EICGF and basic BCGF existed in BCGF, and they were further divided into Grosp
Two types of FGF isolated by Odarowtz et al.

That is, it was found that it corresponds to the above-mentioned basic FGF and acidic PGF. Acidic FGF is also (aye-de
derived growth factor II ) and (a-retina-derived grow
It is known that it is the same as th factor). Acidic FGF is a substance similar to the above-mentioned basic FGF, exhibits strong affinity for heparin, and is easily separated by heparin affinity column chromatography. However, the vascular endothelial cell proliferation effect of acidic FGF is promoted in the presence of heparin, whereas basic FGF
It is known that the action of GF is not affected by the presence or absence of heparin, and that acidic FGF, unlike basic FGF, exists only in nerve tissue.

Human acidic FGF cDNA 8 encodes 155 amino acids. On the other hand, purified bovine acidic FGF has an amino acid composition starting from the 10th Phe, αFGF-1.
β-EICGF), and those with amino acid composition starting from the 22nd Asn (αF G ti-2, α[ICG
F) has also been reported, and no obvious signal peptide has been observed. α-FGF is interleukin-1
In addition to showing approximately 30% homology with , it also has homology with neuromelin, bouvesin, and substances.

[Problem to be solved by the invention]

Vascular endothelial cell growth factor is expected to be a therapeutic agent for vascular diseases such as arteriosclerosis, and is also important as a reagent for clinical tests and research.

Therefore, the development of a new vascular endothelial cell growth factor and a technique for efficiently obtaining it has been eagerly awaited.

[Means to solve the problem]

Under such circumstances, the present inventor has conducted intensive research to solve the above problem, and has found that a substance obtained from the culture supernatant of human placenta, which is easy to use, has excellent vascular endothelial cell growth promoting activity. They discovered that it is a novel vascular endothelial cell growth factor, and completed the present invention.

That is, the present invention provides a vascular endothelial cell growth factor having the following physical and chemical properties and a method for producing the same.

(a) Molecular weight: Approximately 45,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (b) It has the following amino acid composition.

Amino acid content per 100 residues Aspartic acid (D) 6.2 Threonine (T)
3.5 Serine (S) 6.1 Glu9mi”JH<E) 11.5 Proline
(P) 8.2 Glycine (G)
11.7 Alanine (A) 13
．． 2 Cysteine (%) (C) 2.5 Vualin (V) 6.9 Methionine (
M) 1.5 isoleucine (1)
2.40 Ishin (L) 14.
5 Phenylalanine (F) 1.4 Histidine (H) 1.2 Lysine (K)
2.5 Arginine (R) 6.6 (c) It has a partial structure of the following amino acid sequence at or near the N-terminus.

APPAPGDFSGEG Vascular endothelial cell growth factor of the present invention (hereinafter, "factor of the present invention")
For example, human placenta is cultured in a serum-free medium, and the culture supernatant is subjected to ■ ammonium sulfate precipitation, ■ anion exchange chromatography, ■ gel filtration, ■ hydroxyapatite gel chromatography,
It is then subjected to (1) alkyl superose column chromatography and a fraction having vascular endothelial cell proliferation promoting activity is collected.

The culture of human placenta in the present invention can be carried out under the same conditions as those for normal human tissue culture.

The medium is a serum-free medium, in particular a mixture of equal volumes of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 1 insulin.
μg/ml, ) Lanceferrin 1 μg/ml! , a medium supplemented with 10 ng/ml epidermal growth factor and antibiotics is preferred. Examples of antibiotics include benylicin potassium, amphotericin B1, and gentamicin. The culture temperature is 36-37.5℃, and the pH of the medium is
is preferably 6.8 to 7.5. Furthermore, static culture is preferred as the culture method. It is preferable to continue the culture, usually collecting a portion of the culture supernatant every 3 days.

Next, a method for collecting the factor of the present invention from the obtained culture supernatant will be explained. First, the culture supernatant was filtered through an ultrafilter to obtain a
After concentrating 0 to 5,000 times, ammonium sulfate (50%) is added to the supernatant to separate the precipitate. This is adsorbed onto an ion exchange chromatography carrier and fractionated using a sodium chloride concentration gradient. Here, examples of the anion exchange chromatography carrier include DBAIE Sepharose and the like.

Next, the obtained fractions were dialyzed against Tris-HCl & l equilibration solution, lyophilized, and then gel filtrated on a high performance liquid chromatography device (effective fraction range i, oo).

~300,000 Daltons).

