JPH0413696A - New bioactive substance - Google Patents

Abstract

NEW MATERIAL:A bioactive substance containing protein having 87+ or -10 kDa molecular weight measured by SDS-polyacrylamide electrophoresis method in reduced state as constituting protein. USE:Used as a curing drug of a disease requiring reproduction or regeneration of hepatic cell or a disease requiring improvement of hepatic function. PREPARATION:Cell derived from human (e.g. FERM P-11048) is cultured and resultant cultured solution is recovered, then the cultured solution is purified. Besides, in the purification, at least one method selected from anion-exchange chromatography, heparin-affinity chromatography, pigment affinity chromatography and reverse-phase chromatography is preferably used.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a novel physiologically active substance and a method for producing the same. The present invention also relates to a pharmaceutical composition containing the novel physiologically active substance as an active ingredient.

<Prior art> Growth factors for hepatocytes can be obtained using a rat hepatocyte primary culture system established in recent years (Nakamura T. et al., Journal of Biochemistry Vol. 94, 1029-1).
035, 1983). As a result, it was reported that hepatocyte growth factors were present in normal human serum and rat serum, as well as in hepatitis patient serum and rat platelets, including HPTA and HGF, respectively.
(Reza Zarnegar et al., Biochemical and Biophysical Research Communications, Vol. 163, pp. 1370-1376,
1989; Keiji MiyBzawa et al., Biochemical and Biophysical Research Communications, Vol. 163, pp. 967-973, 1
989; and Toshikazu Nakamu
ra et al., Nature vol. 342, pp. 440-443, 1
989), both of which are approximately 70
kDa and approximately 35 kDa subunits (as described by Eiji Miyazawa et al.,
Foshikazu Nakamura et al. (already published),
In addition, TGF, a protein with a molecular weight of approximately 7.4 kDa,
-〇, EGF with a molecular weight of about 6 kDa has also been found to have proliferative activity on hepatocytes. By the way, as you know,
There are many growth factors that have similar effects in vivo, and it is also known that there are multiple different factors that have cell proliferation effects on the same tissue. Liver tissue is the most regenerative tissue in the body, and it is well known that there are many growth factors for hepatocytes, and these growth factors are balanced with growth inhibitory factors, etc. It is thought that it regulates proliferation. Therefore, in order to elucidate the proliferation mechanism of hepatocytes in vivo and apply it to the medical field, it is necessary to discover and identify new substances that are different from the hepatocyte growth factors that have already been identified. It is considered.

<Problems to be Solved by the Invention> An object of the present invention is to provide a novel physiologically active substance and a method for producing the same. The novel physiologically active substance provided by the present invention has a proliferation-promoting effect on hepatocytes.
Another object of the present invention is to provide a pharmaceutical composition containing the novel physiologically active substance as an active ingredient.

Means for Solving the Problems The present inventors isolated a new physiologically active substance from the culture supernatant of a cell line derived from human liver cancer, and determined that this new physiologically active substance has an effect of promoting the proliferation of hepatocytes. The present invention has been completed by confirming that the present invention has the following characteristics.

That is, in the first aspect of the present invention, the molecular weight in SDS-polyacrylamide gel electrophoresis under reduction is 87.
The present invention provides a novel physiologically active substance characterized in that its constituent protein is a protein having a size of ±1 okDa.

The physiologically active substance preferably has a hepatocyte proliferation-promoting effect. Further, the physiologically active substance is preferably a human-derived protein, and particularly preferably a protein produced by human-derived cells.

A second aspect of the present invention provides a method for producing the novel physiologically active substance shown in the first aspect of the present invention, particularly a method for producing the novel physiologically active substance using human-derived cells.

The production method preferably includes the steps of culturing human-derived cells, collecting a culture solution containing the novel physiologically active substance, and purifying the culture solution.

For purification, it is preferable to carry out at least one purification method selected from the following.

(a) Anion exchange chromatography (b) Heparin affinity chromatography (c) Dye affinity chromatography (d) Reversed phase chromatography The heparin affinity chromatography is a heparin affinity chromatography using a hydrophilic polymer adsorbed with heparin, and the dye affinity chromatography is an anion exchange chromatography in which a nal-aminoethyl group is introduced. It is particularly preferable that the dye affinity chromatography is a dye affinity chromatography that uses a hydrophilic polymer to which a dye is adsorbed, and that the reversed phase chromatography is characterized in that the reversed phase chromatography uses a polymer to which an octadecyl group is bonded.

