JPH0472840B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides hepatocyte growth factor (hepatocyte growth factor).
growth factor (HGF), specifically a protein with 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 glandular organ in the body. It mainly has a variety of important functions such as processing (metabolism), storage, detoxification, decomposition, and excretion of various nutrients (carbohydrates, proteins, lipids, vitamins, hormones, etc.), and among them, intermediary metabolism in the body. is known to play a central role. Furthermore, it is known that each hepatic parenchymal cell that constitutes the liver is responsible for the functions of the liver. However, in vivo, the liver is placed in an extremely complex environment containing various hormones, making it extremely difficult to study the functions of the liver parenchymal cells that make up this environment. Therefore, the present inventor believes that if it is possible to reproduce the above-mentioned hepatocytes in a simple in vitro system while maintaining the same function as in vivo, it will be possible to research the above-mentioned liver function or to use various hormones. From the viewpoint that this system is extremely useful for research on the effects of drugs and the like on hepatocytes, intensive research has been carried out with the aim of establishing an in vitro culture system that can stably subculture the above-mentioned hepatocytes. However, with the rapid development of tissue culture technology in recent years, hepatic parenchymal cells can be grown even in the presence of mammalian serum, where various established cell lines actively proliferate.
No growth was observed, and sloughing usually occurred in about one week, making in vitro long-term subculturing impossible.
In addition, through research by the present inventors, factors such as growth factors that support the proliferation of various known cell lines,
For example, fibroblast growth factor (fibroblast growth factor)
factor；FGF, Gospodarowicz, D., J.Biol.
Chea. 250 , 2515 (1975)), platelet-derived growth factor (PDGF),
Ross, R., et al, Proc. Natl. Acad. Sci. USA.
71, 1207 (1974)), somatomedines; Van Wyk, JJet al., Rec.
Prog. Hormone Res. 30 , 259 (1974)), insulin like growth factor:
IGF, Rinderknecht, E. et al., J. Biol. Chem.,
253, 2769 (1978), multiplication stimulating activity (MSA),
Dulak, NC et al., J. Cell, Physiol. 81 , 153
(1973)), thrombin, transferrin, etc., were confirmed to have no effect on the proliferation of the above-mentioned hepatic parenchymal cells. Slightly insulin and epidermal growth factor (EGF)
Carpenter, G. et al., Ann.Rev.Biochem., 48 ,
193 (1979)) showed the activity of promoting DNA synthesis in hepatic parenchymal cells, but it was also 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 relates to hepatocyte growth factor (hereinafter referred to as "HGF"), which is derived from mammalian serum 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 10mM phosphate buffer (PH = 7.0) and eluted with the same buffer containing 0.2M sodium chloride. c) Does not adsorb to CM-cellulose equilibrated with 10mM phosphate buffer (PH = 7.0); d) Adsorbs to heparin-Sepharose CL-6B equilibrated with 10mM phosphate buffer (PH = 7.0). , does not elute with the same buffer containing 0.5M sodium chloride, but elutes with the same buffer containing 1.5M sodium chloride, e) has activity to proliferate hepatic parenchymal cells in vitro, f) acetic acid treatment (1N , room temperature, 12 hours) and formic acid treatment (1%, 4°C, 12 hours). g) Heat treatment at 100°C for 90 seconds causes the above activity to be inactivated. h) Trypsin treatment deactivates the above activity. i) Ultraviolet/visible absorption spectrum: λ _nax = 278 nm, j) Gel filtration of mammalian serum with a gel filtration agent whose fractionation range includes proteins with a molecular weight of about 150,000, and heparin affine. It can be obtained by subjecting it to at least one treatment step of take chromatography. The HGF of the present invention is derived from mammalian serum,
It is characterized by having the above characteristics. Although various studies have been conducted on mammalian serum components, it is completely unknown that the serum contains factors that have the above-mentioned proliferation activity of hepatic parenchymal cells.
Of course, there are no examples of such factors being isolated from serum, and known components isolated from serum do not have the above-mentioned activity. The above characteristics and other properties of the HGF of the present invention will be described in detail in Examples below. By using HGF of the present invention, hepatic parenchymal cells derived from various animals including humans can be extremely easily grown and maintained in vitro. The hepatic parenchymal cells grown and maintained in this way can be used for basic research on liver function, for research on the effects of various hormones or drugs on hepatic parenchymal cells, and for screening tests for drugs to treat 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 any serum derived from humans, horses, cows, pigs, sheep, rabbits, mice, etc. can be used. Such serum is separated from the mammalian serum from which it is derived according to a conventional method. It is preferable that the above-mentioned serum is undenatured fresh serum. In particular, the serum used in the present invention may be normal serum, which does not undergo any special treatment on the animal or blood before it is collected and isolated from the source animal; however, 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 the 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. It can be carried out according to various processing operations (for example, "Biochemistry Data Book" P1175-1259, 1st edition, 1st printing, June 1980)
23rd of May, published by Tokyo Kagaku Doujin Co., Ltd.). Specifically, the method includes treatment with a common protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), centrifugation, electrophoresis, affinity chromatography, dialysis, and combinations thereof. can be mentioned. In general, serum is subjected to gel filtration using a gel filtration agent whose fractionation range includes proteins with a molecular weight of about 150,000.
