JPH03178996A - Growth factor - Google Patents

Abstract

NEW MATERIAL:A growth factor contained in the culture supernatant of a fibroblast-like cell originated from chronic rheumatism synovial cell and having the following properties. The mol.wt.: 50000-70000, not having affinity to heparin, stable on thermal treatment at 56 deg.C for 30min and promoting the growth of vascular endothelial cell. USE:A treatment-promoting drug for burns and wounds, a remedy for angiopathy, or a remedy or diagnostic agent for chronic rheumatism and solid tumors. PREPARATION:For example, synovial cell is collected from the knee arthrosis of a chronic rheumatism patient, finely cut and treated with trypsin in a buffer solution. Cells contained in the supernatant are collected, cultured in a medium and cloned by a limiting dilution analysis. The fibroblast-like cells are cultured and the supernatant of the cultured solution is concentrated and purified by a high performance liquid chromatography to provide the growth factor having a mol.wt. of 50000-70000.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to growth factors. More specifically, the present invention relates to a growth factor that has a significant proliferation-promoting effect on vascular endothelial cells and is useful as a medicine, clinical test reagent, etc.

<Prior Art and Problems to be Solved by the Invention> It is known that living tissues, organs, body fluids, etc. contain various growth factors, and a large number of growth factors have been isolated. Such examples include, for example, fibroblast growth factor (Fi
broblast Growtl+Factor.

(hereinafter referred to as FGF), transforming growth factor (Transf'orming Growth fac)
tor) S platelet-derived growth factor (Platelet-d
Epidermal Growth Factor), Epidermal Growth Factor
or), etc. (for example, the book “Cell Growth Factor J
(1984) and the same book Part I [(1987)
, both published by the Japanese Society of Tissue Culture, published by Shokuhon Shoten).

The above-mentioned growth factors exhibit various effects, such as cell proliferation, hypertrophy and elongation, and regulation of the expression of differentiation functions, based on their physiological activities. Some of these growth factors are known to have vascular endothelial cell proliferation-promoting activity, and these include endothelial growth factors (Endothe! i alcell gro
It is summarized by the concept of ``wth factor''.

Endothelial cell growth factor has been shown to be involved in the formation of capillary networks in solid tumor growth, embryogenesis, luteinization, wound healing processes, immune responses, etc. In the pathological analysis of RA (hereinafter referred to as RA), it is considered to be an important factor in studying the mechanism of vascular proliferation during inflammation. Therefore, endothelial cell growth factors are closely related to physiological and pathological processes such as wound healing, growth of solid tumors, chronic inflammation, and arthritis, and are expected to be used as medicines.

As the fibroblast-derived endothelial cell growth factor, the above-mentioned F
GF is known. FGF includes basic FGF, which is present in the pituitary gland, brain, corpus luteum, adrenal gland, placenta, etc., and consists of 146 amino acids and has a pI of 9.6, and basic FGF, which is present in the brain, retina, etc., and has a pI of 9.6. Acidic FGFs consisting of amino acids and having a pI of 5 to 7 are known, and all of these have an affinity for heparin.

Furthermore, as an endothelial cell growth factor that does not have affinity for heparin, platelet-derived endothelial cell growth factor with a molecular weight of 45,000 has been reported (P, lshik
awa et al., Nature, 333: 5
57-562゜1, 989).

As a result of intensive research on RA slip cells, the present inventors found that the culture supernatant of fibroblast-like cells cloned from slip cells has a strong proliferation-promoting effect on vascular endothelial cells and is resistant to heparin. The present invention was completed by discovering that there are growth factors for which there is no affinity, and by isolating and purifying the growth factors.

That is, the present invention provides a novel fibroblast-like cell-derived growth factor that has vascular endothelial cell growth-promoting activity and has no affinity for heparin.

Means for Solving the Problems> The growth factor of the present invention that has been made to solve the above problems is as follows:
It has the following properties.

(1) Molecular weight +50,000 to 70,000 (2) No affinity for heparin.

(3) Stable to heat treatment at 56°C for 30 minutes.

(4) Promote proliferation of vascular endothelial cells.

The growth factors of the present invention can be obtained by the following method, but are not limited to those obtained by this method. That is, the growth factor of the present invention is contained in the culture supernatant of RA demyelinated cell-derived fibroblast-like cells, and can be obtained by isolating the supernatant.

RA synovial cells used in the above method can be obtained from the knee joints of RA patients, and the collected synovial cells can be obtained using the method of Goto et al.
.