The present invention also provides, as a third aspect, a pharmaceutical composition containing the novel physiologically active substance as an active ingredient.

The configuration of the present invention will be explained in detail below.

A novel physiologically active substance provided as a first aspect of the present invention is characterized in that its constituent protein is a protein having a molecular weight of 87±10 kDa as measured by SDS-polyacrylamide gel electrophoresis under reduction. That is, the physiologically active substance of the present invention has, as at least a portion thereof, a protein that exhibits a molecular weight of 87±10 kDa by SDS-polyacrylamide gel electrophoresis under reduction.

The novel physiologically active substance of the present invention may be any substance having the above-mentioned protein as a constituent protein, but it is particularly preferable to have a hepatocyte proliferation-promoting effect.

Although the origin of the novel physiologically active substance of the present invention is not limited, it is preferably derived from humans.

The novel physiologically active substance of the present invention can be obtained by appropriate means from human-derived tissues and cells that produce it, human body fluids such as human urine and human blood, but preferably from human cancer-derived cells, especially human cancer-derived cells. Preferably, it is produced by cancer-derived cells isolated from human liver, and furthermore,
It is preferably produced by the human metastatic liver cancer-derived cell line SPT-6M. Furthermore, the present inventors have deposited the human metastatic liver cancer-derived cell line SPT-6M, which produces the novel physiologically active substance of the present invention, on October 12, 1989 (Feikoken Bibori No. 11048). (FERM P-1
1048)).

The novel physiologically active substance of the present invention may be produced by any method, but it may be produced by using human-derived cells, preferably human cancer-derived cells, especially cancer-derived cells isolated from human liver. It is preferably produced using the human metastatic liver cancer-derived cell line SPT-6M.

The method for producing a novel physiologically active substance provided as a second aspect of the present invention is characterized in that it utilizes human-derived cells.

The production method of the present invention uses human-derived cells,
Any method may be used as long as the novel physiologically active substance of the present invention can be recovered in the end, but as a preferred example, human-derived cells are cultured and cultured containing the novel physiologically active substance of the present invention. Examples include a method that includes the steps of collecting a liquid and purifying the culture liquid.

Next, a production method using the human metastatic liver cancer-derived cell line SPT-6M, which is a cell line derived from human cancer and is particularly preferably applied to the production method of the present invention, will be described.

The human metastatic liver cancer-derived cell line SPT-6M is cultured under culture conditions that allow it to proliferate. That is, SPT-6
At 37°C in a medium containing serum and growth factors such as fetal bovine serum and insulin at concentrations suitable for growing M.
Cultivate at The medium is, for example, DMEM (Dulbeccoos Mo.
dividedEagle's Medium), R
The medium may be appropriately selected and used from media such as PMI1640.

The novel physiologically active substance of the present invention can be obtained by recovering and purifying the culture supernatant of SPT-6M cultured using a method similar to commonly used cell culture techniques. In order to carry out the process more efficiently, it is preferable to culture the cells under conditions suitable for recovery, for example, by transferring them to a completely synthetic medium that does not contain serum or the like.

Further, the cells can also be cultured using individual animals. That is, the cells can be transplanted into an animal, or cultured using the animal's body fluid in a diffusion chamber installed inside or outside the animal. Furthermore, the cells cultured using an individual animal can be taken out from the individual animal, dispersed, and further cultured in an appropriate growth medium.

Any animal can be used as long as the cells can proliferate, but mice, rats, hamsters, etc. whose thymus has been removed or treated with anti-thymus antibodies, or nude mice or rats should be used. is preferred.

When recovering the novel physiologically active substance of the present invention from a liquid containing the novel physiologically active substance of the present invention, for example, a culture supernatant, a method generally used for purifying proteinaceous physiologically active substances is used. Simply select an appropriate method and combine them; for example, salting out method, ultrafiltration method,
Isoelectric precipitation method, various affinity chromatography such as antibody affinity chromatography, gel filtration chromatography, ion exchange chromatography,
Hydrophobic chromatography chromatofocusing method, adsorption chromatography, reversed phase chromatography,
By selecting a method that does not impair the activity of the novel physiologically active substance of the present invention from methods such as high-performance liquid chromatography, and combining these methods and performing them in an appropriate order, the desired novel substance of the present invention can be obtained. Physiologically active substances can be concentrated and purified.