HGF is obtained. The gelling agent is not particularly limited and includes, for example, ordinary dextran gel, polyacrylamide gel, agarose gel,
Any material made of polyacrylamide-agarose gel, cellulose, etc. can be used. Specific examples of these include Cephadex G type, Cephadex LH type, Cepharose type, Cephacryl type (Pharmacia), Cellulofine (Chitsuso Co., Ltd.), and Biogel P.
Examples of commercially available products include Type A (Bio-Rad), Ultrogel AcA type (LKB), and TSK-G type (Toyo Soda Kogyo Co., Ltd.). Although serum is not particularly necessary for the gel filtration, it may be partially purified in advance into protein components containing the relevant molecular weight fraction. This partial purification includes, for example, treatment using a protein precipitant such as acetone, methanol, ethanol, propanol, dimethylformamide (DMF), treatment using a salting-out agent such as ammonium sulfate, sodium sulfate, sodium phosphate, etc., and/or dialysis membrane, This is carried out by ultrafiltration treatment using a flat plate membrane, hollow fiber membrane, etc. The operations and conditions for each of these treatments may be the same as those for ordinary methods of this type. Although the HGF thus obtained is present in the raw serum, the serum itself generally has weak hepatocyte proliferation activity due to growth inhibitory 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 Wistar male rats (200-250 g) and Higgins and Anderson method [Higgins, GM
and Anderson, RM, Arch. Pathoi., 12, 186.
202 (1931)], blood was collected from each abdominal aorta of the same rats 2 days after partial hepatectomy, and serum was separated. 30 ml of each serum obtained was diluted 1.5 times with phosphate buffered saline (PBS), and then added to a Sephadex G200 column (Pharmacia, Inc., equilibrated with the same PBS).
4.4φ x 85cm) and fractionated into 15ml fractions. The security index
Figure 1 shows the fractionation pattern in terms of absorbance (A280 nm) during gel passage using G200. In the figure, the horizontal axis shows the fraction number, and the vertical axis shows the 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 the same gel filtration are indicated with arrows. IgG...Molecular weight; 150000 Hb...... 〃; 64500 Cyt.C... 〃; 13800 Void volume (Vo) is Blue Dextran 2000, total volume (Vt) is DNP
-Alanine was determined as a marker. As a result of the above gel filtration, the second protein peak fraction (fraction No. 40) that elutes in the void
~50) and transfer it to the Amicon Diaflow membrane.
Concentrate to 15ml using PM30 (Amicon),
HGF of the present invention was obtained from each serum. below,
The serum obtained from normal serum is abbreviated as "HGF-1", and the serum obtained from regenerated liver serum is abbreviated as "HGF-2". Example 2 Using 20 ml of post-coagulated serum collected from a healthy adult, fractionation was performed in the same manner as in Example 1 above.
Fractions No. 40 to 50 were collected and similarly concentrated to 15 ml to obtain human-derived HGF. Hereafter, this will be referred to as "HGF-3"
It is abbreviated as Below, 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 male Wistar rats (180-200 g), collagenase perfusion method [Tanaka, K, et al.
al., J.Biochem. (Tokyo), 84, 937-946
(1978)], hepatic parenchymal cells were isolated and prepared.
These hepatic parenchymal cells were suspended in Williams E medium (Flow Laboratory) supplemented with 5% bovine serum and 10 ^-8 M insulin, and placed in a 12-well multiplate (Linbro) at a low concentration of 3.3 x 10 ⁴ cells/cm ^{2 .} The cells were cultured in the presence of 5% carbon dioxide gas and 30% oxygen gas. After 4 hours of culture, the medium was
% bovine serum and 5 x 10 ^-7 M dexamethasone, and at the same time a predetermined amount of the test sample (HGF of the present invention or various other growth factors).
was added. After 20 hours, the same medium exchange and sample addition were performed again. After 22 hours, 1.25 μCi/ml of ³ H-thymidine
(0.4 μCi/μmol) and cultured for 4 hours.
