JPS6045534A - Hepatocyte growth factor - Google Patents

Abstract

PURPOSE:The titled factor, protein derived from mammalian serum, having specific physical and chemical properties, and physiological activity. CONSTITUTION:A hepatocyte growth factor, protein [estimated molecular weight by gel filtration: 150,000; adsorbed on DEAE-cellulose equilibrated with 10mM phosphoric acid buffer solution (pH=7.0), eluted with the buffer solution containing 0.2M NaCl; not adsorbed on CM-cellulose equilibrated with 10mM phosphoric acid buffer solution (pH=7.0); adsorbed on heparin-Sepharose CL-6B equilibrated with 10mM phosphoric acid buffer solution (pH=7.0), not eluted with the buffer solution containing 0.5M NaCl, eluted with the buffer solution containing 1.5M NaCl; having activity to multiply hepatocyte growth factor in vitro] obtained from mammalian (e.g., human, horse, bovine, etc.) serum by combining treatment with protein-precipitating medium with ultrafiltration, molecular sieve chromatography, centrifugation, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides hepatocyte growth factor (hepatocyte growth factor).
growth factor (HGF), more specifically, it relates to a protein that has a new physiological activity that allows hepatic parenchymal cells to be cultured in vitro, thereby making it possible to maintain and proliferate the cells.

The liver is the largest and most highly differentiated organ in the body. It mainly has various important functions such as processing (metabolism), storage, detoxification, decomposition, and excretion of various nutrients (carbohydrates, proteins, lipids, vitamins, hormones, etc.).
Among these, it is known to play a central role in in vivo intermediate metabolism.

Furthermore, it is known that the functions of the liver are carried out by each hepatic parenchymal cell that constitutes the liver. However, in vivo (1
The liver is placed in an extremely complex environment including various hormones, and it is extremely difficult to study the functions of the hepatic parenchymal cells that make up this environment.

Therefore, the present inventors believe that if it is possible to reproduce the above-mentioned hepatic parenchymal cells in a simple in vitro system while maintaining functions equivalent to those in the living body, the above-mentioned liver function research or various hormones, From the viewpoint of being extremely useful for research on the effects of drugs etc. on hepatic parenchymal cells, the above-mentioned hepatic parenchymal cells,
We have been conducting extensive research with the aim of establishing an in vitro culture system that allows stable subculturing.

However, with the rapid development of tissue culture technology in recent years, hepatic parenchymal cells do not proliferate at all even in the presence of mammalian serum, in which various established cell lines actively proliferate, and usually within about one week. Shedding occurred and its in vitro long-term subculturing was impossible.

In addition, the research conducted by the present inventors has revealed factors such as growth factors that support the proliferation of various known cell lines.
Examples, t, fibrobl, spore growth factor,
ast arrowfactor: F G F
, Gospodarowiczll),,J.

3 iol, c het 250.2515 (197
5)), platelet-derived growth factor (platelet-d
erive l1lrOWthfactor: PD
G.F., Ross, R., et at, Proc.
.

N atl, A cad, sci, USA.
721, 1207 (1974)), '/v Tomedin (
somatomedtnes: Van Wyk, J.
J,et al.,,Rec,prog.

Hormone Res, 30, 259 (1974)
), insulin-like growth factor (insulin 1lke
growthfactor: IGF, Rlnder
knot, E. et al.

J. Biol. Chem., 253. 2769 (19
78), Multiplication Stimrating Activities
imulating acNvity: M3A
, Dulak, N.

C, et al., J. Ce1l, Physio.
l, 81.

153 (1973)), thrombin (throIllb
in), transferrin
) etc. were confirmed to have no effect on the proliferation of the above-mentioned hepatic parenchymal cells. Slight insulin (ins
ulin) and epidermal growth factor (epidermal
growth ractor: EGF, Carpen
ter, Q. et al., Ann, Rev.

