JPH083195A - Tissue injury healing factor - Google Patents

Abstract

PURPOSE:To provide a new tissue injury healing factor originated from human or animal tissue or blood component, increasing by the injury of human or animal tissue and active to promote the production of hepatocyte growth factor(HGF) of an HGF-producing cell. CONSTITUTION:This new tissue injury healing factor indulin is originated from the tissue or blood component of human or animal, increases by the injury of human or animal tissue, exhibits a molecular weight of 10-30kD by the gel column treatment with biogel P-60 and Sephadex G-150 and a molecular weight of 40-60kD by SDS polyacrylamide electrophoresis under non-reducing condition and 10-20kD under reducing condition and is active to promote the production of hepatocyte growth factor (HGF) of an HGF-producing cell, stable by heat- treatment at 100 deg.C for 5min, acid treatment at pH1.0, reducing treatment with 1mM dithiothreitol and heparinase treatment at 0.31mU/ml and 37 deg.C for 1hr and inactivated by trypsin treatment at 100mug/ml and 37 deg.C for 3hr.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a tissue injury healing factor, and more particularly, it is derived from a human or animal tissue or blood component, and has a hepatocyte growth factor (Hepatocyto Growth Factor).
ctor (hereinafter, referred to as HGF)).

[0002]

2. Description of the Related Art HGF is a protein found by the present inventors as a factor for proliferating mature hepatocytes from regenerated liver rat serum in vitro (Biochem Biophys Re.
S Commun, 122, 1450, 1984). We further
Succeeded in isolating HGF from rat platelets (Pro
c. Natl. Acad. Sci, 83, 6489,1986, FFBS Letter, 2
2,311, 1987), and its amino acid sequence was partially determined. Furthermore, the present inventors succeeded in cloning human and rat-derived HGF cDNA based on the elucidated HGF amino acid sequence, and recombining this cDNA into animal tissues to obtain hepatocyte growth factor as a protein ( Human HGF: Nature, 342, 440, 1989; Rat HGF: Pr
oc. Natl. Acad. Sci, 87, 3200, 1990).

The molecular weight of HGF is 82-85 kD by SDS-polyacrylamide gel electrophoresis. Rat HG
The F molecule has a heterodimer structure in which an α chain consisting of 463 amino acid residues and a β chain consisting of 233 amino acid residues are crosslinked by one disulfide bond, and both α and β chains have 2 chains.
There are individual glucosamine-type sugar chain binding sites. Human HG
F also has almost the same physiological activity and consists of an α chain consisting of 463 amino acid residues and a β chain consisting of 234 amino acid residues. There are four kringle structures in the α chain, which are similar to the fibrinolytic enzyme plasmin, and the amino acid sequence of the β chain is about 3 times that of the B chain of plasmin that has serine protease activity.
It has a homology of 7%. Rat HGF and human HGF
The amino acid sequence homology is 91.6% in α chain, β
It has a very high homology of 88.9% in the chains and its activities are completely compatible.

HGF, which was discovered as a factor that specifically proliferates liver parenchymal cells, has been shown to exhibit various activities in vivo according to recent research results by the present inventors and other researchers. However, expectations are growing for its application not only as a research target but also as a therapeutic drug for humans and animals. The present inventors have clarified that HGF acts widely as a growth factor not only on hepatocytes but also on epithelial cells, and achieved several inventions. In Japanese Patent Application No. 2-158841, HGF promotes the proliferation of renal proximal tubule cells, so that the application development as a therapeutic agent for renal diseases is also described.
-419158, HGF is melanocyte,
By promoting the growth of normal epithelial cells such as keratinocytes, it can be used as an epithelial cell promoter to treat wounds and skin ulcers.
The application and development of hair root cells as a proliferative agent were accomplished and the details thereof were disclosed. In particular, HGF is more suitable for practical use because it does not have the canceration action and cancer cell growth activity found in many other growth factors such as EGF. Furthermore, the present inventors have disclosed that in Japanese Patent Application No. 3-140812, HGF human liver cancer-derived HepG2 cell line and lymphoblastic cancer-derived IM9.
It has been disclosed that it can be used as an antitumor agent by utilizing the cancer cell growth inhibitory activity of cell lines and the like.