A group to which hydroxyurea (25 mM) was added 15 minutes before the above-mentioned labeling with ³ H-mitidine was used as a control group. After labeling by incubation for 4 hours, the cells were washed with PBS and fixed with cold 10% trichloroacetic acid (TCA) aqueous solution. The cells were solubilized with 0.5 ml of 1N aqueous sodium hydroxide solution per well, and aliquots were taken to measure protein content according to the Lowry method. TCA in residual liquid
The TCA-insoluble fraction was collected by centrifugal sedimentation, washed with 5% TCA aqueous solution, added with 1 ml of 10% TCA aqueous solution, and incubated at 90°C for 15 minutes.
The mixture was boiled for a minute, and the radioactivity of the supernatant fraction was measured using a toluene-ethanol scintillator. The amount of ³ H-mitidine incorporated into the DNA of hepatocytes by the test sample was determined as the difference in counts from the control, and this was calculated as the amount of protein per mg of hepatocytes and per hour, and the DNA synthesis activity (dpm/mg Protein/hr) 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>Thrombin; (Sigma) FGF; (Collaborative Res. Inc.) HGF-1; Obtained in Example 1 HGF-2; 〃 1 〃 HGF-3; 〃 2 〃 <Test>PDGF; (Collaborative Res.Inc.) Insulin (Ins); (Sigma) Ins+EGF; (Collaborative Res.Inc.) The results of each test are shown in Table 1 below. In addition, the amount of the test sample used in the table is expressed as the final concentration of the test system. [Table] [Table] From Table 1 above, all currently available growth factors with well-defined physical properties are
No DNA synthesis promoting activity could be detected. In contrast, the HGF of the present invention has been found to have a strong activity of promoting DNA synthesis in hepatocytes; for example, when HGF-2 is used at a dose equivalent to 20% of the original serum, Approximately 22 times more DNA synthesis activity is obtained,
This value 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 to 80% of the total number of hepatocytes.
This corresponds to the value when 90% of cells start DNA synthesis, and in fact, the number of nuclei labeled with ^3H -thymidine (%
This has been confirmed by measurements of labeling nuclei. In addition, HGF-1 is approximately 55% of HGF-2.
This suggests that partial hepatectomy increases serum HGF activity approximately twice. (2) Using the HGF-2 obtained in Example 1, the DNA synthesis activity was determined in the same manner as in (1) above, and a volume dependence curve was created. The results are shown in Figure 2. In Figure 2, the vertical axis is DNA synthesis activity (dpm/mg
The horizontal axis shows the amount of HGF-2 added as the final concentration of the test system (mg protein amount ^/ ml). In addition, each mark in the figure is as follows. (1)...HGF-2 used alone (2)...HGF-2+Ins10 ^-7 M (3)...HGF-2+EGF20ng/ml Test Example 2 Affinity test of HGF for ion exchanger Using HGF-2, diethylamino The affinity of ethyl (DEAE) and carboxymethyl (CM) to ion exchangers was investigated. That is,
2.5ml of HGF-2 (14.2mg protein amount/ml),
DEAE-cellulose (Watmann) or
CM-Cellulose (Watmann) column (1φ
×5 cm), washed with the same buffer (bypass fraction), and then washed with the same buffer containing 0.2M sodium chloride.
Then, elution was performed with the same buffer containing 0.5 M sodium chloride (flow rate 22 ml/hr). The total amount of eluted protein is shown in Table 2 below. [Table] 2.5 ml of each fraction in Table 2 above was added using Amicon Diaflo membrane PM-10 (Amicon).
Concentrate it to
DNA synthesis activity was measured. The results are shown in Table 3 below. [Table] Equivalent.
From Table 3, it is estimated that the HGF of the present invention is an acidic protein with an isoelectric point of 5 to 6 because it is neutral and adsorbed to DEAE and eluted with 0.2M NaCl. Test Example 3 HGF Stability Test Using 1 ml of HGF-2 (13.3 mg protein amount/ml), the DNA synthesis activity was measured in the same manner as in Test Example 1-(1) above after each of the following treatments. and tested the stability of activity. Acetic acid treatment: Add acetic acid to 1N, leave at room temperature for 12 hours, dialyze against PBS, centrifuge (105000 x g, 30 minutes), collect the supernatant (7.23 mg protein amount/ml), This was used for testing. Formic acid treatment: Add formic acid to 1%,
After being left for 12 hours at ℃, it was dialyzed against PBS, and after centrifugation (105,000 xg, 30 minutes), the supernatant (9.35 mg protein/ml) was collected and used for the test. 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. b) After heat treatment at 100° C. for 90 seconds, the mixture was centrifuged (105,000×g, 30 minutes), and the supernatant (11.7 mg protein/ml) was collected and used for the test. Trypsin treatment: Trypsin 2 on HGF-2
After adding trypsin inhibitor (Sigma NOT-825, 20 BAEE units) and incubating at 37°C for 2 hours, trypsin inhibitor 4 mg (Sigma NOT-9378,
The reaction was stopped by adding 64 units of inhibitor activity, and this was used for the test. The results are shown in Table 4. [Table] Equivalent.