Invest, 80: 7gB-796, 1987)
can be cultured and cloned according to the following. The cloned slipped cells are morphologically classified into three main types of cells: fibroblast-like cells, macrophage-like cells, and dendritic cell-like cells, and the growth factor of the present invention can be applied to fibroblast-like cells. Contained in cultured slides. S fibroblast-like cells are cultured in conventional media (e.g. HAM F12,
RPM11640, etc.), but usually serum components (e.g., fetal bovine serum, etc.) are added to about 3 to 15%.
Preferably it contains about 10%, and if necessary, about 100 U/xI of penicillin and 1 liter of streptomycin.
It is carried out in a medium containing about 00 μg/11.

As for culture conditions, it is preferable to adopt conditions such as humidity, temperature of about 37° C., and carbon dioxide concentration of about 5%.

The growth factor of the present invention is contained in the culture supernatant of the above-mentioned fibroblast-like cells, and after concentrating the culture supernatant by ultrafiltration, ammonium sulfate salting out, etc. as necessary, The growth factor of the present invention can be obtained by subjecting it to conventional separation and purification means such as gel filtration, affinity chromatography, and ion exchange chromatography. The growth factor of the present invention thus isolated and purified has the following properties.

(1) Molecule i: 50,000 to 70,000 (2) No affinity for heparin.

(3) Stable to heat treatment at 56°C for 30 minutes.

(4) Promote proliferation of vascular endothelial cells.

Although the growth factor of the present invention has various physiological activities, it exhibits particularly strong proliferation-promoting activity against vascular endothelial cells. Vascular endothelial cell proliferation promoting activity (hereinafter referred to as ECGF) of the growth factor of the present invention
activity) can be measured by a known activity measurement method. For example, a sample is placed between the blood vessels of the chorioallantoic membrane of a fertilized chicken egg, and the capillaries are guided in an axle shape around the sample. Measuring method (CAM method): A method in which a polymer pellet containing a sample is implanted in the cornea of a rabbit to measure new capillary formation; An air bladder is created under the skin of a mouse.
Examples include a method of administering a sample and performing an autopsy to determine the formation of capillary vessels (S, Taylor et al., N.
ture, 297: 307.1982, J.F.
Olkman et at Science, 221:
719.1983, M. A. Gimbrone
et at, J, Na11. Cancer 1n
st,, 52: 413.1974, G, A, P
Owner Ctal, Invest, Op
hthalmol.

Visual Set, 21: 351.1981
, R.Ranger et al.

Nature, 2H: 797.197Ei, J.
B, Hurray et a, l.

In VHro 19: 74B, 1983, Mitsui et al. 1 Japan Clinical, 44:L [see i9.1988, etc.]. More preferably, human venous vascular endothelial cells (HUV-
An example of this method is to measure the proliferation of HUV-EC using the amount of 3H-thymidine incorporation as an indicator using EC (M
, Kan etat, J., Ce11. Phys.
iol, 111: 155.1982).

<Effects of the Invention> Since the growth factor of the present invention has a strong proliferation-promoting activity on vascular endothelial cells, it is useful as a treatment promoter for burns, wounds, etc., and a therapeutic agent for diseases such as vascular disorders. Furthermore, the growth factors and their inhibitors or antibodies of the present invention can be expected to be applied to the development of therapeutic and diagnostic agents for RA, solid tumors, and the like. Furthermore, as an in vitro application of the growth factor of the present invention,
It can be used as a cell growth promoter in the production of non-thrombotic artificial blood vessels obtained by adhering and proliferating vascular endothelial cells on the inner surface of a tubular polymer material.

<Examples> Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of Human Slipped Cells Slipped cells were obtained from the knee joints of five RA patients using an arthroscope. These five patients were receiving nonsteroidal anti-inflammatory drugs, and some were also receiving low doses of corticosteroids. After removing adipose tissue, the slipped cells were cut into small pieces, transferred to a sterile plastic tube, and soaked in PBS (phosphate-buffer).
d 5aline) three times.

The sliced sloughed cells were treated with a PBS + 0.25% trypsin solution at 37°C for 40 minutes, and the cells in the supernatant were collected. Cells were washed three times with PBS and RAMF-12
The cells were dispersed in culture medium (Gibco Laboratories). The HAM F-12 medium contained 10% fetal bovine serum (FBS), 5X10-5M 2-mercaptoethanol, 1000/11f penicillin, and 100% fetal bovine serum (FBS).
Contains μg/'11 streptomycin.

Example 2 Cloning of Human Slipped Cells Cloning of human slipped cells was performed by the limiting dilution method. The cells were dispersed in the above-mentioned medium at a ratio of 2 cells/111 cells. ↓ Divide the 50μρ sample into 96-well microplates (0.6 pieces/well), carbon dioxide concentration 5%, humidity 1
00% and cultured at 37°C. The presence of only one cell in each formula was confirmed using a microscope, and if multiple cells were present, they were removed as polyclones. After 30 days of primary culture, each cloned cell was recloned at a ratio of roughly 0.3 cells/well. Thereafter, cells were treated with 0.05% trypsin for 5 minutes at room temperature once every 2 to 4 weeks, and after washing, cells were divided into 5 cells/well and passaged repeatedly.
A cell line exhibiting stable proliferation ability was obtained.