More important point in considering the practicality of HGF as a medicine is that HGF promotes the growth of only cells that have entered the G1 phase, that is, the growth phase, and acts on the cells in the G0 phase, that is, the quiescent phase. Do not do it. This is
It means that proliferation and regeneration of injured tissue is promoted, but it has no effect on undamaged tissue. Therefore, it is considered that even if HGF is excessively administered or HGF reaches the non-affected part via blood or the like, it does not induce canceration in normal tissues or cause excessive proliferation.

[0006]

As described above, HGF promotes the proliferation of not only hepatocytes but also epithelial cells in a wide range and has an activity of suppressing the proliferation of cancer cells.
It is expected that GF acts in healing tissue damage. H
GF-producing cells are not epithelial cells themselves, but Kup in the liver
It is known to be produced mainly by mesenchymal cells such as ffer cells and sinusoidal endothelial cells, capillary endothelial cells in the kidney, and alveolar macrophages and vascular endothelial cells in the lung. It has been clarified that a so-called paracrine mechanism in which HGF is supplied according to the above is established. However, when the liver or kidney is injured, HGF production is increased even in an uninjured organ, such as the lung, and therefore it is considered that HGF is also supplied by the so-called endocrine mechanism. This fact means that there is a substance that transmits production promotion from the injured tissue to the HGF-producing cells.

That is, a substance that promotes the production of HGF is secreted from the injured tissue, and HG is produced through blood and the like.
It is considered that it has a function of reaching the F-producing cell and releasing HGF accumulated in the cell, or initiating new production. It is needless to say that HGF is administered for the purpose of wound healing and promotion of renal regeneration, but it is expected that the same effect will be obtained with a smaller dose and administration frequency if a substance that promotes HGF production is administered. More specifically, compared with the case of directly administering HGF as a tissue injury treatment method, HGF
It is expected that administration of the production-promoting substance can maintain the blood concentration of HGF in the blood for a long period of time, and the dose per administration and the administration frequency can be reduced.

On the other hand, the living body is constantly injured by physical, chemical, or microbial in all tissues and organs including respiratory organs, skin, digestive organs, both externally and internally. On the other hand, HGF, FGF, EGF, PDG
Mobilize all functions including cell growth factors such as F,
Repair of functional tissue cells, matrix cells, etc. of organs is one of the important factors of homeostasis, but it is considered that the central factor that activates and regulates the compensation function network exists in the tissue. Came. The tissue injury healing factor is not only useful for studying the compensatory function of organ injury such as liver and kidney and the expression mechanism of HGF, but also a factor that heals all tissue and organ injury in the living body and is a therapeutic drug. Obviously it will be very useful. Considering the liver, for example, in the case of liver resection or organ injury due to hepatitis, only HGF administration can regenerate hepatic parenchymal cells, whereas administration of a tissue injury healing factor that controls the entire repair function of organ injury. This accelerates not only parenchymal cells but also the whole injury site including supporting tissues such as perisinusoidal connective tissue which is a non-parenchymal cell, and true repair is accelerated.

[0009] That is, if the tissue injury healing factor can be used as a therapeutic drug, the injury is markedly and promptly treated as compared with the case of using various conventionally found cell growth factors alone. And its usefulness is immeasurable. In order to trace the tissue damage healing factor and elucidate its main body, it is most efficient to make the cell growth factor production promoting activity Mercury, and in fact, the HGF production promoting substance is the main body of the tissue damage healing factor. There is no doubt that

[0010]

[Means for Solving the Problems] The present inventors have found that HG is present in the blood of rats artificially injured in the liver or kidney.
It was found that there is an action of stimulating F-producing cells and increasing the production ability, and the agent was named a tissue injury healing factor "indullin" (also in the present specification, for the sake of convenience, indulin is the name of the tissue injury healing factor. use).
Further, as a result of detailed examination using the production amount of HGF mRNA or HGF as an index, the inventor established the purification method and elucidated its molecular weight and physicochemical properties, and completed the present invention.