Biochem, 48, 193 (1979)).
Although the activity of promoting DNA synthesis in hepatic parenchymal cells was observed, it was difficult to subculture the hepatic parenchymal cells in vitro due to their utilization.

However, in subsequent research, the present inventors found that although hepatocytes cannot proliferate at all in the presence of serum itself, as mentioned above, in the presence of a specific protein component contained in the serum, hepatocytes do not proliferate. They found that the serum proliferated very well in the outside world and could be subcultured, and also succeeded in isolating the specific serum component.

The present invention was completed based on the above findings.

That is, the present invention provides hepatocyte growth factor (rl (hereinafter referred to as GFJ)), which is derived from mammalian serum and is a protein having the following physicochemical properties and physiological activities.
Pertains to.

a) Estimated molecular weight by gel filtration method is approximately 150,000, b) 10mM JJ acid buffer (1)H-7,0)r adsorbed on equilibrated DEAE-cellulose and containing 0.2M sodium chloride. Elute with buffer, c) 10RI M! J > Flifm solution (+) H
= 7.0) r not adsorbed on equilibrated CM-cellulose, d) adsorbed on heparin-Sepharose CL-6B equilibrated with 101IIM phosphate buffer (pH = 7.0), 0.5M sodium chloride Same N! It did not elute with solution ii, but the same all! containing 1.5M sodium chloride! elutes in liquid i); e) has activity to proliferate hepatic parenchymal cells in vitro;

The HGF of the present invention is derived from mammalian serum and is characterized in that it has the above-mentioned properties. Conventional mammal If
Although various studies have been conducted on iI-digesting components, it is unknown that the factors that have the above-mentioned proliferation activity of hepatic parenchymal cells exist in the serum, and of course, there are no cases in which such factors have been isolated from serum. Moreover, known components isolated from serum do not have the above-mentioned activity.

The above properties and other properties of the HGF of the present invention will be explained in detail in Examples below.

According to the use of HGF of the present invention, hepatic parenchymal cells derived from various animals including humans can be grown extremely easily in vitro.
can be maintained. The hepatic parenchymal cells proliferated and maintained in this way can be used, for example, for basic research on liver function, for research on the effects of various hormones or drugs on the liver parenchyma, and for screening tests for therapeutic drugs for liver diseases. Furthermore, they are extremely useful as host cells for carcinogenesis tests and in vitro culture of hepatitis viruses. According to the present invention, such useful physiologically active substances can be provided.

Hereinafter, the method for producing HGF of the present invention will be described in detail.

The HGF of the present invention is isolated from mammalian serum.

The mammalian serum used as a raw material here is not particularly limited, and includes, for example, human, horse, cow, pig, sheep,
Any serum derived from rabbits, mice, etc. can be used. Second grade serum is separated from the blood of the mammal from which it originates according to a conventional method. The serum is preferably undenatured and fresh. In particular, the serum used in the present invention may be ordinary serum from second-class animals or blood that is not subjected to any special treatment in advance when blood is collected and separated from the source animal, but serum from animals other than humans may be used. In this case, it is more preferable to use serum from the regenerated liver after partial hepatectomy in terms of HGF yield.

