WO1993006853A1

WO1993006853A1 - Cell damage repairing factor

Info

Publication number: WO1993006853A1
Application number: PCT/JP1992/001286
Authority: WO
Inventors: Toshikazu Nakamura; Kunio Matsumoto
Original assignee: Toshikazu Nakamura
Priority date: 1991-10-04
Filing date: 1992-10-05
Publication date: 1993-04-15
Also published as: JPH083195A

Abstract

A cell damage repairing factor which can promote repair of damages to the tissues and organs of a living organism. This factor (indulin) originates in a human or animal tissue or blood component, increases in response to the extent of damages to a human or animal tissue, and has an activity of promoting the production of a hepatocyte growth factor (HGF) in a HGF-producing cell. Since the repairing factor of the invention promotes the production of HGF having an activity of repairing tissue damages, it is useful as a tissue damage repairing agent.

Description

Description Tissue injury healing factor Technical field

TECHNICAL FIELD The present invention relates to a tissue injury healing factor, and more specifically, to promote the production of HGF by Hepatocyto Growth Fact or (HGF) -producing cells derived from arbor or animal tissue or blood components. Regarding substances. Background art

HGF is a protein found by the present inventors as a factor that allows mature liver parenchymal cells to proliferate in vitro from regenerated liver rat serum (Biochem Biophys Res Commun, 122, 1450, 1984). The present inventors have further succeeded in isolating HGF from rat platelets (Proc. Natl. Acad. Sci, 83, 6489, 1986, FFBS Letter, 22, 311, 1987), and Partially decided. Furthermore, the present inventors cloned human and rat-derived HGF cDNA based on the elucidated HGF amino acid sequence, and recombined the cDNA into animal tissues to obtain hepatocyte growth factor as a protein. (Human HGF: Nature, 342, 440, 1989; Rat HGF: Proc. Natl. Acad. Sci, 87, 3200, 1990).

The molecular weight of HGF is 82-85 kD by SDS-polyacrylamide gel electrophoresis. Rat HGF molecules have a heterodimer structure in which an α chain consisting of 463 amino acid residues and a / 9 chain consisting of 233 amino acid residues are bridged by a single disulfide bond. Each chain has two glucosamine-type sugar chain binding sites. Human HGF also has almost the same physiological activity, and is composed of an α chain composed of 463 amino acid residues and a ^ chain composed of 234 amino acid residues. The α-chain has four kringle structures similar to the fibrinolytic enzyme plasmin, and the / 9-chain amino acid sequence has about 37% homology with the plasmin Β chain having serine protease activity. Rat HGF The homology between the amino acid sequences of human and human HGF has a very high homology of 91.6% in the α chain and 88.9% in the ^ chain, and their activities are completely compatible.

HGF, which was discovered as a factor that specifically proliferates hepatocytes, has been shown to show various activities in vivo based on recent research results by the present inventors and other researchers. Expectations are rising not only for application to humans and animals but also to therapeutic drugs.

The present inventors have achieved several inventions by elucidating that HGF acts not only on hepatocytes but also on epithelial cells widely as a growth factor. In Japanese Patent Application No. 15881041Z1990, HGF promotes the proliferation of proximal tubule cells of the kidney. In application no. 4 191 588 X 199, HGF promotes the proliferation of normal epithelial cells such as melanocytes and keratinocytes, and is used as a therapeutic agent for epidermal cells to treat wounds, skin ulcers, and hair roots. He achieved the development of application to cell growth agents and disclosed the details. In particular, HGF- is suitable for practical use because it does not have the canceration activity and cancer cell proliferation activity found in many other growth factors such as EGF. Furthermore, the present inventors have disclosed in Japanese Patent Application No. 140812/1991 that cancers such as HepG2 cell line derived from human liver cancer of HGF, and IΜ9 cell line derived from lymphoblastoid cancer. It disclosed that it can be used as an anticancer agent by utilizing its cell growth inhibitory activity.