That is, a laparotomy is performed on a normal rat,
After 70% partial hepatectomy or partial ligation of the portal vein to create an ischemic state and give liver injury, blood was collected over time and injected intraperitoneally into normal rats, 6 to 18 hours later. It was found that the expression level of HGF mRNA in the lung increased remarkably over time (see Experimental Example 1). Similarly, in vitro, human fetal lung-derived fibroblast MRC-5
Blood of a rat with 70% partial hepatectomy, carbon tetrachloride administration, liver injury due to hepatic ischemia treatment, or nephrectomy, mercuric chloride administration, and kanamycin administration renal injury by HGF production quantification using a cell line. It was confirmed that some of them have an action of promoting HGF production (see Experimental Example 2). From these results, it was expected that an agent that promotes HGF production in response to the injury is present in the blood of an animal injured in the liver, kidney, or the like. The present inventors have used Biogel P-60 as a starting material from the serum of carbon tetrachloride-administered rats.
The agent was successfully purified using a gel column for each molecular sieve of Sephadex G-150, ion-exchange FPLC, reverse phase HPLC, preparative SDS-PAGE and the like (see Examples 2 and 4). Regarding the obtained tissue injury healing factor indurin, SDS-PAGE
The molecular weight was measured in more detail by means of various methods, and various physicochemical treatments were performed to examine the stability (see Examples 1, 3, 4).

That is, the present invention relates to: 1. In a hepatocyte growth factor (HGF) -producing cell, which is derived from a human or animal tissue or blood component and has a characteristic of increasing in response to injury of human or animal tissue. H
Injurin, a tissue injury healing factor having an activity of promoting GF production. 2. Injulin, a tissue injury healing factor which retains its activity by the treatments 1) to 4) below and is inactivated by the treatment 5). 1) Heat treatment at 60 ℃ for 5 minutes or 100 ℃ for 5 minutes. 2) Acid treatment at pH 1.0, 3.5 and 5.0. 3) Reduction treatment of 1 mM dithiothreitol. 4) Heparinase treatment at 0.31mU / ml at 37 ° C for 1 hour. 5) Trypsin treatment at 100 µg / ml at 37 ° C for 3 hours. 3. Biogel P-60 and Sephadex G-150
Indulin, a tissue injury healing factor showing a molecular weight of 10 kD to 30 kD by each gel column treatment. 4. Injurin, a tissue injury healing factor showing a molecular weight of 10 kD to 20 kD by SDS polyacrylamide gel electrophoresis under non-reducing conditions. 5. Injurin, a tissue injury healing factor showing a molecular weight of 40 kD to 60 kD by SDS polyacrylamide gel electrophoresis under non-reducing conditions. Regarding

As a method for obtaining the tissue injury healing factor and indulin of the present invention, tissues and blood components of animals such as rats subjected to carbon tetrachloride treatment or unilateral nephrectomy surgery, or hepatitis, nephritis patients or immediately after surgery It can be purified using a human tissue or blood component as a raw material by the gel filtration method shown in Example 2, FPLC as shown in Example 4, or SDS polyacrylamide gel electrophoresis. In addition, when the indulin activity of various porcine organ extracts was measured by the in vitro assay system shown in Experimental Example 2, the activity was found in many organs as shown in Table 1 (see Experimental Example 5). In particular, high activity was observed in the cerebellum, cerebrum, liver, and lung, and it became clear that the indulin of the present invention was accumulated at a high concentration. MR used in this embodiment
Similar activity was observed in rats, pigs, and humans (see Experimental Example 4) in the C-5 cell assay system, indicating that, like HGF, the activity of indulin is at least compatible between mammals. There is.