The production of HGF of the present invention is basically carried out using the physical and chemical properties of the target HGF in the same manner as the usual method used for separating proteins from biological materials such as serum of this species. It can be carried out according to various processing operations (for example, "Biochemistry Data Book I [J Pl 175-125
9, 1st edition, 1st printing, June 23, 1980, published by Tokyo Kagaku Dojin Co., Ltd.). Specific examples of the method include treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), centrifugation, electrophoresis, affinity chroma-1-graphy, dialysis, and combinations thereof. Can be mentioned. Generally, the desired HGF is obtained by subjecting serum to gel filtration using a gel filtration agent whose fractionation range includes proteins with a molecular weight of about 150,000. The gel filtration agent is not particularly limited, and includes, for example, ordinary dextran gel, polyacrylamide gel,
Any material made of agarose gel, polyacrylamide-agarose gel, cellulose, etc. can be used. Specific examples of these include Sephadex G type, Sephadex LH type, Sephadex 0-S type, Sephacryl type (Pharmacia), Cellulofine (Chisso ■), Bioglu P type, and Sephadex A type (Bio-Rad). ), Ultrogel ACA type (LK
Examples include commercially available products such as Company B) and TSK-G type (Toyo Soda Kogyo ■). Although it is not particularly necessary for the serum to be subjected to the above-mentioned gel filtration, it is also possible to partially purify the serum in advance into a protein component containing both of the molecular weights. This partial purification may be carried out by, for example, treatment using a protein precipitant such as acetone, methanol, ethanol, propatool, or dimethylformamide (DMF), treatment using a salting-out agent such as ammonium sulfate, sodium sulfate, or sodium phosphate, and/or treatment using a dialysis membrane. This is carried out by ultrafiltration using a flat plate membrane, hollow manifold, or the like. The operations and conditions for each of these treatments may be the same as those for ordinary methods of this type.

Although HGF thus obtained is present in the raw serum, the serum itself generally has weak hepatocyte proliferation activity due to growth-inhibiting factors and cytotoxic factors that coexist in the serum. Its strong activity can only be obtained by fractionating it as described above, and it is specified by the above-mentioned physicochemical properties and physiological activity.

The present invention will be described in more detail with reference to Examples and Test Examples below, but the present invention is not limited thereto.

Example 1 Female Wistar rats (200-250 g) and the method of Higgins and Anderlin (H1QQins).

G., Iyl, and Anderson, R.
M., Arch.

Pathof, 12.186-202 (1931
2 days after partial hepatectomy was performed, blood was collected from the aorta of each of the same rats and serum was separated.

Thirty centimeters of each serum obtained was added to phosphorus 9M% saline (
Sephadex 0200 column (Pharmacia, 4.4φ) diluted 1.5 times with PBS and equilibrated with the same PBS.
The gel was separated into 15 fractions.

Absorbance during gel filtration with Sephadex G200 (
The fractionation pattern at 280 nm) is shown in FIG.

In the figure, the horizontal axis shows fraction NO, and the vertical axis shows absorbance. In the figure, (1) shows the case of normal rat serum, and (2) shows the case of regenerated liver rat 48 hours after partial 2/3 hepatectomy. Further, in FIG. 1, as a guide to the molecular weight of each fraction, the elution positions of the following molecular weight markers (Pharmacia) during gel filtration are indicated with arrows.

IfllG...Molecular weight: 150000Hb...
・・・ 〃 ; 64500 Cyt, C-・ 〃 :
13800 In addition, void volume (■0) is blue dextran 2
000. l-1 Talborium (V) was determined using DNP-alanine as a marker.

The second protein peak fraction (7 runs N040 to 50) eluted following the void was collected by the above gel filtration, and concentrated to 15 mQk using an Amicon Diaflo membrane PM30 (Amicon). Invention 1-IGF was obtained from each serum.

Hereinafter, the product obtained from normal serum will be abbreviated as rHGF-1J, and the product obtained from regenerated liver serum will be abbreviated as rl-IGF-2J.

Example 2 Using 20 hours of post-coagulated serum from a healthy adult,
In the same manner as in Example 1 above, fraction No. 40 to
Both 50 parts were collected and similarly concentrated to 15 parts to obtain human-derived HGF. Hereinafter, this will be abbreviated as rHGF-3J.

Hereinafter, test examples will be given to clarify the physiological activity and physicochemical properties of HGF obtained in the above examples.

