JPH02196798A - Growth factor of vascular endothelial cell - Google Patents

Abstract

NEW MATERIAL:A protein as growth factor of vascular endothelial cell adsorbing to heparin-immobilizing gel and able to be eluted with >=0.8M sodium chloride, having 38,000-48,000 molecular weight in non-reductive condition and 4.0-5.0, 5.5-7.0 and 8.5-9.0 isoelectric points. USE:Curing accelerator of heat injury and wound, remedy agent of angiopathy or diagnostics and remedy agent of malignant tumor or chronic rheumatism, etc. PREPARATION:For instance, human diploid fibroblast is cultured in a medium and supernatant solution of said fibroblast is collected and concentrated, then resultant concentrated solution is flowed through heparin-agarose column, concentration of sodium chloride in 200mM acetic acid buffer solution (pH4.5) is gradually increased, thus adsorbed substance is eluted from the column at >=0.8M sodium chloride concentration and the eluate is introduced into reverse-phase column chromatography and purified to afford the aimed growth factor of vascular endothelial cell.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel vascular endothelial cell growth factor that is useful as a medicine or diagnostic agent, and furthermore, in research on vascular endothelium.

[Prior Art] In recent years, aging, damage, and functional decline of blood vessels are thought to be the main causes of stroke and heart disease, which account for the highest mortality rates among Japanese people. Vascular endothelial cells are cells that form a monolayer that covers the lumen of blood vessels, and are thought to be involved in lipid metabolism, which is a cause of arteriosclerosis, which causes aging and damage to blood vessels. Furthermore, the proliferation of malignant tumors is closely related to angiogenesis, and proliferation of vascular endothelial cells is an essential condition. Furthermore, angiogenesis is also required for the healing of burns and wounds, and the involvement of vascular endothelial cell growth factor is clear.

As mentioned above, vascular endothelial cell growth factor (endothelial cell growth factor), which is thought to exist naturally in living organisms,
If growth factor (hereinafter abbreviated as ECGF) can be isolated and utilized, it can be expected to serve as a protective and therapeutic drug for vascular endothelial damage associated with arteriosclerosis, and a healing promoting drug for burns, wounds, and the like. Furthermore, ECGF can be used as a vascular endothelium-forming agent when creating a biocompatible artificial blood vessel coated with vascular endothelium. Furthermore, E
Since antagonists of CGF are thought to inhibit angiogenesis, ECGF can be expected to be an extremely useful material for the development of anti-malignant tumors and therapeutic agents for rheumatoid arthritis and retinopathy. In addition, vascular endothelium proliferates due to arteriosclerosis and malignant tumor growth, resulting in blood, urine,
If ECGF is present in stool, etc. at a higher than normal physiological concentration (an increase in specific hormones is often observed in various diseases), antibodies against ECGF can be created,
It becomes possible to develop diagnostic agents for the above-mentioned diseases using the antibodies. In this way, the medical value of ECGF is extremely large, and by the way, it has been shown to be effective in preventing malignant tumors, stroke, and heart disease.
The usefulness of ECGF's use and application in medical care is clear, considering that it ranks among the top three in terms of mortality rate among Japanese people (as of 1986).

Based on the background and expectations described above, the search for ECGF has been vigorously carried out in recent years.
00, protein rLob with an isoelectric point of about 5 or about 10
b et al., Anal, Biochem.

154, 1. (198B)] was found. Recently, platelet-derived ECGF has also been discovered, which does not adsorb to heparin-immobilized gel and has a molecular weight of 45°000.
, isoelectric point4. 6 [Miyazono et al., J, B
iol, Chem, 262.4098. (19
87) It has been reported that this protein is a cocoon protein.

[Problems to be Solved by the Invention] When extracting and utilizing biological substances, it is important to use materials and methods that have excellent productivity and safety. E
Considering the application of CGF from this point of view, it seems that brain and cancer cells are somewhat problematic, and platelets are difficult to obtain on a regular basis in large quantities. In addition, various molecular types of ECGF have been discovered to date, and it is unclear which molecular types are suitable for actual medical application, or the possibility of application of undiscovered ECGF derived from other tissues. These issues remain as issues for future consideration.