[0014]

EFFECTS OF THE INVENTION The tissue injury healing factor of the present invention is a novel factor, and the tissue injury healing factor of the present invention has the effect of promoting healing of injuries of tissues and organs of a living body. .

[0015]

EXAMPLES Examples will be given to explain the present invention in more detail, but the present invention is not limited thereto. Further, all the rats used in the following experimental examples and examples were Wistar adult male normal rats (130
~ 150g).

Experimental Example 1 A 70% partial hepatectomy was performed on a normal rat by laparotomy, or a portal vein was partially ligated to an ischemic state to injure the liver. 2m serum from these liver-injured rats over time
l was collected and administered intraperitoneally to each normal rat. As a control, normal rat peripheral blood and physiological saline were administered, and 6 hours after the administration, the rat was dissected and the lungs were excised.
It was subjected to F mRNA measurement. More specifically, the acid-guanidine-phenol-chloroform method (AGP) from isolated lungs
RNA is extracted by the C method) and poly-treated with Oligotex dT-30.
(A) RNA was purified. Denature 2 μg of poly (A) RNA with formaldehyde and then add 0.7% formaldehyde
It was electrophoresed on a 1.2% agarose gel. After migration, Hybond-N
Transferred to a filter and hybridized with a 1.4 kb RBC-1 fragment labeled with [α-P ³² ] -CTP. Hybridization is 20 μg / ml salmon sperm DN
A, 50% (V / V) formaldehyde, 0.5% (W / V) SDS, 0.9
It was carried out at 42 ° C. for 17 hours using a buffer solution containing M NaCl, 50 mM NaH ₂ PO ₄ , 5 mM EDTA and 2 × Denhardt's solution. 0.36M NaCl, 20mM NaH ₂ PO ₄ , 2mM
It was washed twice with a buffer solution consisting of EDTA and 0.5% SDS at 65 ° C. for 15 minutes and photographed with an X-ray film (Fuji Film Co., Ltd.). The result is shown in FIG. In both cases of partial hepatectomy and liver ischemia as shown in FIG.
It was confirmed that the expression of F mRNA was promoted.

Experimental Example 2 It was confirmed by an in vitro assay system that the HGF-inducing active substance was present in the serum of liver-injured or renal-injured rat by the following procedure. Liver-damaged rats were subjected to 70% partial hepatectomy, carbon tetrachloride-administered rats (2 mg CCl ₄ / g body weight) and rats that had been in hepatic ischemia, and kidney-damaged rats in which only one kidney had been removed, chloride Mercury administration (2mg HgCl
₄ / g body weight rats and kanamycin administration (0.3 mg
Serum was collected over time from each of the rats (kanamycin / g body weight). Human fetal lung-derived fibroblast cell line MRC
-5 cells are 10% fetal calf serum (FCS), penicillin 1
The cells were cultured in Dulbecco's Eagle medium (DMEM) supplemented with 00 IU / ml and streptomycin 100 IU / ml. Inulin activity was measured by quantifying HGF produced by MRC-5 cells. That is, MRC-5 cells were cultured in a 24-well microplate (manufactured by Corning) and
When the cell density reached ˜90%, the medium was replaced with DMEM without FCS, the sample serum was added to each well, and the culture was continued for another 24 hours. The unit of indulin activity is 1 mU (1 / m) with an activity that gives 50% of the maximum concentration of HGF expressed when an acidic extract of pig lung is added.
1000 units of activity).