Test Example 1 Hepatic parenchymal cell proliferation activity (1) Using Wistar male rats (180-200 rats), collagenase reflux method (Tanaka, K., e.g.
tal, J.Biochem, (Tokyo),
84.937-946 (197B)), hepatic parenchymal cells were isolated and prepared. These hepatic parenchymal cells were mixed with 5% bovine l1i
1 supernatant and 10-8M insulin (O-Laboratory), and plated in a 12-well multiplate (Linpro) at 3.3 x 104/cells.
Infused with a low concentration of Cl112, 5% carbon dioxide gas and 30
% oxygen gas. After 4 hours of incubation, the medium was changed to Williams E medium containing 5% bovine serum and 5X10-'M dexamethasone, and at the same time a predetermined amount of the test sample (
HGF of the present invention or various other growth factors) were added. 20
After an additional period of time, the same medium exchange and sample addition were performed.

After 22 hours, 1.25 μCi/4 (0
, 4 μCi/μmol) and cultured for 4 hours.

In addition, a group in which hydroxyurea (25mM) was added 15 minutes before the labeling with 3H-thymidine was treated as control 0-
group. After labeling by incubation for 4 hours, the cells were washed with PBS and fixed with cold 10% aqueous trichloroacetic acid (TCA). The cells were solubilized with 0.5 mQ of 1N aqueous sodium hydroxide solution per well, and aliquots were taken to measure protein content according to the O-ri method. Add TCA to the remaining solution to make it 20%, collect the TCA-insoluble fraction by centrifugation, wash with 5% TCA aqueous solution, and add 10% TCA.
Add 1 ml of aqueous solution A, boil at 90°C for 15 minutes,
The radioactivity of the supernatant fraction was measured using a toluene-ethanol scintillator.

3 incorporated into liver parenchymal cell DN by the test sample
- Determine the amount of thymidine as the difference in counts from the control, and calculate this as the difference in counts from the control.
DNA synthesis activity (dawn/a+g
protein, 'hr), and this was used as an index of the hepatocyte proliferation activity of the test sample.

The above test was conducted twice (Test ■ and Test ■) using each of the following test samples.

<Test engineer> ■Thrombin: (Sigma) ■FGF: (Collaborative RGS
, Inc. ) ■ HGF-1; obtained in Example 1 ■ HGF-2: 〃 1 ■ 1-(GF-3: 〃 2 <Test ■> ■ PDGF; (Collaborative Re
s, Inc, ) ■ Insulin (Ins,; (Sigma)
■I ns+ E G F: (Co11abora
tive Res.

Inc.) The results of each test are shown in Table 1 below. In the table, the final concentration of the test sample used is expressed as the final concentration of the test system.

Table 1 Test sample DNA synthesis activity used ffi (dpH/III++ protein/hr) <Test ■) ■Thrombin IU/11112 6140.5U/m
Q 315 0.1U/IRQ 506 ■FGF 20no/mQ 998 10no/mQ 258 20g/Tsuru 405 Test sample DNA synthesis activity used fi (dpm/m<+ protein/l+r) ■HGF-
11mo proteinffi/+nQ 6089 (10V/V%
Serum equivalent) 2mo protein amount/Tsuru 12036 (20V/V% serum equivalent) ■HG F-21raa proteinffi/m12 110
71 (equivalent to IOV/V% serum) 2u protein 伶/噌 21884 (equivalent to 20V/V% serum) ■HGF-31,05no+protein amount/m92282 (1
0V/V% serum equivalent) 2.1mg protein m/MU 3057 (20V/V% serum equivalent) - (No addition) -999 <Test ■> ■PDGF 2.5U/4 781 1U/Peng 987 0.25U/ mQ 893 ■Ins 10-7M 127゜Test test FI DNA synthesis activity jar (du+/mg protein/h'') ■Ins+ 1
0-'M EGF 20ng/1Ili2 3738-(no additive)
733 From Table 1 above, none of the currently available growth factors with well-defined physical properties can detect DNA synthesis promoting activity in hepatic parenchymal cells. On the other hand, the HGF of the present invention has
A strong activity to promote DNA synthesis in hepatic parenchymal cells has been observed; for example, when HGF-2 is used at a dose equivalent to 20% of the original serum, DNA synthesis activity is approximately 22 times that of the control (no additive). , which is about 5.8 times higher than the maximum DNA synthesis activity when insulin and EGF are used together. The DNA synthesis activity that occurs at this concentration is approximately 80% of the total number of hepatocytes.
This corresponds to the value when ~90% of the cells have started DNA synthesis, and in fact, the number of counts labeled with 3H-thymidine (%lab
This has been confirmed by measurements of celluloid cells (elling nucleus). In addition, HGF-1 is about 55% of HGF-2.
% DNF synthesis activity, which suggests that partial hepatectomy increases serum HGF activity approximately twice.