Furthermore, it is subjected to hydroxyapatite gel chromatography in a high performance liquid chromatography device and fractionated using a phosphate concentration gradient. Next, the mixture was adsorbed onto an alkyl super gel, which is a hydrophobic chromatography method, in an ammonium sulfate solution, and eluted by gradually decreasing the concentration of ammonium sulfate. The vascular endothelial cell growth factor active fraction was collected, salted, and lyophilized. If carried out, the factor of the present invention can be obtained.

The method for measuring the activity of vascular endothelial cell growth factor used in the present invention is as follows. After resecting the human vein or porcine aorta, both ends were tied with thread, and a 0.2% collagenase solution (manufactured by Worthington, dissolved in Ham's F12 medium) was injected into the lumen using a syringe. After incubation at 37° C. for minutes, the injected collagenase solution is collected and centrifuged (100×g) to obtain vascular endothelial cells. The obtained vascular endothelial cells are dispersed in an F12 medium to which 10% tallow serum and 5 μg/rnl of fibroblast growth factor (FGF) are added, and cultured at 37° C. in a 5% carbon dioxide gas incubator. The vascular endothelial cells thus obtained were dispersed in a 0.25% trypsin solution (manufactured by Gibco) using a conventional method, collected by centrifugation, and then placed in ham F12 supplemented with 0.5% tallow serum. Distribute and place 5×10 3 cells (1.0 ml) in each well of a 24-well culture plate (manufactured by Falcon), and culture in a 5% carbon dioxide gas incubator at 37° C. for 24 hours. After 24 hours, a sample to be measured is placed in each well, and cultured in a 5% carbon dioxide incubator at 37°C for an additional 16 hours. After a further 16 hours, 0.5 μCi of tritium MA thymidin (manufactured by Jungland Nuclear) was added to each well, and the cells were cultured under the same conditions for a further 4 hours. After culturing, add 1 omi of 10% trichloroacetic acid cooled to 4°C to each well and leave at room temperature for 1 hour. Discard the liquid from each well, wash the well with 5% trichloroacetic acid, dry it, add 1N sodium hydroxide solution, let it stand for 10 minutes, neutralize with 1N hydrochloric acid, and add a certain amount of the well. was added with 10 times the amount of Aquasol, and the radioactivity was measured using a liquid scintillation counter.The DNA synthesis promoting activity of vascular endothelial cells in each well was measured. activity of cell growth factors).

The properties of the thus obtained factor of the present invention are as follows.

(a) Molecular weight The molecular weight is approximately 45,000.

The purified factor of the present invention is separated by dodecyl sulfate (IJ) polyacrylamide gel electrophoresis, and when its molecular weight is measured, it shows a single band around 45,000.

(b) Amino acid composition The results of amino acid analysis of the factor of the present invention are shown in Table 1 below.

Table 1 (c) Amino acid sequence The following amino acid sequence has a partial structure at or near the N-terminus.

APPAPGDFSGEG (d) Isoelectric point The isoelectric point shows pl+4.0 to 4.8.

(e) Temperature stability Stable between 0 and 56°C, inactivated above 70°C.

(f) pH stability Stable at pH 5-9, inactivated below 2113.

(g) Stability to enzymes Inactivated by trypsin and pronase, but not inactivated by RN, s, and neuraminidase.

(h) Stability towards enzymes It shows no affinity for heparin and does not promote the proliferation of fibroblasts. Furthermore, it does not lose its activity even if it is subjected to a reduction treatment using dithiothreitol or the like.

〔Example〕

The present invention will be described in more detail by giving examples below.

Example 1 Primary culture of human placenta A normal full-term placenta was aseptically cut into pieces of 2 to 3 m+a squares using scissors, and a culture medium (a mixture of equal volumes of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1 insulin, ug/mf
, transferrin lug/mj', epidermal growth factor <EGF) 10 ng/tr#l, penicillin potassium 100 units/ml, amphotericin 82 μg/mi
! , with 50 ttg/mal of gentamicin added)
Wash with
Carbon dioxide incubator at 7℃ (carbon dioxide concentration 5%)
After incubation for 3 days in a 100% humidity environment, half of the culture medium was replaced. Furthermore, the culture solution was exchanged three times every 3 days, and the collected culture solution was stored at -20°C. The culture vessel is a plastic flask (culture area: 150 cffl)
) with 10mj of culture solution! I used it with

Example 2 Isolation of placenta-derived vascular endothelial cell growth factor Approx.
Concentrate twice and transfer this to a Vericon lab cassette using PTI.
After concentrating 100 times using a device equipped with an O9000 filter (Millipore Il), it was purified in the following order.