HGF in the culture supernatant of MRC-5 cells was quantified by enzyme-linked immunosorbent assay (ELISA method). The anti-human HGF polyclonal antibody was obtained by immunizing a rabbit with recombinant human HGF and purifying IgG from the obtained serum using protein A sepharose gel (Pharmacia). The obtained antibody did not cross-react with rat HGF. 20 μg / ml of anti-human HGF antibody in 50 mM carbonate buffer
The solution was dissolved at a concentration of, and dispensed into a 96-well microplate (manufactured by Coaster Co., Ltd.), and allowed to stand at 37 ° C for 15 hours in a saturated humidity constant temperature room to prepare a plate solid phase. Phosphate buffered saline (PB
Bovine serum albumin (BS) prepared at a concentration of 3% in S)
After blocking with A), the culture supernatant was added to the wells,
Incubated at 37 ° C for 2 hours. 0.025 for each well
Biotin-conjugated anti-human HG dissolved in PBS-Tween20 after washing 3 times with PBS containing% Tween20 (PBS-Tween20)
F antibody was added and incubated at 37 ° C for 2 hours. P
After washing 3 times with BS-Tween20, Horse Radish Peroxid
An ase-labeled streptavidin was added, the mixture was incubated at 37 ° C. for 1 hour, and washed again with PBS-Tween20 three times. 50 mM citric acid, 100 mM sodium phosphate, 2.5 mg / m
An enzyme reaction was initiated by adding a color former consisting of l-o-phenylenediamine and 0.015% H ₂ O ₂ . After the reaction was stopped by adding 1.5 M sulfuric acid, the absorbance at 490 nm was measured. The result is shown in FIG. In FIG. 2, (A): Carbon tetrachloride administration
(●), partial hepatectomy (○), hepatic ischemia treatment (▲), (B): Rats subjected to treatments of hemi nephrectomy (△), mercuric chloride administration (■), and kanamycin administration (□). The result is shown. As shown in FIG. 2, indulin activity was greatly increased after 3 hours of each treatment and reached the maximum after 6 to 12 hours.

Experimental Example 3 According to the method of Experimental Example 1, mRNA of MRC-5 cells caused by tissue injury healing factor in serum of carbon tetrachloride-administered rat.
The change in expression over time was examined. 10% FC of MRC-5 cells
The cells were cultured in SMEM-containing DMEM until the cell density reached 80 to 90%, and the medium was replaced with a medium containing FCS-free DMEM supplemented with normal rat serum and rat serum 12 hours after carbon tetrachloride administration. After 3 to 48 hours, sample the medium and
RNA was extracted by the GPC method, and Northern hybridization was performed according to the method of Experimental Example 1 to obtain HGF mRNA.
The expression level of was monitored over time. As a control, an example in which the medium is replaced with serum-free medium is shown. The result is shown in FIG. As shown in FIG. 3, HGF mRNA of MRC-5 cells
It was confirmed that the expression level was extremely increased after 3 hours, and was decreased again and then increased again 24 hours later.

Experimental Example 4 The following experiment was carried out in order to investigate the indulin activity in human serum. Serum inducin activity, HGF, during surgical treatment of two patients diagnosed with liver cancer
The change in production-inducing activity was followed. Four days before surgery 14
Serum was collected from the patient several times until the day after, and the indulin activity was measured according to the method of Experimental Example 2 using MRC-5 cells. FIG. 4 shows the results. As shown in FIG. 4, inducin activity was 2 to 2.5 immediately after surgery.
It doubled, then gradually decreased, and returned to the normal value in about 2 weeks.

Experimental Example 5 The indulin activity present in various pig organ extracts was measured by the following method. Each of the excised pig organs was added with 5 times amount (5 ml / g. Sample) of 1 M acetic acid (pH 3.5) per weight, and homogenized for 2 minutes at 0.degree. C. using a Polytron homogenizer. The disrupted product was centrifuged at 100,000 xg for 1 hour, and the resulting supernatant was neutralized to adjust the pH to 7.0. After centrifugation at 100,000 xg for 20 minutes, the supernatant was dialyzed against PBS and filtered through a 0.22 µm mesh filter. The obtained extract was subjected to MR according to the method of Experimental Example 2.
Indulin activity was measured by C-5 cell HGF production stimulating activity. The protein concentration was measured using a BCA protein assay kit (Pierce Chemical Co.). Table 1 shows the results. As shown in Table 1, the indulin activity per protein was high in the cerebellum, lung and cerebrum, and the total activity per organ was significantly higher in the lung.