(2) Using the HGF-2 obtained in Example 1, the above (
In the same manner as in 1), a capacity dependence curve was created for DNA synthesis activity. The results are shown in Figure 2.

In Figure 2, the vertical axis represents DNA synthesis activity (dplIl/m
.

The horizontal axis shows the amount of HGF-2 added as the final concentration of the test system (II1g protein amount/Peng).

In addition, each mark in the figure is as follows.

(1)...HGF-2 used alone (2)...HGF-2+In510-' M(
3) ・・・-・1-IGF-2+EGF20na/mQ
Test Example 2 Affinity test of 1-IGF for ion exchanger Using HGF-2, diethylaminoethyl (DEAE>
The affinity of HGF-2 and carboxymethyl (CM) for ion exchangers was investigated.
(14,211g protein!/mQ) was equilibrated with 10++M phosphate buffer (pH=7.0).
It was applied to a column (1φx5c+n) of AE-cellulose (Whatman) or CM-cellulose (Whatman), washed with the same buffer (bypass fraction), and then washed with the same buffer containing 0.2M sodium chloride, followed by 0.2M sodium chloride. Elution was performed with the same buffer containing 5M sodium chloride (flow rate 22w+Q/hr
).

The total amount of eluted protein is shown in Table 2 below.

Table 2 DEAE CM (mu protein) ([l1g protein) Bypass fraction 5.39 32.7 0.2M Na C129,72,63 elution fraction 0.5M NaCl O,040,16 elution fraction Above 2nd Both parts of the table were concentrated to 2,5111Q using Amicon Dai V Flow Membrane PM-10 (Amicon), and the DN
A synthesis activity was measured.

The results are shown in Table 3 below.

Table 3* Each amount used corresponds to 10V/V% serum.

From Table 3, the present invention 1-IGF is neutral and DEAE
It is estimated that it is an acidic protein with an isoelectric point of 5 to 6 because it is adsorbed to and eluted with 0.2M NaCl.

Test Example 3 Stability test of 8GF Using HGF-'2 1a [(13,3u protein length/reed), the DNA synthesis activity was determined after each of the following treatments.
The stability of the activity was tested in the same manner as in Test Example 1-(1) above.

■ Acetic acid treatment: Add acetic acid to 1N and bring to room temperature at 1N.
After leaving it for 2 hours, it was dialyzed with PBS and centrifuged (105,000
x 30 minutes) The supernatant (7,23io protein amount/mQ)
was collected and used for testing.

■ @Acid treatment: Add *m to 1% and heat to 4℃.
After leaving it for 12 hours, it was dialyzed with PBS and centrifuged (10500
(0XIJ, 30 minutes) The supernatant (9.35 u protein amount/unit) was collected and used for the test.

(2) Heat treatment: (a) After heat treatment at 55°C for 30 minutes, no precipitate was observed, and this was used for the test as it was.

The mixture was centrifuged by heat treatment at 100°C for 90 seconds (105,000 x a, 30 minutes), and the supernatant (11,7111 (1 protein amount/mQ)) was collected and used for the test.

■ Trypsin treatment: )-Trypsin 21 to IGF-2
11G (Sigma N0T-825, 20BAEE unit)
After incubating at 37°C for 2 hours, trypsin inhibitor 4+g (Sigma N0T-9378, 64 units of inhibitor activity) was added to stop the reaction.
This was used for testing.

The results are shown in Table 4.

Table 4* Each amount used corresponds to 10V/V% serum.