The present invention aims to solve the above-mentioned problems by obtaining ECGF, which has the potential for pharmaceutical application, by a method with excellent productivity and safety.

The present inventors completed the present invention as a result of intensive research in accordance with this objective.

[Means for Solving the Problems] The present invention provides: a) adsorption to heparin-immobilized gel;
eluted with 8M or higher sodium chloride, b) Molecular weight under non-reducing conditions is 38.000-48°000, C) Isoelectric point is 460-5.0, 5.5-7°0 and 8.5 ~9
．． The present invention relates to a novel ECGF having a property of 0. The cells used for the production of this ECGF include this ECGF.
Any cell may be used as long as it has the ability to produce GF protein, but human diploid fibroblasts are preferred. Human diploid fibroblasts are highly safe because they are derived from normal tissues, and can be cultured for long periods of time (about 50 generations) in various large-scale culture systems, and have high productivity. Furthermore, human diploid fibroblasts exist in various vascular-rich tissues such as connective tissues and lungs, and therefore, this ECGF may actually be locally involved in angiogenesis and vascular endothelial cell metabolism in vivo. There is a high possibility that

The ECGF of the present invention can be obtained by the following method. The present ECGF is preferably contained in the culture supernatant of human fibroblast cells or the cell extract.

Human fibroblasts are normally cultured at 37°C in Eagle's MEM medium containing 5% tallow serum, but if the culture supernatant is to be used as a purification material, the culture medium should be changed to a serum-free medium after the cells have grown sufficiently. It is preferable. The culture supernatant or cell extract containing the present ECGF is concentrated by ultrafiltration, ammonium sulfate salting out, etc. as necessary, and then subjected to various chromatographies, from which the present ECGF can be separated. Any chromatography can be used for purification as long as it has affinity for the present ECGF.

Examples include chromatography using columns bound with heparin, columns with hydrophobic groups as ligands, columns made of calcium phosphate or silicon dioxide, ion exchange columns, and columns with molecular sieving ability. It is desirable to adsorb to the heparin column under neutral conditions. Further, it is also possible to further purify by SDS polyacrylamide electrophoresis or isoelectric focusing. The ECGF of the present invention thus obtained is adsorbed on a heparin-immobilized gel and eluted with 0.8M or more thorium chloride, but preferably 1.0 to 1.5M sodium chloride is used. . Also, under non-reducing conditions 38,000
It has a molecular weight of ~48,000 with an isocenter of 4.0-5.
0.5.5-7°0 and 8.5-9.0.

The activity of ECGF of the present invention can be determined by a known activity measurement method. In other words, Chor of chicken fetus
ioal Iantoic membrane (CA
How to use M) [Taylor and Folkman
, Nature, 297.307 (1982) and Folkman et al., 5science, 221.7
19 (1983) or rat or rabbit c.
Orneal m1cropocket method [Gimbr
one et al., J. Natl. CanCer In5t
,, 52.413 (1974) and Fourni
er et al., Invest.

Ophthalmal, Visual Sci.
, 21. 351 (1981), also 5ustained-release poly
How to use r1nplants [Ranger and Polkman, 5science, 263.797
(1976) and Murray et al., In Vitr.
o, 19.743 (1983)].

To perform this process simply and quickly, human venous endothelial cells (
The proliferation promoting activity against HUV-EC) was determined in 24 wells.
Kan and Yanane's method of measuring the proliferation amount of HUV-EC implanted in plastic dates as an index [J, cent, Physiol, 111.1
55 (1982)] is desirable.

The ECGF of the present invention is directly useful as a healing promoter for wounds, burns, post-operative tissues, etc., and as a therapeutic agent for cardiovascular disorders. It can also be used as an endothelium-forming agent for artificial blood vessels. Indirectly, the coexistence of this factor enables long-term culture of vascular endothelial cells, and it can be used as an essential reagent for vascular endothelial cell research. Also, this ECGF
Antibodies and inhibitors are useful as therapeutic or diagnostic agents for malignant tumors, retinopathy, and chronic rheumatoid arthritis.