An even more important point in considering the practicality of HGF as a drug is that HGF promotes growth only in cells that have entered the G1 phase, that is, the growth phase, and has an effect on cells in the GO phase, that is, the stationary phase. That is not to do. This means that the growth and regeneration of damaged tissue is promoted, but has no effect on undamaged tissue. Therefore, even if HGF is excessively administered, or -HGF reaches non-affected areas via blood or the like, it is considered that it does not induce canceration in normal tissues or cause excessive proliferation. It is possible.

As described above, HGF promotes the proliferation of epithelial cells as well as hepatocytes, and has the activity of inhibiting the growth of cancer cells. It is expected that healing works on tissue injury. HGF-producing cells are not epithelial cells themselves, but are mesenchymal in the liver, such as Kuffff cells and sinusoidal vascular endothelial cells in the liver, capillary endothelial cells in the kidney, and alveolar macrophages in the lung It has been elucidated that it is produced by cells, and it has been revealed that the so-called paracrine mechanism, in which HGF is supplied from neighboring cells as needed, has been established.

However, when liver and kidneys are injured, HGF production is also increased in non-injured organs such as the lungs, so it is considered that HGF is supplied by the so-called endocrine mechanism. This fact means that there is a substance that transmits production promotion from the injured tissue to HGF-producing cells.

In other words, substances that promote HGF production are secreted from injured tissues, reach HGF-producing cells via blood, etc., and release HGF stored in the cells or start new production. It is thought to have a function.

HGF is of course administered for the purpose of wound healing and renal regeneration, but if a substance that promotes HGF production is administered, the same effect is expected to be obtained with a smaller dose and frequency of administration. . More specifically, compared with the case of directly administering HGF as a treatment for tissue injury, administration of an HGF production-promoting substance can maintain the blood concentration of HGF for a longer period of time. It is expected that the amount and frequency of administration can be reduced. On the other hand, living organisms are constantly affected by physical, chemical, or microbial injuries, whether external or internal, in all tissues and organs, including the respiratory system, skin, and digestive system. On the other hand, recruiting all functions, including cell growth factors such as HGF, FGF, EGF, and PDGF, to perform repair work on functional tissue cells of organs and matrix cells' is one important homeostasis. As a factor, it has been thought that there is a central factor in the tissue that activates and regulates the compensation network.

The tissue injury healing factor is useful not only for studying the compensation function of organ injury such as liver and kidney and for studying the mechanism of HGF expression, but also for any tissue of the living body, It is a factor that cures organ damage and is obviously very useful as a therapeutic agent. For example, in the case of the liver, in the case of organ injury due to hepatectomy or hepatitis, administration of HGF can regenerate only hepatic parenchymal cells, while administration of a tissue injury healing factor that controls the overall repair function of organ injury This accelerates regeneration of not only parenchymal cells but also non-parenchymal cells, such as supporting tissues such as peri-sinusoidal connective tissue, and the entire site of injury, thereby promoting true repair.

In other words, if a tissue injury healing factor can be used as a therapeutic agent, the injury can be treated much more mildly and promptly compared to the conventional use of various cell growth factors used alone. However, its usefulness is immense.

The most efficient way to track and elucidate the tissue injury healing factor is to make the production promoting activity of cell growth factor Merckmar the most efficient.In fact, the HGF production promoting substance is a tissue injury healing factor. It must be thought of as the main body.

The present inventors have found that the blood of rats artificially injured to the liver or kidney has the effect of stimulating HGF-producing cells and increasing their production ability. The wound healing factor was named "Indulin" (for convenience, indulin is used as the name of the tissue wound healing factor in this specification). Furthermore, as a result of detailed examination using the amount of HGF mRNA or HGF produced as an indicator, the inventors established a purification method for indulin and elucidated its molecular weight and physicochemical properties, thereby completing the present invention.