[0022]

Example 1 In accordance with the method of Experimental Example 1, the physical properties of the tissue injury healing factor indulin in the serum of liver injury rat were examined. Serum of a normal rat and serum of a normal rat after 70% hepatectomy 3 hours after surgery were used as samples. To investigate the acid stability of this factor, add 1M acetic acid to adjust the pH to 3.5 and
After incubating at 12 ° C. for 12 hours, centrifugation was performed at 100,000 × g for 1 hour, and the supernatant was subjected to the following test. Next, in order to examine the stability against heat, the sample subjected to the above-mentioned acid treatment was heat-treated at 100 ° C. for 5 minutes, filtered through a 0.22 μm mesh filter, and then the filtrate was subjected to the following test. The sample was ultrafiltered using an Amicon YM10 membrane filter to determine whether the molecular weight was 10 kD or more, and the solution that passed through the filter (including those with a molecular weight of less than 10 kD) remained in the filter (molecular weight of 10 kD Each of these solutions (including the above) was subjected to the following tests.

2 ml each of normal serum, acid-treated serum, acid- and heat-treated serum, and a filtrate and residue obtained by filtering 2 ml of normal serum with a 10 kD filter were injected into the peritoneal cavity of a normal rat, and 6 hours later, the lung was excised. RNA extraction, purification, and Northern hybridization were performed according to the method of Experimental Example 1. The result is shown in FIG. As shown in FIG. 5, acid treatment,
The heat treatment did not attenuate the indulin activity, revealing that the indulin of the invention is acid and heat resistant and does not pass through a filter with a molecular weight of 10 kD.

Example 2 Injulin, a tissue injury healing factor of the present invention, was purified from the serum of a carbon tetrachloride-administered rat by molecular sieve chromatography. (1) Carbon tetrachloride was inoculated into the abdomen of the rat, and 15 hours later, serum was collected. The obtained serum was adjusted to pH 3.5 with formic acid, stirred at 4 ° C. for 2 hours, and then filtered with a 0.22 μm mesh filter. 20 ml of the obtained acid-treated serum was equilibrated with 100 mM ammonium formate (pH 3.5) to obtain Biogel P-
It was applied to a 60 (manufactured by Bio-Rad) column (19.6 cm ² x 60 cm). Separated into 8 ml fractions, the obtained fractions were added to the cell culture medium, so after freeze-drying,
Dissolve in 50 mM HEPES-NaOH buffer (pH 7.4),
It was sterilized by filtration. Inulin activity was quantified using MRC-5 cells according to the method of Experimental Example 2. The result is shown in FIG. As shown in FIG. 6, it was revealed that indurin was eluted in fractions Nos. 43-60.

(2) In order to measure the molecular weight in more detail,
Sephadex G-150 obtained by collecting the active fraction of indulin obtained in the above (1) and equilibrating it with 10 mM HEPES-NaOH buffer (pH 7.2) containing 0.2 M NaCl.
It was applied to a column (5.3 cm ² x 90 cm, manufactured by Pharmacia). As a molecular weight marker, aldolase (158kD),
BSA (67 kD), chymotrypsin A (25 kD) and ribonuclease A (14 kD) were used and shown in the figure. Fractions were separated by 6 ml, and the indulin activity was measured for the even-numbered fractions obtained. The activity was measured according to the method of Experimental Example 2 by measuring the HGF production stimulating activity of MRC-5 cells. FIG. 7 shows the result. As shown in FIG. 7, the indulin of the present invention is about 10,000 to 30,000 (10 kD-
It was revealed that it showed a molecular weight of 30 kD).