(1) Ammonium sulfate precipitate Add ammonium sulfate (50%) and leave at 4°C for 3 hours while keeping ρ11 at 7.0.
Centrifuge for 1 minute, collect the sediment and add 0.02% Tween 20.
and Tris-HCl buffer containing 0.02% azide nitride (
0,OLM, pH7,2) 50 mj! It was dissolved in

(2) The active fraction obtained by anion exchange chromatography and ammonium sulfate precipitation was applied to a Sephas CL-6B column (5 (1 mf!)) to DB8.

(manufactured by Pharmacia) and Tris-hydrochloride buffer (
After washing the column with 150 ml of OIM, pH 7,2), apply a sodium chloride concentration gradient from 0 to 300-M
Elution was performed by increasing the concentration linearly to .

Elution was performed at 4°C at 2.5+nf per minute. The active fraction is 1
Obtained corresponding to the position of sodium chloride concentration from 00 to 150 mM.

(3) Gel filtration OF! The active fraction obtained by AH Sepharose chromatography was mixed with Tris hydrochloride buffer ([], 01 M,
pt17.2), lyophilized, dissolved in 100 ml of distilled water, and applied to a Superose 12 column (
The mixture was subjected to gel filtration chromatography on HflIO/30 (Pharmacia). The buffer solution was Tris-HCl buffer (0
,01M, pH 7,2) with 150d of 1-IJ chloride.
The column flow rate was 500 μl/roll. Calibration of the column was performed in advance with bovine serum albumin (molecule i67, ooO), ovalbumin (molecular weight 45.000), cytochrome C (molecule, 1
112.400). The active fraction has a molecular weight of 45
, 000 position.

(4) Hydroxylapatite chromatography A hytroxylapatite column 0.7825 m+e (Toa Fuel) where the active fraction obtained by gel filtration chromatography was further attached to a high performance liquid chromatography device.
4°C (0.5mM phosphate buffer) at 0.5m/mn
The phosphate was adsorbed to the solution and eluted using a linear gradient of phosphate temperature from 0.5 to 50 mM. Active fraction is approximately 40mM
Lin? Il! 2 concentrations were eluted.

(5) Alkylsuperose column chromatography The active fraction obtained by hydroxylapatite chromatography is further bound to an alkylsuperose column (manufactured by Pharmacia) with ammonium sulfate (2M), and this is mixed from 1.2M to 0. When eluted with a concentration gradient of ammonium sulfate up to .5M, an active fraction is obtained at a position of about .069M.

Both portions were desalted and lyophilized to obtain the final specimen.

Example 3 Physical and chemical properties In order to examine the physical and chemical properties of the vascular endothelial cell growth factor of the present invention obtained in Example 2, the following analysis was performed.

(1) Molecular weight Analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5O5-PAGIE). The electrophoresis device is GE2/4 (manufactured by Pharmacia) and the gel is T=
15%, C = 2.6% polyacrylamide slab gel (70 x 0 x 3 mm) and concentrated gel (T = 3%,
C=20%) was used. The sample is 0.5M2-
After boiling for 3 minutes in a solution of mercaptoethanol and sodium dodecyl sulfate at a concentration of 2%, 2 μg of the product was denatured by adding urea to a concentration of 4M. After running at 120V for 3 hours, the gel was taken out and mixed with methanol:acetic acid:water (4:1:
5) and fixed with silver staining (Silver 5tain).
: manufactured by Bio-Rad).

Bovine serum albumin (molecular weight 67.
000), ovalbumin (molecular weight 45.000)
, cytochrome C (molecular weight 12.000) was treated in the same manner and used. As a result, a single band with a molecular arrangement of 45,000 was observed. The measurement results are shown in Figure 1.

(2) Amino acid composition The sample was hydrolyzed in vacuo with 6M hydrochloric acid at 110°C for 20 hours, followed by amino acid measurement. As a result, the new active substance had the amino acid composition (percentage of total) listed in Table 1 above.
showed that.

(3) Temperature stability sample 0. OIM) Lis-HCl buffer (pt17.2)
It was dissolved in 1, 0 μg/lnl protein*, and 1 degree.

This sample was treated at 0° C., 37° C., 45° C., 56° C., 70° C. and 100° C. for 30 minutes each, and then the remaining vascular endothelial cotton 1 growth-promoting activity was examined.

As a result, it was stable at 0 to 56°C and deactivated at 70°C or higher.