In other words, laparotomy was performed on a normal rat, the liver was injured by 70% partial hepatectomy or partial ligation of the portal vein to cause liver injury, and blood was collected over time, and the abdominal cavity of the normal rat was collected. When injected downward, it was found that the expression level of HGF mRNA in the lung was significantly increased from 6 to 18 hours (see Experimental Example 1).

Similarly, in vitro, 70% partial hepatectomy, administration of carbon tetrachloride, and HGF production assay using human embryonic lung-derived fibroblast MRC-5 cell line were performed. It was confirmed that the blood of rats that had liver injury due to hepatic ischemia treatment or kidney injury due to hemi-nephrectomy, mercury chloride administration, or kanamycin administration had the effect of promoting HGF production (see Experimental Example 2). ). From these results, it was anticipated that an agent that promotes the production of HGF in response to the injury was present in the blood of an animal having an injury to the liver or kidney.

The present inventors have developed a biogel P-600 and Cefadettas G-150 molecular sieve gel column using the serum of a carbon tetrachloride-administered rat as a raw material, as well as ion-exchange FPLC, reverse-phase HPLC, The active substance was successfully purified using SDS-PAGE or the like (see Examples 2 and 4). The molecular weight of the obtained tissue injury healing factor indulin was measured in more detail by SDS-PAGE and subjected to various physicochemical treatments to examine the stability (see Examples 3 and 4). Disclosure of the invention

The present invention

1. Promote HGF production in hepatocyte growth factor (HGF) -producing cells, which are derived from human or animal tissues or blood components and have characteristics that increase in response to human or animal tissue damage. Injurin, an active tissue injury healing factor.

2. Injurin, a tissue injury healing factor that retains its activity by the following treatments 1) to 3) and is inactivated by the treatment of 4).

1) Heat treatment at 60 ° C for 5 minutes or 100 ° C for 5 minutes.

2) H 1.0, 3.5, 5.0 acid treatment.

3) 0. Reduction treatment of ImM dithiothreitol.

4) lOOAg / ml, trypsinization for 3 hours at 37 ° C.

3. Injurin, a tissue injury healing factor that exhibits a molecular weight of 10 kD to 30 kD when treated with each of gel columns of Biogel P-600 and Sephadex G'-150.

4. Indulin, a tissue injury healing factor that exhibits a molecular weight of 10-20 kD by SDS polyacrylamide gel electrophoresis under non-reducing conditions. 5. Indulin, a tissue injury healing factor with a molecular weight of 40 kD to 60 kD on SDS polyacrylamide gel electrophoresis under non-reducing conditions.

To review. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the results of tracing the expression of HGF mRNA in the lungs of serum from rats that had liver injury due to partial hepatectomy and ischemic conditions over time and injected into the peritoneal cavity of normal rats. Show.

FIG. 2 shows the results of time-sequential collection of serum from liver injury and kidney injury rats and measurement of indulin activity by induction of HGF expression in MRC-15 cells. In the figure, (A): administration of carbon tetrachloride (g), partial hepatectomy (〇), hepatic ischemia treatment (盍;), (B): heminephrectomy (Δ), administration of mercuric chloride (（), The results of rats treated with kanamycin administration (mouth) are shown.

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

FIG. 4 shows the results of time-course sampling of human serum subjected to liver surgery and measurement of indulin activity by induction of HGF expression in MRC-5 cells. 〇 and 示し indicate two patients, and ◊ indicates the serum (normal serum) of a healthy person. FIG. 5 shows the results of measurement of indulin activity when physicochemical treatment was performed on serum from rats subjected to partial hepatectomy. (A) shows the results of HGF mRNA measurement when acid-heat treatment was performed, and (B) shows the results of HGF mRNA measurement after ultrafiltration.

FIG. 6 shows the results of separating indulin from the serum of rats fed with carbon tetrachloride by biogel P-60 column chromatography.會 indicates the measured value of the insulin activity, and 〇 indicates the absorbance at 280 nm.