Example 3 In order to examine the physical properties of the cytotoxic healing factor indullin of the present invention, various treatments were carried out using the inducin active fraction obtained in Example 2. To check the heat resistance,
Two types of heat treatment were performed at 60 ° C. for 5 minutes and 100 ° C. for 5 minutes. To check acid resistance, use acetic acid at pH 5.0, pH 3.5,
The pH was adjusted to 1.0 with hydrochloric acid. As trypsin treatment, trypsin was added to 100 μg / ml to the indulin fraction with a protein concentration of 470 μg / ml,
Incubated at ℃ for 3 hours. The enzymatic reaction was stopped by adding soybean-derived trypsin inhibitor at a concentration of 200 μg / ml. To examine the stability against reducing agents, dithiothreitol was added at a concentration of 1 mM. As the heparinase treatment, heparinase was added to the inducin fraction having a protein concentration of 470 μg / ml so as to be 0.31 mU / ml, and the mixture was incubated at 37 ° C. for 1 hour, and the enzymatic reaction was stopped by adding 1M hydrochloric acid. Using the sample treated as described above, the MRC-5 cell HGF production stimulating activity was examined by comparison with the untreated inducin fraction according to the method of Experimental Example 2. The results are shown in Table 2. As shown in Table 2,
The indulin of the present invention is stable to heat, acid and reducing agent,
In addition, since it was inactivated by trypsin treatment and was not affected by heparinase, it was assumed that at least the part responsible for the activity is not a heparin-like polysaccharide but a protein.

[0028]

Example 4 The indulin of the present invention was further purified, and in order to determine the molecular weight in more detail, the indulin fraction was subjected to electrophoresis. The indulin active fraction obtained in Example 2- (2) was purified by ion exchange FPLC, reverse phase HPLC, preparative SDS-PAGE and then 10-2 under non-reducing conditions.
Electrophoresis was performed on a 0% gradient polyacrylamide gel. The following five proteins were used as molecular weight markers. Phospholipase b (94 kD), BSA (67 kD), ovalbumin (43 kD), soybean-derived trypsin inhibitor (21 kD), lysozyme (14 kD). After the completion of SDS electrophoresis, the gel was cut into small pieces and crushed with a Teflon homogenizer. Put each gel piece into a test tube and
After adding S, the mixture was shaken at 4 ° C. for 15 hours. After centrifugation at 1,000 xg for 20 minutes, BSA was added to the supernatant to give a final concentration of 25 μg / ml.
It was adjusted to become. The extract obtained is purified water at 4 ° C 12
Dialyzed for 10 hours, freeze-dried and then containing 0.15M NaCl 10
It was dissolved in mM HEPES-NaOH buffer (pH 7.2).
BSA was added to a final concentration of 4 mg / ml, cold ethanol (-20 ° C) was added, and the mixture was incubated on ice for 30 minutes. Centrifuge at 15,000 xg for 10 minutes and collect the sediment for 10
It was dissolved in mM ammonium acetate and lyophilized again. Freeze-dried product containing 10 mM HEPES-Na containing 0.15 M NaCl
It was dissolved in an OH buffer solution (pH 7.2) and subjected to indulin activity measurement. The inducin activity was measured by the MRC-5 cell HGF production stimulating activity according to the method of Experimental Example 2. FIG. 8 shows the result. As shown in FIG. 8, it became clear that the indulin of the present invention has a molecular weight of about 10,000 to 20,000. Also, the molecular weight is about 40,000 to 60,000 (40kD-60
Indulin activity was also observed in the kD) fraction.

Example 5 Indulin in human serum was obtained by purification by the following method. Serum obtained from a patient diagnosed with liver cancer obtained in Experimental Example 4 was subjected to Biogel P-6 according to the method of Example 2.
0, purified by Sephadex G-150, followed by ion exchange FPLC, reverse phase HPLC, according to the method of Example 4.
Purified by preparative SDS-PAGE, the resulting inducin active fractions were electrophoresed on a 10-20% gradient polyacrylamide gel under non-reducing conditions. Gel slices were cut according to the method of Example 4, and the inducin active fraction was purified by using the HGF-inducing activity of MRC-5 cells as an index to obtain the tissue injury healing factor of the present invention, indulin.