In the present invention, any of the above methods can be applied, but it is preferable to carry out an appropriate combination of two or more purification methods including at least one of the following purification methods.

(a) Anion exchange chromatography (b) Heparin affinity chromatography (c) Dye affinity chromatography (d) Reversed phase chromatography Note that anion exchange chromatography uses quaternary aminomethyl groups and/or Anion exchange chromatography with a quaternary aminoethyl group introduced is preferable, and heparin affinity chromatography is preferably heparin affinity chromatography using a hydrophilic polymer adsorbed with heparin. , dye affinity chromatography is preferably dye affinity chromatography that uses a hydrophilic polymer adsorbed with a blue dye, and reverse phase chromatography is a reverse phase chromatography characterized by using a polymer to which an octadecyl group is bonded. Phase chromatography is preferred.

The activity of the novel physiologically active substance of the present invention is 1. For example, hepatocyte-
This can be confirmed by measuring the growth-promoting effect on. In recent years, a primary culture system for hepatocytes has been established (Nakamura T.

et al., Journal of Biochemistry 94, 1
029-1035, 1983), the hepatocyte proliferation effect of various substances has been measured using primary cultured hepatocytes collected from rats and using the effect on hepatocyte DNA synthesis as an indicator. The hepatocyte proliferation promoting effect of the novel physiologically active substance of the present invention can also be determined in vitro according to the above method.
The physiologically active substance is applied to hepatocytes at o, and the DNA
The effect on synthesis can be used as an indicator to confirm the effect.

The present invention also provides, as a third aspect, a pharmaceutical composition characterized by containing the novel physiologically active substance of the first aspect of the present invention as an active ingredient.

The pharmaceutical composition of the present invention may be obtained by simply subjecting the novel physiologically active substance shown in the first aspect of the present invention to a pharmaceutically necessary treatment selected from freeze-drying, filter sterilization, etc. , can sufficiently provide its effects to the medical field, but in addition to the above-mentioned physiologically active substances, the pharmaceutical composition can also contain various pharmaceutically acceptable auxiliary ingredients. be.

These auxiliary ingredients include bases, stabilizers, preservatives, preservatives, emulsifiers, suspending agents, solvents, solubilizing agents, lubricants, flavoring agents,
Colorants, fragrances, soothing agents, excipients, binders, thickeners,
It refers to buffering agents, etc., specifically calcium carbonate,
Lactose, sucrose, sorbitol, mannitol, starch.

Amylopectin, cellulose derivatives, gelatin, cocoa butter, distilled water for injection, aqueous sodium chloride solution, Ringer's solution, glucose solution, human serum albumin (HSA), etc., pharmaceutical additives list (Tokyo Pharmaceutical Industry Association Pharmaceutical Affairs and Regulations Committee) and published by Osaka Pharmaceutical Association Pharmaceutical Affairs Law Research Committee). In the present invention, it is possible to appropriately select and use auxiliary ingredients with reference to this list of pharmaceutical additives. The type and amount of the substance to be used may be appropriately selected from a pharmaceutically acceptable range depending on the drug form of the pharmaceutical composition.

The dosage of the pharmaceutical composition of the present invention is appropriately determined depending on the condition, age, sex, weight, etc. of the patient receiving treatment.

The pharmaceutical composition of the present invention can be administered orally, depending on the condition of the patient.
It can be used in various administration methods such as intramuscular administration, intraperitoneal administration, subcutaneous administration, intravenous administration, intraarterial administration, and intrarectal administration, but intravenous administration is particularly preferable. .

The pharmaceutical composition provides an effective means for treating diseases that require proliferation or regeneration of hepatocytes or diseases that require improvement of liver function.

<Examples> The present invention will be explained in more detail below using Examples, but these are shown as examples of implementation, and the present invention is not limited to the same extent by these. Also,
The abbreviations used in the following description are based on common abbreviations in the field.

(Example 1) Production of a novel physiologically active substance ■ Subculture of human metastatic liver cancer-derived cell line SPT-6M The human metastatic liver cancer-derived cell line SPT-6M was subcultured by the following method.