FIG. 7 shows the results of the separation of indulin from the active fraction after separation on the Biogel P-60 column by Sephadex G-150 gel column chromatography. Hata indicates the measured value of indulin activity, and 〇 indicates the absorbance at 28 Onm. FIG. 8 shows the results of separation by SDS-PAGE using the active fraction after gel filtration. References indicate the measured values of indulin activity of the gel extract, and the numbers in the figure indicate the elution positions of the molecular weight markers. BEST MODE FOR CARRYING OUT THE INVENTION

Methods for obtaining the tissue injury healing factor, indulin, of the present invention include tissue and blood components of animals such as rats subjected to carbon tetrachloride-treated heminephrectomy surgery, or patients with hepatitis, nephritis or immediately after surgery. It can be purified using the tissue or blood components of a mouse as a raw material by the gel filtration method shown in Example 2 or the FPLC or SDS polyacrylamide gel electrophoresis shown in Example 4.

When the indulin activity of various organ extracts of ribta was measured by the in vitro Atsusei 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, indicating that the indulin of the present invention was accumulated at a high concentration.

Similar activity was observed in rats, puta, and human (see Experimental Example 4) in the atsey system of MRC-5 cells used in this example. It indicates that they are compatible. '

The tissue injury-healing factor of the present invention, indurin, is useful for healing injuries to tissues and organs of humans and other mammals, and is usually prepared in the form of general pharmaceutical preparations, and Can be administered topically or orally. The pharmaceutical preparation used in the above administration may contain an effective amount of the active ingredient indulin and a pharmaceutically acceptable carrier or excipient.

That is, for example, preparations for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules) And syrups, milks, suspensions and the like. Such preparations are produced by a method known per se, and are used in the field of preparations as carriers or excipients that are practically used. It contains a fixative. For example, carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate and the like.

Formulations for parenteral administration include, for example, dosage forms such as injections, intramuscular injections, and infusions. Such injections are prepared by a method known per se, that is, by dissolving, suspending or emulsifying indulin in a sterile aqueous or oily liquid commonly used for injections. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and the like and other scavengers, and suitable dissolution aids such as alcohol (eg, ethanol) and polyalcohol (eg, Propylene glycol), a nonionic surfactant (for example, polysorbate 80), a preservative, and a soothing agent may be used in combination. Examples of the oily liquid include sesame oil and soybean oil. Benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizer. The prepared injection solution is usually filled in an appropriate ampoule and sterilized by a conventional method such as high-pressure steam sterilization.

The above-mentioned oral or parenteral pharmaceutical preparation is preferably adjusted to a dosage unit form suitable for the dose of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections, and lotions.

The amount of indulin which should be contained in the above-mentioned pharmaceutical preparation is not particularly limited and may be selected in a wide range, but usually 5 to 95%, particularly 10 to 70% is appropriate in the whole composition. .

The administration method of the above-mentioned preparation is not particularly limited, and is administered by a method according to various preparation forms, patient's age, gender and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of an injection, it is administered intravenously, alone or in combination with a normal liquid sampling solution such as glucose or amino acid.If necessary, it is administered alone intramuscularly, intradermally, subcutaneously or intraperitoneally. You. The dosage of the pharmaceutical preparation is appropriately selected depending on the usage, the age of the patient, gender and other conditions, the degree of the disease, and the like. The product should be administered in 2 to 4 divided doses per day Can be.

The present invention will be described in more detail with reference to Examples, but it should not be construed that the invention is limited thereto.

In addition, all rats used in the following experimental examples and examples are mature male normal rats of the Wistar strain (130 to 150 g).