(4) Dissolve the pH stability sample in distilled water to a concentration of 10 μg/ml,
pl containing 0.2% bovine serum albumin) 1.3.5.
7.9 was diluted 10 times with each buffer solution and treated in water for 4 hours. After treatment, 1 ml of each is 0.0
The mixture was dialyzed against 1M 1-Lis-HCl buffer (pH 7.2) for 24 hours, and the remaining vascular endothelial cell growth-promoting activity was measured.

As a result, pll,3 was inactivated, but PH5,? ,
9 was stable.

(5) Stability of the sample against enzymes at a concentration of 1 μg/ml. OIM) dissolved in Lys salt W1 buffer (pH 6,5) and added with trypsin (Tryps
in), pronase, neuraminidase, and RNA5e. ugi was added, and the mixture was reacted at 37°C for 1 hour. After the reaction was completed, 0.1 mA was applied to each, 0.5% bovine serum albumin was added, and after dilution with 1 ml of Ham's F12 medium, the vascular endothelial cell proliferation promoting activity of each was measured. As a result, the vascular endothelial cell growth factor of the present invention was inactivated by trypsin and pronase, but not by RNA5e and neuraminidase.

(6) Determination of amino acid sequence The sample was subjected to Edman digestion using a gas phase sequencer (manufactured by Applied Biosystems),
The obtained PTH amino acid was transferred to a high performance liquid chromatography device (manufactured by Beckman Instruments) and an Ultr
Analysis was performed using an ophhere-ODS column (manufactured by Beckman Instruments) in a conventional manner. column(
5μm diameter 4.6mm, length 250mm) starting buffer (15mM Mi!8 acid) IJum buffer.

After equilibration with pH 4, 5, aqueous solution containing 40% acetonitrile), sample (dissolved in 20 μl of starting buffer)
injection and separation by isocratic elution with starting buffer. The flow rate was 1.4 mβ/min, and the column temperature was 1.4 mβ/min at 40°C. Confirmed. The elution of PTH amino acid is 2
Ultraviolet absorption at 69 nm and 320 nm was used. Standard PTH amino acids (manufactured by Sigma) 2n each
The mol was separated using the same system, the retention time was determined, and identification was performed from the retention time of the test specimen. As a result, the amino acid sequence from the N-terminus to the 15th residue was determined as follows.

APPAPGDFSGEG**G Here, * indicates unidentified.

Example 4 Measurement of specific activity value The specific activity value of the vascular endothelial cell growth factor of the present invention obtained in Example 2 against human vascular endothelial cells was measured according to the method for measuring the proliferation promoting activity of vascular endothelial cells described above. As a result, the maximum stimulation activity is approximately 20ng/n+tlS, and 50% of the maximum activity is 5ng/+++j! Met.

〔Effect of the invention〕

Growth factors for vascular endothelial cells include basic FGF and acidic FGF.
Two factors with heparin affinity have been isolated and their structures have been clarified, but the factor of the present invention has a molecular weight of 45.000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an amino acid composition of FG.
It is different from F, and the amino acid sequence of the N-terminal part is FG.
It is a novel vascular endothelial cell growth factor because it is different from other reported human proteins, including F.

The factors of the present invention can be used as reagents for clinical tests and as research reagents. Furthermore, this factor is useful as a prophylactic and therapeutic agent for diseases such as arteriosclerosis and hypertension, the onset of which is said to be associated with damage to vascular endothelium. Furthermore, it is useful as a tissue repair agent that is administered locally in the event of a bone fracture or tissue injury.

[Brief explanation of drawings]

FIG. 1 is a drawing showing the relationship between migration distance and molecule l in electrophoresis of the factor of the present invention. Above Figure 1 1 2 3 4 5 (c!rL) Migration distance ◎ 2 Vascular endothelial cell base length factor of the present invention

Claims (1)

[Claims] 1. A vascular endothelial cell growth factor having the following physical and chemical properties. (a) Molecular weight: Approximately 45,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (b) It has the following amino acid composition. ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (c) Has a partial structure of the following amino acid sequence at or near the N-terminus. APPAPGDFSGEG 2. Human placenta was cultured in serum-free medium, and the culture supernatant was subjected to (1) ammonium sulfate precipitation, (2) anion exchange chromatography, (3) gel filtration, (4) hydroxyapatite gel chromatography, and then (5) The method for producing vascular endothelial cell growth factor according to claim 1, which comprises subjecting it to alkylsuperose column chromatography to collect a fraction having vascular endothelial cell proliferation promoting activity.