First, Dulbecco's modified Eagle medium (hereinafter referred to as D) containing 10% bovine serum (manufactured by Irvine Scientific) was used.
GIBCO is abbreviated as MEM.

The human metastatic liver cancer-derived cell line SPT-6M was added to 1.6 x 10' cells/50mβ of Life Chiknology Seeds Co., Ltd. 11J).
Plant to a concentration of mI2, base area 150cm"
5% CO in a roof flask (manufactured by Iwaki Glass Co., Ltd.)
The cells were cultured at 37°C for 3 days in the presence of 2. After culturing, remove the medium and add 20ml of trypsin solution containing EDTA.
Add j+ to detach the cells, then add 10% bovine serum.
DMEM medium containing was added to prepare a cell suspension. Collect this cell suspension with a pipette, add DMEM medium containing 10% bovine serum, and adjust the concentration of the cell suspension to 1.6X10.
' cells/mi and cultured again at 37°C in a roof flask. Thereafter, subculture was performed in the same manner.

■Culture of human metastatic liver cancer-derived cell line SPT-6M for recovery of physiologically active substances In order to recover the novel physiologically active substances of the present invention from the culture supernatant of human metastatic liver cancer-derived cell line SPT-6M, the following The human metastatic liver cancer-derived cell line SPT-6M was cultured using the method.

First, DMEM medium containing 10% bovine serum 31! , to,
Human metastatic liver cancer-derived cell line SPT-6M? i-0,6x
The cells were implanted at a concentration of 10' to 0.7 x 10' cells/mβ, and cultured by rotation at 37° C. for 3 days at 10 revolutions for 7 hours using a roller bottle (manufactured by Suzuki Seiko Co., Ltd.) with a capacity of 20 J2. After the culture was completed, the medium was removed, and DMEM medium 3I2 containing 10% bovine serum was newly added, and the cells were further cultured for 2 days at 37° C. with 10 rotations. After the culture was completed, the medium was removed, the inside of the bottle was washed twice with PBS-212, and then serum-free DMEM medium lβ was added and cultured at 37°C for 2 days to produce the novel physiologically active substance of the present invention. I let it happen.

(2) Recovery of the novel physiologically active substance of the present invention from the culture supernatant The novel physiologically active substance of the present invention was isolated from the culture supernatant obtained in (2) by the following method.

First, the culture supernatant obtained in step (3) is passed through a filter with a pore size of 5 μm (polypropylene PP cartridge, manufactured by Millibore) and then through a filter with a pore size of 0.22 μm (Duravore TP cartridge, manufactured by Millibore) to remove cells, etc. Sterilization was performed. This filtrate has a molecular weight cutoff of 20
After passing through a 0 kDa ultrafiltration III (Prostack, manufactured by Millibore), an ultrafiltration membrane with a molecular weight cutoff of 6 kDa (Morsep Phi Hermosul type II FS-
10 membranes (manufactured by Daicel Chemical Industries, Ltd.) for 50 m.
Desalting and concentration were performed using M Tris-HCl buffer (pH 8,0).

Next, the obtained desalted concentrate was heated at 4°C for 50 minutes.
0mM) Q equilibrated with Lis-HCl buffer (pH 8,0)
- Sepharose fast flow column (φ5cm x 24
．． 4 cm, manufactured by Pharmacia) at a flow rate of 600 mβ/hour, and anion exchange chromatography was performed. After washing the column with the same buffer, elution was performed using a 50 mM) Lis-HCl buffer (pH 8,0) containing NaCβ at a flow rate of 980 mI2/h using a linear concentration gradient method with NaCl concentration oM to IM 75 hours. I did it. After the elution was completed, the effect of each fraction on the proliferation of hepatocytes was measured by the method described in Example 2. As a result, NaCj
Hepatocyte proliferation activity was observed at both concentrations of 2, approximately 0.08M to 0.34M.