Experimental example 1

A normal rat underwent a 70% partial hepatectomy by laparotomy or partially ligated the portal vein blood vessel to make it ischemic and injured the liver. 2 ml of blood was collected over time from these liver injury rats and administered intraperitoneally to normal rats. As controls, normal rat peripheral blood and physiological saline were administered. Six hours after the administration, the rats were dissected, the lungs were excised, and subjected to HGF mRNA measurement. More specifically, RNA was extracted from the excised lung by the acid-guanidine-phenol-chloroform method (AGPC method), and poly (A) RNA was purified using Oligotex dT-30. After poly _(Α) ΚΝΑ2μ δ denatured with formaldehyde and electrophoresed on a 1.2% Agarosugeru containing 0.7% formaldehyde. After electrophoresis, transferred to Hybond-N filters one, [- P ^a2] - and the 1.4 kb RB C-1 fragment labeled with CT P is high Buridizu.. Bruno, I pre die See Chillon is 20 g / ml salmon sperm DNA, 50% (V Hachiri Horumua Rudehi de, 0.5% (W / V) SDS, 0.9 N a C _{Interview, 50mM N a H 2 P 0} _ 5mM

Using a buffer containing EDTA and 2X Denhardt's solution, the reaction was performed at 42 ° C for 17 hours. 0.36MN a C 1, 20mM N a H 2 P 0 4, 2mM EDTA, 65 ° in a buffer consisting of 0.5% SDS (:, washed twice with for 15 minutes, X-rays films

(Manufactured by Fuji Film).

Figure 1 shows the results. As shown in FIG. 1, it was observed that the expression of HGF mRNA in the lung was promoted in both cases of partial hepatectomy and hepatic ischemia. '

Experimental example 2

The presence of an HGF-inducing substance in the serum of a liver injury or kidney injury rat was confirmed in an in vitro system by the following procedure.

Rats with 70% partial hepatectomy as liver injury rats, administration of carbon tetrachloride (2 mg CGlJg body weight), rats in hepatic ischemia, rats with only one kidney removed as kidney injury rats, rats with mercuric chloride (2 mg HgCl ₄ / g body weight) and kanamycin (O.Smg kanamycin / g body weight) Serum was collected from each rat over time.

Human fetal lung-derived fibroblast cell line MRC-5 cells are 10% fetal serum (

The cells were cultured in Dulbecco's Eagle's medium (DMEM) supplemented with FCS), penicillin 100 IU / IR1, and streptomycin 100 IU / ml.

Indulin activity was measured by quantifying HGF produced by MRC-5 cells. In other words, MRC-5 cells were cultured in a 24-well microplate (manufactured by Koingen Co., Ltd.), and when the cell density reached 80-90% saturation, the medium was replaced with DMEM without FCS. Sample serum was added to each well and the culture was continued for an additional 24 hours. The unit of indulin activity was ImU (1/1000 unit activity), which was the activity that gave the maximum HGF concentration of 50 expressed when an acidic extract of porcine lung was added.

HGF in the culture supernatant of MRC-5 cells was quantified by enzyme immunoassay (ELISA). An anti-human HGF polyclonal antibody was obtained by immunizing rabbits with recombinant human HGF and purifying IgG from the resulting serum using Protein A Sepharose gel (Pharmacia). The obtained antibody did not cross-react with rat HGF. The anti-human HGF antibody was dissolved at a concentration of 20 iig / ml in a 50-carbonate buffer solution, dispensed into a 96-well microplate (manufactured by Costar), and incubated at 37 ° C and 15 ° C in a constant-humidity chamber with saturated humidity. The plate was allowed to stand for a period of time to prepare a plate solid phase. After blocking with bovine serum albumin (BSA) prepared at a concentration of 3% in phosphate buffered saline (PBS), the culture supernatant was added to the wells and incubated at 37 ° C for 2 hours. Each well was washed three times with PBS containing 0.025% Tween20 (PBSTeen20), and a biotin-conjugated anti-human HGF antibody dissolved in PBS-Tween20 was added and incubated at 37 ° C for 2 hours. After washing with PBS-Tween20 three times, streptavidin labeled with Horse Radish Peroxidase was added, incubated at 37 ° C for 1 hour, and washed again with PBS-Tween20 three times. 50mM Kuen acid, sodium lOOroM phosphate, 2.5uig / na o- phenylene Renjiamin, 0.015% H _z 0 ₂ Was added to initiate the enzymatic reaction. After the reaction was stopped by adding 1.5 M sulfuric acid, the absorbance at 490 nm was measured.