From Table 4, it can be seen that HGF of the present invention is a proteinaceous growth factor that is unstable to acid and heat. A growth factor that has a strong hepatocyte proliferation activity and exhibits such properties has never been seen before.

Test example 4 (ultraviolet/visible absorption spectrum) LJV250
(Shimadzu Corporation), HGF-2 (0,831B
The ultraviolet/visible absorption of the protein amount was measured. The results are shown in FIG. In the figure, the main peak at 278 nm is a typical absorption spectrum of proteins due to aromatic amino acids.

The peak in the visible region of 420 nm is the Soret band of hemoprotein, and the reason why HGF-2 exhibits a pale brown-red color is based on this absorption, and this is thought to be due to the hemoprotein mixed in HGF-2.

Test Example 5 1-IGF affinity test H for heparin
Using GF-2, the affinity for heparin-Sepharose CL-0B and the possibility of purification using this were investigated.

That is, 4WfI (12,6+no protein amount/-) of HGF-2 was added to 1011M phosphate buffer (at-1=7.
Heparin-Sepharose CL-6B equilibrated with 0)
(Pharmacia) column (1φ
．． It was separated into 911Q fractions.

Subsequently, proteins that were not eluted up to 0.5M sodium chloride were eluted with 1.5M sodium chloride and fractionated in the same manner. The fractionation pattern is shown in FIG. In FIG. 4, the vertical axis shows the absorbance at 280 nm (←-1) and the conductivity (----), and the horizontal axis shows the fraction number.

All the obtained fractions were divided into fractions (fraction N00
2-12), fraction ■ (fraction NO.

13-20), Fraction ■ (Fraction No. 21-30)
), fraction ■ (fraction No. 0.31 to 40) and fraction V (fraction No. 41 to 50), and both fractions were separated using Amicon Dia 70 membrane PM-10 for 4 m
12, and after dialysis with PBS, the DNA synthesis activity of each fraction was measured in the same manner as in Trial Example 1-(1). The results are shown in Table 5 below.

Table 5* Each amount used corresponds to IOV/V% serum.

Table 5 shows that the HGF of the present invention is strongly adsorbed to heparin-Sepharose CL-6B, is not eluted with 0.5M sodium chloride, but is eluted with 1.5M chloride. This confirms that HGF, like many blood coagulation system proteins, is a protein that exhibits a strong affinity for heparin. In addition, IGF-2 was purified approximately 66 times by hepatic isomer affinity chromatography using heparin-Sepharose CL-6B, and strong hepatocyte proliferation activity was observed at a dose of 19 μg protein.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the fractionation pattern during gel filtration with Sephadex G-200 according to Example 1, and FIG.
) is a graph showing the hepatocyte proliferation activity (DNA synthesis activity) of the same HGF-2, FIG. 3 is an ultraviolet/visible absorption spectrum analysis diagram of the same HGF-2, and FIG. It is a graph showing the results of an affinity test for 0L-6B. (That's all) 1 Agent Patent Attorney Eiji Saegusa 11. . Figure 1 Fukushoji No. Figure 2

Claims (1)

[Scope of Claims] (1) A hepatocyte growth factor, which is derived from a mammal and is characterized by being a protein having the following physicochemical properties and physiological activities. a) Estimated molecular weight by gel filtration method is approximately 150,000 b) Adsorbed on DEAE-cellulose equilibrated with 10111M phosphate buffer (pH = 7.0) and containing 0.2M sodium chloride. c) Does not adsorb to CM-cellulose equilibrated with 10mM phosphoric acid mild I acid solution (al-1 = 7.0), d) 10I
Adsorbed onto heparin-Sepharose CL-6B equilibrated with IIM phosphate buffer (t+H=7.0), 0.5M
It did not elute with the same buffer containing sodium chloride, and was 1.5M.
The same containing sodium chloride! ! Elute with lfi solution, e
) It has the activity of proliferating hepatic parenchymal cells in vitro.