Next, the obtained active fraction was subjected to heparin affinity chromatography. That is, 50mM Tris-HCl buffer (pH 8,0)/
TS equilibrated at 0,5 M Na CIt
K Gel AF-Heparintotovar 650M column (
115 times the amount of 50 mM Tris-HCl buffer (pH
8.0)/2M NaCl added at a flow rate of 80
m 12/h. After loading, the column was diluted with 50 mM) lithium-HCl buffer (pH 8,0) at 10.
Washed with 5M NaCρ80mff at a flow rate of 80mI2/h. After washing, using 50mM) Lis-HCl buffer (pH 8.0) containing NaCβ, NaCj2 concentration is 0.
5M to 2M/8 hours linear concentration gradient method, flow rate 20m
Elution was performed at β/hour. After the elution was completed, the effect of each fraction on the proliferation of hepatocytes was measured by the method described in Example 2. As a result, the NaCJ2 concentration was approximately 0.5M~
Hepatocyte proliferation activity was observed in the fraction around 1.4M.

The obtained active fraction was subjected to dye affinity chromatography in the following manner. That is, an equal amount of distilled water was added to the above active fraction, and it was added at a flow rate of 60 mg/hour to a TSK Gel Blue SPW column (φ7.5 mm x 75 mm, manufactured by Tosoh Corporation) that had been equilibrated with PBS- beforehand. After addition, PBS-/1.
Wash with 4M NaCl2 (hereinafter referred to as solution A), then add solution A and 10mM Hepes (pH 7,4)/2M
Elution was performed using guanidine hydrochloride (hereinafter referred to as solution B) using a linear concentration gradient method over 72 minutes with a concentration of solution B ranging from 0 to 100%, followed by elution with solution B at a flow rate of 30 mJ2/hour. After the elution was completed, the effect of each fraction on the proliferation of hepatocytes was measured by the method described in Example 2.

The active fraction eluted with solution B was subjected to the following reversed phase chromatography. That is, TSK gel octadecyl-NPR equilibrated with 0.05% trifluoroacetic acid.
Column (φ4.6mm x 35mm, manufactured by Tosoh Corporation)
The above active fraction was added at a flow rate of 60 mβ/hour, and 0.
After washing with 0.05% trifluoroacetic acid at a flow rate of 60 mβ/hour, elution was performed using 0.05% trifluoroacetic acid containing acetonitrile. The elution was performed using a linear concentration gradient method of acetonitrile concentration 0 to 100%/20 minutes at a flow rate of 6.
It was performed at 0 mβ/hour.

After the elution was completed, the effect of each fraction on the proliferation of hepatocytes was measured by the method described in Example 2. As a result, a proliferation-promoting effect on hepatocytes was observed in fractions with acetonitrile concentrations of approximately 46% to 55%.

The obtained active fraction was again applied to a TSK gel octadecyl-NPR column equilibrated with 0.05% trifluoroacetic acid and eluted in the same manner.

After the elution was completed, the effect of each fraction on the proliferation of hepatocytes was similarly measured. As a result, the acetonitrile concentration was approximately 46% to 55%.
A proliferation-promoting effect on hepatocytes was observed in the vicinity.

■ SDS polyacrylamide gel electrophoresis Using a part of the active fraction with high proliferative activity obtained by the method shown in ■, SDS was performed according to the method of Laemmli (Nature vol. 227, p. 680, 1970). Polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE)
) was carried out.

First, the active fraction was dried under reduced pressure overnight, and 2% S
DS10.05% BPB/20% glycerol 15%2
-Mercaptoethanol 762.5mM) was dissolved in Lis-HCl buffer (pH 6,8). After dissolving, at 100℃
Heat treatment was performed for 1 minute, and the entire sample was collected by centrifugation. The collected samples were placed on a 12.5% gel (1 mm x 14
cm x 14 cm) was subjected to SDS-PAGE.

As a molecular weight standard, LMW Kit E (manufactured by Pharmacia) was used, and electrophoresis was performed on IQmA for 4 hours.

After the electrophoresis was completed, staining was performed using a silver staining kit (manufactured by Dai-Kagakuyaku Co., Ltd.). As a result of staining, the molecular weight was 87±10kD.
A band was observed near a.

(Example 2) Measurement of hepatocyte proliferation activity of the novel physiologically active substance of the present invention■ Isolation of hepatocytes from rats First, as the first reflux solution, 0.5mM glycol ether diamine tetraacetic acid (Wako Pure Chemical Industries, Ltd. (manufactured by) /
Hanks' BSS as the second reflux liquid, 0.0
5% collagenase (manufactured by Sigma) 15 m M Ca
”/Ranks' BSS was prepared.