Figure 2 shows the results. In Fig. 2, (A): administration of carbon tetrachloride (parable), partial hepatectomy (〇), hepatic ischemia treatment (▲), (B): heminephrectomy (Δ), administration of mercury chloride (picture), The results are shown for rats treated with kanamycin (mouth). As shown in FIG. 2, indulin activity was very high 3 hours after each treatment and peaked after 6-12 hours.

Experiment 3

According to the procedure of Experimental Example 1, the change over time in the mRNA expression of MRC-5 cells due to the healing factor of the tissue injury in the serum of the rats administered with carbon tetrachloride was examined.

Culture MRC-5 cells in DMEM containing 10% FCS to 80-90% saturation cell density, normal rat serum in DMEM without FCS and rat serum 12 hours after carbon tetrachloride administration The medium was replaced with a medium containing. After 3 to 48 hours, the medium was sampled, RNA was extracted by the AGPC method, and Northern hybridization was performed according to the method of Experimental Example 1 to track the expression level of HGF mRNA over time. As a control, an example in which the medium was replaced with a serum-free medium is shown.

Figure 3 shows the results. As shown in FIG. 3, it was confirmed that the expression level of HGF mRNA in the MRC-5 cells was extremely increased after 3 hours, and was once increased and then increased again 24 hours later.

Experiment 4

The following experiment was performed to examine the indulin activity in human serum. Changes in serum indulin activity, ie, HGF production-inducing activity, were followed during surgical treatment of two patients diagnosed with liver cancer. Serum was collected from the patient several times from 4 days before the operation to 14 days after the operation, and the indulin activity was measured using MRC-5 cells according to the method of the experiment described in Example 2.

Fig. 4 shows the results. As shown in Fig. 4, indulin activity increased 2-2.5 times immediately after the operation, then gradually decreased, and returned to a normal value in about 2 weeks. Experimental example 5

Indulin activity present in various organ extracts of pigs was measured by the following method.

Each of the excised septum organs was added with 5 volumes (5 ml / g-sample) of 1 M acetic acid (pH 3.5) per 1 weight, and homogenized at 0 ° C for 2 minutes using a polytron homogenizer. The crushed material was centrifuged at 100,000 xg for 1 hour, and the obtained supernatant was neutralized and adjusted to pH 7.0. After further centrifugation at 100, OOOxg for 20 minutes, the supernatant was dialyzed against PBS and filtered through a 0.22 μιιι mesh filter. According to the method of Experimental Example 2, the obtained extract was measured for lindulin activity by MRC-5 cell MGF production stimulating activity. The protein concentration was measured using a ΒCA protein assay kit (Pierce Chemical). The results are shown in Table 1. As shown in Table 1, the indulin activity per protein was high in the cerebellum, lung, and cerebellum, and the total activity per organ was significantly higher in the lung.

table 1

Example According to the method of Experimental Example 1, the physical properties of the tissue injury healing factor indulin in the serum of the liver injury rat were examined.

Serum from a normal rat and serum 3 hours after the operation of a normal rat with a 70% hepatectomy were used as samples. To examine the stability of this factor against acid, add 1M acetic acid to pH 3.5, incubate at 4 ° C for 12 hours, centrifuge at 100,000 xg for 1 hour, and discard the supernatant as follows. Tested. Next, in order to examine the stability to heat, the acid-treated sample was heat-treated at 100 ° C for 5 minutes, filtered through a 0.22 μιη mesh filter, and the filtrate was subjected to the following test. .