The ECGF of the present invention can be used directly as a powder as a protein preparation, or with pharmacologically acceptable carriers, excipients,
It can be safely administered parenterally or orally to warm-blooded animals such as humans as a pharmaceutical composition (eg, injection, tablet, capsule, liquid, ointment) together with a diluent.

In this way, the present ECGF can be provided as a new and useful drug in this field, where there have been few effective drugs until now.

[Examples] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Human diploid fibroblasts were sufficiently grown in Eagle's MEM medium containing 5% tallow serum, and after reaching confluence, they were grown in Eagle's MEM medium without serum for 2 to 3 days.
The cells were cultured at 7°C, and the supernatant was collected. 50% of this culture supernatant
When the concentration was doubled and a dose-effect curve was drawn, as shown in FIG. 1, a clearly concentration-dependent ECGF activity was observed.

40ml (1.6x20cm) of this culture supernatant 20α
Heparin Sepharose CL-68 (Pharma
CIA). The ECGF active component in the culture supernatant is adsorbed by this column, and as shown in Figure 2, when the sodium chloride concentration is increased in 20mM acetate buffer (pH 4, 5), the ECGF active component in the culture supernatant is absorbed by the column at a concentration of 0.8M or higher. eluted from the column. This active fraction was treated with TSKgel Ethe
r-5PW (2.15X15cm, Toyo Jozo) 1.
Adsorption was carried out in the presence of 5M ammonium sulfate. As the ammonium sulfate concentration was gradually lowered, ECGF activity was eluted at 1.2-0.5M ammonium sulfate. Furthermore, this active fraction was applied to a C18-reversed phase column (6X
250mm, Senshu Science), 0.1%
The ECGF active component was eluted with a 0-70% acetonitrile concentration gradient in the presence of trifluoroacetic acid (pH 2,0). This purified ECGF fraction was subjected to SDS polyacrylamide gel electrophoresis without a reducing agent, and the activity was extracted from the gel and the molecular weight was measured, as shown in Figure 3.
Activity was detected at molecular weights of 38,000 to 48,000. In addition, when the above-mentioned ECGF purified fraction is subjected to polyacrylamide gel isoelectric focusing and the activity is extracted in the same manner,
As shown in Figure 4, pH 4.0-5.0, pH 5.5
Peaks of ECGF activity were observed at pH ~7.0 and pH 8.5-9.0. All of the above ECGF activities are Ka
It was measured by the method of n and Yamane.

From the above results, the E produced in human diploid fibroblasts
CGF is a) adsorbed to the heparin-immobilized gel, and 0.
eluted with 8M or higher sodium chloride, b) molecular weight under non-reducing conditions of 38.000-4s, ooo, C) isoelectric point of 4.0-5.0, 5.5-7.0 and 8.0. 5-9.
It was found that it is a protein with the property of 0.

[Effects of the Invention] The ECGF of the present invention is useful as a healing promoter for burns, wounds, etc., and as a therapeutic agent for cardiovascular disorders.

Furthermore, it can be an extremely useful material as a therapeutic or diagnostic agent for malignant tumors, retinopathy, chronic rheumatism, etc. Furthermore, the ECGF of the present invention can be obtained by a method with much higher productivity and safety than conventional methods.

[Brief explanation of the drawing]

FIG. 1 shows the concentration dependence of ECGF activity. Figure 2 shows the ECGF elution curve from the heparin column. FIG. 3 shows the results of molecular weight measurement of the ECGF active ingredient by SDS polyacrylamide gel electrophoresis. FIG. 4 shows the results of isoelectric point measurement of the ECGF active ingredient. Patent Applicant Azuma Co., Ltd. 5O of ECGF
S-polyacrylamide gel electrophoresis (activity extraction) dilution factor Concentration dependence of ECGF activity Figure 1 Percentage number Eζ (7F:Li1/1 fraction Figure 3

Claims (1)

[Claims] (1) A vascular endothelial cell growth factor protein having the following properties. a) Adsorbs to heparin-immobilized gel and elutes with 0.8M or higher sodium chloride b) Molecular weight under non-reducing conditions is 38,000 to 48,00
0 c) Isoelectric points of 4.0-5.0, 5.5-7.0 and 8
．． 5-9.0