In addition, Hanks' BSS is Ha that does not contain Ca'' or Mg''.
nks 'BSS (Hanks' balance)
d 5alt 5solution, Gibco, manufactured by Life Chikunolose Co., Ltd.) was used.

Next, 7-week-old male Wistar rats were given 0.3 mβ/Neptal (manufactured by Dainippon Pharmaceutical Co., Ltd.) and heparin sodium injection (manufactured by Mochida Pharmaceutical Co., Ltd.), respectively.
were administered intraperitoneally at a dose of 0.2 mβ/individual and 0.2 mβ/individual.

After disinfecting the rat's skin with 70% ethanol, the rat's abdomen was opened. While the first reflux fluid was flowing, a cannula was inserted into the portal vein, and the abdominal caval vein was cut to release the reflux fluid. Next, the chest was incised, a cannula was inserted into the thoracic vena cava, and the cut abdominal vena cava was ligated. After running the first reflux for about 5 minutes, the liquid was exchanged with the second reflux, which was refluxed for about 15 minutes. After reflux, remove the liver into a Petri dish and place the hepatocytes in cold Hanks' B55.
(Already mentioned). This hepatocyte suspension was collected with a pipette, filtered with a 150-mesh cell filter (manufactured by Ikemoto Rikagaku Kogyo Co., Ltd.), and the filtrate was heated at 4°C and 16×g
The mixture was centrifuged for 5 minutes. Discard the supernatant, resuspend the precipitated cells in Ranks' BSS, and incubate again at 4°C at 16 x g.
The mixture was centrifuged for 5 minutes. The same operation was repeated two more times, and hepatic parenchymal cells were collected. The separated hepatic parenchymal cells were divided into 5
% fetal bovine serum (manufactured by Japan Biological Materials Center Co., Ltd.)/
1 μM dexamethacin (manufactured by Sigma) / '0.1 μM insulin (manufactured by Sigma) / 1llilliaa+'
s E medium (manufactured by Flow Laboratory).

■Measurement of growth-promoting effect on rat hepatocytes The active fraction obtained by method (■) of Example 1 was immediately added with 10 times the amount of 0.05% bovine serum albumin (hereinafter abbreviated as BSA, manufactured by Sigma)/PBS- was added. Next, a dialysis tube with a molecular weight cutoff of 3500 (SPECTRA/PO
PBS-
Dialyzed overnight against After dialysis, molecular weight cutoff is 5000
ultrafiltration membrane (Ultrafree CL).

Millibore) and a sterile filtration membrane (pore size 0).
．． It was sterilized by filtration using a 22 μm filter (manufactured by Millibore). This was serially diluted with 0.1% BSA (already mentioned)/PBS- to prepare a sample, and the proliferation-promoting effect of the novel physiologically active substance of the present invention on hepatocytes was measured.

First, the rat hepatocyte suspension prepared in ① was added to a 24-well collagen-coated dish (manufactured by Corning) so that LmQ/well. This dish was placed in a CO□ incubator (manufactured by Tabai Espec Co., Ltd.) and cultured for 5 hours at 37° C. and 5% COt, and then the medium was replaced. For medium exchange, remove the medium from each well and use serum-free Will containing lOK I U/mβ aprotinin (manufactured by Mochida Pharmaceutical Co., Ltd.).
This was carried out by adding 500 μβ of iam's E medium (previously described) to each well. After culturing for another 18 hours, remove the medium again and use 5% fetal bovine serum (already listed).
0.1μM insulin (already mentioned) /William's
E medium (described above) was added to each well at 400 μC.

Then, the experimental group received the above sample and the control group received 0
．． 100 μβ of 1% BSA (already described)/PBS- was added and cultured at 37°C. After 6 hours, each well was given [m
ethyl-”Hl-Thymidine (hereinafter [”H
(abbreviated as lTdR, manufactured by Ama Jam Japan Co., Ltd.) is 0
．． The mixture was added at 5 μCi/well and further cultured for 22 hours. After culturing, remove the medium and wash 3 times with 1 mβ/well of ice-cold PBS-, then add 1 ml of water-cooled 10% trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) to each well.
Cells were fixed by adding 2 mJ each and leaving at 4°C for 15 minutes. After cell fixation, add 0.5N NaOH to each well.
Solubilize the cells by adding 500 μm of
．． The sample was collected in a scintillation counter vial to which 325 μρ of 15N HCA had been added. To this, 3 ml of scintillator (Scintisol EX-H1 manufactured by Dojindo Laboratories Co., Ltd.) was added, and [”H] of each well was measured using a liquid scintillation counter (manufactured by Aloka).
The amount of lTdR was measured.