The sample was ultrafiltered using an Amicon YM10 membrane filter to check 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) and remained in the filter 1 (Including those having a molecular weight of 10 kD or more) were subjected to the following tests.

Normal serum, acid-treated serum, acid and heat-treated serum, 2 inl each, and 2 ml of normal serum, 10 kD-filtered filtrate and residue were injected into the peritoneal cavity of a normal rat, and after 6 hours, the lungs were removed and the experiment was performed. According to the method of Example 1, RNA extraction, purification and Northern hybridization were performed.

Figure 5 shows the results. As shown in FIG. 5, the indulin activity was not attenuated by the acid treatment and the heat treatment, and it became clear that the indulin of the present invention was resistant to acid and heat and did not pass through a filter having a molecular weight of 10 kD. Was.

Example 2

The tissue injury healing factor indiulin of the present invention was purified from serum of a rat to which carbon tetrachloride had been administered by molecular sieving chromatography.

(1) The rat abdomen was inoculated with carbon tetrachloride, and serum was collected 15 hours later. The resulting serum was adjusted to pH 3.5 with formic acid, stirred at 4 ° C 'for 2 hours, and filtered through a 0.22 μιη mesh filter. 20 ml of the obtained acid-treated serum was applied to a Biogel P-60 (manufactured by Biorad) column (19.6 cm ² × 60 cm) equilibrated with 10 OmM ammonium formate (PH3.5). Separate fractions by 8 ml each and freeze the obtained fractions for addition to the cell culture. After drying, it was dissolved in 50 mM HEPES-NaOH buffer (ρΗ7.4) and sterilized by filtration.

Indulin activity was quantified using MRC-5 cells according to the method of Experimental Example 2.

Figure 6 shows the results. As shown in FIG. 6, it was found that indulin was eluted in fractions Nos. 43-60.

(2) To measure the molecular weight in more detail, collect the indulin activity fraction obtained in (1) above and equilibrate with 10 mM HEPE S-Na〇H buffer (pH 7.2) containing 0.2 M Na C1. Sephadex G-150 (manufactured by Pharmacia) column (5.3 cm ² × 90 cm). Aldolase (158 kD), BSA (67 kD), chymotribcin A (25 kD), and ribonuclease A (14 kD) were used as molecular weight markers and are shown in the figure.

Fractions were separated by 6 ml each, and 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 activity of stimulating HGF production in MRC-5 cells.

Figure 7 shows the results. As shown in FIG. 7, it was revealed that the indulin of the present invention has a molecular weight of about 10,000 to 30,000 (10 kD to 30 kD). Example 3

In order to examine the physical properties of the cytotoxic healing factor indulin of the present invention, various treatments were performed using the indulin activity fraction obtained in Example 2.

To examine heat resistance, two types of heat treatment were performed at 60 ° C for 5 minutes and at 100 ° C for 5 minutes.

In order to examine acid resistance, the pH was adjusted to 5.0 and 3.5 using acetic acid, and adjusted to pH 1.0 using hydrochloric acid.

As a trypsin treatment, trypsin was added to an indulin fraction having a protein concentration of 470 μ / ιιι1 at a concentration of 100 ig / ml and incubated at 37 ° C for 3 hours. The enzymatic reaction was stopped by adding soybean-derived trypsin inhibitor at a concentration of 200 g / ml.

To examine the stability against reducing agents, dithiothreitol was added to O.lmM. It was added at a concentration.

Using the sample treated as described above, the activity of stimulating the production of HGF in MRC-5 cells was examined in comparison with the untreated indulin fraction according to the method of Experimental Example 2.

The results are shown in Table 2. As shown in Table 2, the indulins of the present invention were stable to heat, acids and reducing agents, and were inactivated by trypsin treatment. Therefore, it was assumed that at least the part responsible for the activity was a protein.