The cloned cells can be morphologically classified into macrophage-like cells, dendritic cell-like cells, and fibroblast-like cells, and among these, fibroblast-like cells were named RAF.

Example 3 Preparation of RAF culture supernatant Using a 966 microplate (manufactured by FALCON),
Culture medium (RPM11
640) was added at 200μ/well and cultured at 37°C for 18 hours in a carbon dioxide incubator.

The culture supernatant was then separated and stored at -20°C.

Example 4 Preparation of HUV-EC The method of Jaf'fe et al.
tal, J, Cl1n.

Invest, 52: 2745-2758.197
According to 3), endothelial cells were isolated from fresh human cord veins by treatment with 0.2% collagenase, and cultured in RPM11640 solution containing 25% F0 BS in a 25 cm flask.

Example 5 Measurement of ECGF activity The ECGF activity of the RAF culture supernatant obtained in Example 3 was measured according to the method of Kan et al., in which the proliferation promoting activity against HUV-EC was measured by the amount of 3H-thymidine taken up. tested (M, Kan et al, j.

Ce11. Physiol, 111: 155.1
982).

That is, HUV-EC in a confluent state is
．． The cells were treated with 0.01% EDTA containing 0.05% trypsin to form floating cells, and the cells were dispensed into a 96-well microplate (manufactured by PALCON) at a ratio of 2×10 3 cells/well. 18
After a period of time, the supernatant was replaced with RPM11640 solution (hereinafter referred to as test medium) containing 50% of the RAF culture supernatant obtained in Example 3. After culturing at 37°C for 48 hours, 3H-thymidine was added, the culture was further incubated for 24 hours, and the amount of 3H-thymidine taken up was measured using a scintillation counter (first
(see figure). As a control, a similar test was conducted using RPM11 1640 solution containing no RAF culture supernatant instead of the above test medium. In addition, as a comparative test (positive control), acidic FGF, an endothelial cell growth factor (product name: ECG5. Distributor:
A similar test was conducted using C0IlabOratiVeRe5earches (derived from bovine brain). Typical test results are shown in Table 1.

As shown in Table 1, in the system using the culture solution containing RAF culture supernatant, the amount of 3H-thymidine uptake increased significantly compared to the control, and the proliferation of vascular endothelial cells was significantly increased. It is clear that this is being promoted. In addition, 3
An increase in the amount of H-thymidine uptake was also observed in acidic FGF, confirming the validity of the above test method.

Further, in the above test, a similar test was conducted with the concentrations of RAF2 culture supernatant in the test medium being 25% and 12.5%. FIG. 2 shows a plot of the amount of 3H-thymidine incorporation versus the concentration of RAF culture supernatant.

As is clear from FIG. 2, as the concentration of the RAF culture supernatant decreased, the amount of 3H-thymidine incorporation decreased, and concentration dependence was observed.

Example 6 Purification of RAF culture supernatant The RAF culture supernatant obtained in Example 3 was purified using Amicon membrane PM.
50, ECGF activity was observed in the non-dialyzed fraction (ie, molecular weight of 50,000 or more).

In addition, the Amicon-enriched fraction was added to Superrose 12
(Superrose 12) to examine the relationship between molecular weight and ECGF activity. The results are shown in FIG. As is clear from Figure 3, the molecular weight is 5.
A peak of ECGF activity was observed between 0,000 and 70,000.

Furthermore, when fractions with molecular weights between 50,000 and 3 and 70,000 separated by HPLC were subjected to affinity chromatography using heparin-Sepharose, ECGF activity was observed in the non-adsorbed fractions. The growth factor was shown to have no affinity for heparin.

In addition, the heparin/Sepharose column non-adsorbed fraction was
Although it was subjected to heat treatment at 6° C. for 30 minutes, ECGF activity was not impaired, indicating that the growth factor of the present invention is heat resistant.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram illustrating the endothelial cell cloning method and the ECGF activity measurement procedure. FIG. 2 is a graph showing the concentration dependence of ECGF activity of RAF culture supernatant. Figure 3 shows protein elution ff1 when RAF culture supernatant was applied to an HPLC column packed with Superrose 12.
(−) and its ECGF activity (−〇−〇−).

Claims (1)

[Scope of Claims] 1. A growth factor contained in the culture supernatant of fibroblast-like cells derived from rheumatoid arthritis synovial cells and having the following properties. (1) Molecular weight: 50,000 to 70,000 (2) No affinity for heparin. (3) Stable to heat treatment at 56°C for 30 minutes. (4) Promote proliferation of vascular endothelial cells.