As a result, the amount of [''Hl TdR in the experimental group was clearly higher than that in the control group, indicating that DNA synthesis was promoted in the cells of the experimental group.

(Example 3) Test on the toxicity of the novel physiologically active substance of the present invention To the active fraction obtained according to method ① of Example 1, 10 times the amount of 0.05% BSA (already mentioned)/PBS- was immediately added. . Next, the membrane was dialyzed against physiological saline overnight using a dialysis tube with a molecular weight cutoff of 3500 (SPECTRA/POR Molecular Borus Membrane, manufactured by Spectrum Medical Induction). After dialysis, it was concentrated using an ultrafiltration membrane (Ultrafree CL, manufactured by Millibore) with a molecular weight cutoff of 5000, and sterilized by filtration using a sterile filtration membrane (pore size 0.22 μm, manufactured by Millibore).

The physiologically active substance solution of the present invention prepared as described above was administered intravenously to a group of 10 ICR mouse mice, and the mice were observed for 2 weeks. Physiological saline was similarly administered to the control group.

As a result, no toxicity was observed in the physiologically active substance of the present invention even when the maximum possible dose was administered to mice.

Therefore, it was proven that the physiologically active substance of the present invention is safe for living organisms, and that the pharmaceutical composition of the present invention containing the physiologically active substance as an active ingredient also has low toxicity for living organisms.

<Effects of the Invention> The present invention provides a novel physiologically active substance and a method for producing the same.

The present invention also provides a pharmaceutical composition containing the novel physiologically active substance as an active ingredient.

Since the novel physiologically active substance provided by the present invention has a proliferation-promoting effect on hepatocytes, a pharmaceutical composition containing the novel physiologically active substance as an active ingredient can be used to treat diseases that require proliferation or regeneration of hepatocytes, or Provides an effective means for treating diseases that require improvement of liver function.

Claims (9)

[Claims] (1) A novel physiologically active substance characterized in that its constituent protein is a protein having a molecular weight of 87±10 kDa as measured by SDS-polyacrylamide gel electrophoresis under reduction. (2) The novel physiologically active substance according to claim 1, wherein the physiologically active substance has an action of promoting proliferation of hepatocytes. (3) The novel physiologically active substance according to claim 1 or 2, wherein the physiologically active substance is a human-derived protein. (4) The novel physiologically active substance according to any one of claims 1 to 3, wherein the physiologically active substance is produced by human-derived cells. (5) A method for producing a novel physiologically active substance, which comprises producing the novel physiologically active substance according to any one of claims 1 to 4 using human-derived cells. (6) The novel method according to claim 5, comprising the steps of culturing human-derived cells, collecting a culture solution containing the novel physiologically active substance according to any one of claims 1 to 4, and purifying the culture solution. Method for producing physiologically active substances. (7) The method for producing a novel physiologically active substance according to claim 5 or 6, wherein at least one purification method selected from the following is carried out. (a) Anion exchange chromatography (b) Heparin affinity chromatography (c) Dye affinity chromatography (d) Reversed phase chromatography (8) The anion exchange chromatography is an anion exchange chromatography in which a quaternary aminomethyl group and/or a quaternary aminoethyl group is introduced as an ion exchange group, and the heparin affinity chromatography is an anion exchange chromatography in which a quaternary aminomethyl group and/or a quaternary aminoethyl group is introduced as an ion exchange group; Heparin affinity chromatography uses an adsorbed hydrophilic polymer, the dye affinity chromatography is dye affinity chromatography using a hydrophilic polymer adsorbed with a blue dye, and the reversed phase chromatography uses octadecyl 8. The method for producing a novel physiologically active substance according to claim 7, which is reversed phase chromatography using a polymer to which a group is bonded. (9) A pharmaceutical composition comprising the novel physiologically active substance according to any one of claims 1 to 4 as an active ingredient.