Table 2

Example 4

To further purify the indulin of the present invention and determine the molecular weight in more detail, the indulin fraction was subjected to electrophoresis.

The indulin activity fraction obtained in Example 2- (2) was purified by ion-exchange FPLC, reverse-phase HPLC and preparative SDS-PAGE, and then 10-20% gradient * polyacrylamide gel under non-reducing conditions I did an electric swim. The following five types of 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 completion of SDS electrophoresis, the gel was cut into small pieces and disrupted with a Teflon homogenizer. Each gel piece was placed in a test tube, PBS was added, and the mixture was shaken at 4 ° C for 15 hours. After centrifugation at l, 000xg for 20 minutes, BSA was added to the supernatant. , _R

1 6

Was added to adjust the final concentration to 25 gAil. The obtained extract was dialyzed against purified water at 4 ° C for 12 hours, freeze-dried, and then dissolved in a 10 mM HEPES-NaOH buffer (pH 7.2) containing 0.15 M NaCl. After 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. After centrifugation at 15,000 xg for 10 minutes, the precipitate was collected, dissolved in lOmM ammonium acetate, and freeze-dried again. The lyophilized product was dissolved in a 10 mM HEPES-NaOH buffer solution (pH 7.2) containing 0.15 M NaCl and subjected to indulin activity measurement.

Indulin activity was measured by MRC-5 cell HGF production stimulating activity according to the method of Experimental Example 2.

Figure 8 shows the results. As shown in FIG. 8, it was revealed that the induulin of the present invention has a molecular weight of about 10,000 to 20,000. Indulin activity was also observed in fractions with a molecular weight of about 40,000 to 60,000 (40 kD to 60 kD). Example 5

Indulin in human serum was purified by the following method.

The serum obtained from the patient diagnosed with liver cancer obtained in Experimental Example 4 was purified on Biogel P-60 and Sephadex G-150 according to the method of Example 2, and then the method of Example 4 was performed. Purified by ion-exchange FPLC, reversed-phase HP LC, and preparative SDS-PAGE according to the procedure described above, and the obtained indulin-active fraction was subjected to electrophoresis with a 10-20% gradient polyacrylamide gel under non-reducing conditions. . The slices were cut into gel slices according to the method of Example 4, and the indulin activity fraction was purified using the HGF-inducing activity of MRC-5 cells as an index to obtain the tissue injury healing factor of the present invention, indulin. Industrial applicability

The tissue injury healing factor of the present invention is a novel factor, and the tissue injury healing factor of the present invention has an effect of promoting the healing of injuries to tissues and organs of a living body.

Claims

1. Hepatocyto Growth Factor (HGF) -producing tissue injury healing factor having activity to promote HGF production of cells.

2. Claim 1 derived from the tissue or blood components of the human or animal

Tissue injury healing factor listed in Indulin.

3. The tissue injury healing factor according to claim 1, which has a characteristic of increasing in response to injury of a human or animal tissue, and indulin.

4. The tissue injury healing factor or indulin according to claim 1, which retains viability by the following treatments 1) to 3) and is inactivated by the treatment of 4).

Enclosure

1) Heat treatment at 60 ° C for 5 minutes or 100 ° C for 5 minutes.

2) pH 1.0, 3.5, 5.0 acid treatment.

3) Reduction treatment of O.lmM dithiothreitol.

4) Tryptic treatment for 3 hours at 37 ° C, lOO ^ g / ml.

5. The tissue injury healing factor according to claim 1, which exhibits a molecular weight of 10 kD to 30 kD by the treatment of each of the gel columns of Biogel P-60 and Sephadex G-150, and indulin.

6. The tissue injury healing factor according to claim 1, which exhibits a molecular weight of 10 kD to 20 kD by SDS polyacrylamide gel electrophoresis under non-reducing conditions, indulin.

7. The 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, indulin.