[Brief description of drawings]

FIG. 1 shows the results of tracing the expression of HGF mRNA in the lung by collecting the serum of a rat in which liver injury was caused by partial hepatectomy and ischemia, and injecting it into the abdominal cavity of a normal rat.

FIG. 2 shows the results of measuring sera of liver injury and renal injury rats over time and measuring inducin activity by inducing HRC expression of MRC-5 cells. In the figure, (A): carbon tetrachloride administration (●), partial hepatectomy (○), hepatic ischemia treatment (▲),
(B): Shows the results of rats subjected to the treatments of single nephrectomy (Δ), mercuric chloride administration (■), and kanamycin administration (□).

FIG. 3 shows the time course of HGF mRNA expression level when carbon tetrachloride-administered rat serum was added to an MRC-5 cell culture system. After agarose gel electrophoresis, it was detected by Northern hybridization.

FIG. 4 shows the results of measuring the indulin activity by inducing HGF expression on MRC-5 cells by collecting human serum subjected to liver surgery over time. ○ and ● indicate two patients, and ◇ indicates serum (normal serum) of a healthy person.

FIG. 5 shows the results of measuring the indulin activity when the serum of a rat subjected to partial hepatectomy was subjected to physicochemical treatment. (A) shows the results of HGF mRNA measurement when acid / heat treatment was carried out and (B) was carried out when ultrafiltration was carried out.

FIG. 6: Biogel P- from carbon tetrachloride-administered rat serum
The result of having isolate | separated indulin by 60 column chromatography is shown. ● indicates the measured value of indulin activity, ○
Indicates the absorbance at 280 nm.

FIG. 7 shows the results of separation of indurin from the active fraction after separation by Biogel P-60 column by Sephadex G-150 gel column chromatography. ● indicates the measured value of indulin activity, and ○ indicates the absorbance at 280 nm.

FIG. 8: SDS using active fraction after gel filtration
-Shows the results of separation by PAGE. ● indicates the measured value of inducin activity of the gel extract, and the numerical value in the figure indicates the elution position of the molecular weight marker.

Claims (7)

[Claims] 1. A hepatocyte growth factor (Hepatocyto G)
Rowth Factor, HGF) Injurin, a tissue injury healing factor having an activity of promoting HGF production in cells. 2. The tissue injury healing factor according to claim 1, which is derived from human or animal tissues or blood components, indulin. 3. The tissue injury healing factor, indulin according to claim 1, which has a characteristic of increasing in response to injury of human or animal tissue. 4. The tissue injury healing factor, indulin according to claim 1, which retains its activity by the treatments 1) to 4) below and is inactivated by the treatment 5). 1) Heat treatment at 60 ℃ for 5 minutes or 100 ℃ for 5 minutes. 2) Acid treatment at pH 1.0, 3.5 and 5.0. 3) Reduction treatment of 1 mM dithiothreitol. 4) Heparinase treatment at 0.31mU / ml at 37 ° C for 1 hour. 5) Trypsin treatment at 100 µg / ml at 37 ° C for 3 hours. 5. The treatment of Biogel P-60 and Sephadex G-150 with each gel column is performed at 10 kD to 10 kD.
The tissue injury healing factor according to claim 1, which has a molecular weight of 30 kD,
Indulin. 6. The tissue injury healing factor, indurin according to claim 1, which exhibits a molecular weight of 10 kD to 20 kD by SDS polyacrylamide gel electrophoresis under non-reducing conditions. 7. The injulin, a tissue injury healing factor according to claim 1, which exhibits a molecular weight of 40 kD to 60 kD by SDS polyacrylamide gel electrophoresis under non-reducing conditions.