It can be seen from Table 4 that the 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) Using UV250 (Shimadzu Corporation), HGF-2
(0.83 mg protein amount/ml) ultraviolet/visible absorption 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 Affinity test of HGF for heparin Heparin-Sepharose CL- using HGF-2
The affinity for 6B and the possibility of its purification were investigated. That is, 4ml of HGF-2 (12.6mg protein amount/
ml) was applied to a column (1φ x 5cm) of heparin-Sepharose CL-6B (Pharmacia) equilibrated with 10mM phosphate buffer (PH = 7.0), washed with the same buffer, and then injected at 15ml/hr. Gradient elution was performed at a flow rate up to 0.5M sodium chloride, and each fraction was fractionated into 1.9 ml. 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 Figure 4, the vertical axis is 280nm.
The absorbance (●――●) and conductivity (...) at
Indicates the number. The obtained total fractions were divided into fractions (fractions No. 2 to 12), fractions (fractions No. 13 to 20),
Fraction (Fraction No. 21-30), Fraction (Fraction No. 31-40) and Fraction (Fraction No.
41 to 50), each fraction was concentrated to 4 ml using Amicon Diaflo membrane PM-10, and after dialysis with PBS, the DNM synthesis activity of each fraction was determined according to Test Example 1-(1). Measurements were made in the same manner. The results are shown in Table 5 below. [Table] Equivalent.
Table 5 shows that the HGF of the present invention is strongly adsorbed to heparin-Sepharose CL-6B, and is not eluted with 0.5M sodium chloride, but eluted with 1.5M sodium chloride. This confirms that, like many proteins in the blood coagulation system, HGF is a protein that exhibits a strong affinity for heparin. Furthermore, HGF-2 was purified approximately 66 times by group-specific affinity chromatography using heparin-Sepharose CL-6B, and strong hepatocyte proliferation activity was observed at a dose of 19 μg protein/ml. HGF-3 obtained in the same manner as in Example 2,
Heparin-cephalose CL was added using the same method as above.
-6B column for purification, collect the fractions eluted with 1.5M sodium chloride, and after dialysis with PBS,
DNA synthesis activity and stability were measured in the same manner as in Test Example 1-(1) and Test Example 3, respectively. The results are shown in Table 6. 【table】

[Brief explanation of the drawing]

FIG. 1 shows a security index G-according to Example 1.
FIG. 2 is a graph showing the hepatocyte proliferation activity (DNM synthesis activity) of the hepatocyte growth factor of the present invention (HGF-2) obtained in Example 1. Figure 3 shows the ultraviolet/visible absorption spectrum analysis of the same HGF-2, and Figure 4 shows the heparin-visible absorption spectrum of the same HGF-2.
It is a graph showing the results of an affinity test for Sepharose CL-6B.

Claims (1)

[Scope of Claims] 1. A hepatocyte growth factor, which is derived from mammalian serum and is 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 10mM phosphate buffer (PH = 7.0) and eluted with the same buffer containing 0.2M sodium chloride. c) Does not adsorb to CM-cellulose equilibrated with 10mM phosphate buffer (PH = 7.0); d) Adsorbs to heparin-Sepharose CL-6B equilibrated with 10mM phosphate buffer (PH = 7.0). , does not elute with the same buffer containing 0.5M sodium chloride, but elutes with the same buffer containing 1.5M sodium chloride, e) has activity to proliferate hepatic parenchymal cells in vitro, f) acetic acid treatment (1N , room temperature, 12 hours) and formic acid treatment (1%, 4°C, 12 hours). g) Heat treatment at 100°C for 90 seconds causes the above activity to be inactivated. h) Trypsin treatment deactivates the above activity. i) Ultraviolet/visible absorption spectrum: λ _nax = 278 nm, j) Gel filtration of mammalian serum with a gel filtration agent whose fractionation range includes proteins with a molecular weight of about 150,000, and heparin affine. It can be obtained by subjecting it to at least one treatment step of take chromatography.