JPH09188697A - Platelet increasing factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a physiologically active substance acting on a megakaryocyte-platelet system and having activities to promote the differentiation and maturation of megakaryocyte and promote the formation of platelet. SOLUTION: This platelet increasing factor contains the amino acid sequence of the formula Xaa Gly Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile (Xaa is an unidentified amino acid) in the molecule and is useful as an active component of a pharmaceutical composition for the treatment of thrombocytopenia. The physiologically active substance is useful as an active component of an agent for the treatment or prevention of thrombocytopenia caused by chemotherapy and marrow transplantation, thrombocytopenic purpura and various diseases accompanying bleeding tendency and suspected to be caused by thrombocytopenia.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active substance (platelet proliferating factor) having an activity of acting on megakaryocyte cells, promoting their differentiation and maturation, and promoting the production of platelets,
And a pharmaceutical composition for treating thrombocytopenia containing the physiologically active substance as an active ingredient. The physiologically active substance (platelet proliferating factor) of the present invention acts on the megakaryocyte-platelet system,
Since it has the activity of promoting its differentiation and maturation and promoting the production of platelets, thrombocytopenia and thrombocytopenic purpura associated with chemotherapy and bone marrow transplantation, and various diseases showing a bleeding tendency that is thought to be caused by thrombocytopenia It is particularly useful in the medical field as an active ingredient of therapeutic agents and preventive agents for

[0002]

2. Description of the Related Art In blood, which is an essential medium for somatic cells constituting a living body, red blood cells, white blood cells,
There are blood cells such as lymphocytes and platelets, and the blood cells share their own functions and play a role of constantly maintaining the living body. By the way, elucidation of the substance such as differentiation, maturation, and proliferation in vivo of the blood cells has been a long-standing research topic in the field of hematology, but in recent years, various blood cells are Differentiation and maturation from one type of multi-functional hematopoietic stem cell in
It was clarified that various in vivo humoral factors are involved in the process of differentiation and maturation.

From these facts, the in vivo humoral factor is expected to be applied to pharmaceuticals such as therapeutic agents for diseases associated with reduction of blood cells, and so far, for example, erythropoietin, G-CSF. , GM-CSF, M-CSF,
Various humoral factors such as interleukins have been found, and some of them have been actually applied as pharmaceuticals having an action of promoting differentiation and maturation of blood cells such as erythroid system, leukocyte system and lymphocyte system. There is.

[0004] By the way, platelets are non-nucleated cells having a diameter of 2 to 3 µm existing in blood, and are a kind of formed elements in blood that play an important role in hemostasis and formation of thrombus in a living body. The platelets, from the multi-functional hematopoietic stem cells in the bone marrow to megakaryocyte blasts through megakaryocyte progenitor cells, further mature megakaryocyte cytoplasm is produced by fragmentation, it is revealed that released into the blood ing.

Recently, various research results on the megakaryocyte-platelet system have been reported. For example, I
It has been reported that L-6 has an action of promoting maturation of megakaryocytes, which are progenitor cells of platelets [Toshiy
uki Ishibashi, et al. , Pro
c. Natl. Acad. Sci. USA. , Vol.
86,5953-5957 (1989), and Tosh.
iiuki Ishibashi, et al. , Bl
good. , Vol. 74, 1241-1244 (198)
9)].

Further, according to the studies conducted so far, it is considered that there are two kinds of factors having different actions in forming a megakaryocyte colony from bone marrow cells [Will].
iams, N.M. , Et al. , J. et al. Cell Phy
siol. , 110 , 101 (1982)]. That is, as the factor, a megakaryocyte colony stimulating factor Meg-CSF that alone forms a megakaryocyte colony and a factor that does not form a megakaryocyte colony by itself are
Megakaryocyte amplification factor M having the activity of increasing the number of megakaryocyte colonies or promoting its maturation in the presence of eg-CSF
The presence of egg-POT has been reported.

[0007] For example, as one having Meg-CSF activity in human, IL-3 [Teramur
a, M. , Et al. , Exp. Hematol. ,
16 . 843 (1988)] and granulocyte / macrophage colony stimulating factor [Teramura, M. et al. , Et
al. , Exp. Hematol. , 17 , 1011
(1989)], c-Mp1 ligand [de Sau.
vage F. J. , Et al. , Nature, 36
9 , 533 (1994), Kaushansky
K. , Et al. , Nature, 369 , 568
(1994)] and the like are reported. In humans, Me
Those having g-POT activity include IL-6 [Te
ramura, M .; and Mizoguchi,
H. , Int. J. Cell Cloning, 8 , 2
45 (1990)], IL-11 [Teramura,
M. , Et al. , Blood, 79 , 327 (19
92)], and erythropoietin [Bruno, E .; ,
et al. , Blood, 73 , 671 (198).
9)] etc. have been reported.

However, most of these are not factors that act specifically on the megakaryocyte-platelet system, but rather act on other hematopoietic cells or cells other than the hematopoietic system to express their activity. Are known. Therefore, temporarily
When these substances are administered as pharmaceuticals in the expectation of their action on the megakaryocyte-platelet system, it is feared that they may exhibit an activity other than the activity. That is, for example, the IL-6 has a wide variety of actions in addition to the above actions, and one example thereof is that it is deeply involved in inducing inflammation as an acute phase reaction protein in vivo. As is suggested by the fact that the IL-6 is known, it is feared that when the IL-6 is used as a drug as it is, it has strong side effects.

From these facts, the factor acting on the megakaryocyte-platelet system has a high activity of specifically acting on the megakaryocyte-platelet system and promoting its differentiation and maturation. It was important to find out the physiologically active substance possessed, and there was a strong demand in the art to develop such a physiologically active substance.

[0010]

Under these circumstances, the present inventors act on the megakaryocyte-platelet system, promote their differentiation, maturation and / or proliferation, and promote the production of platelets. As a result of intensive research aimed at finding a new physiologically active substance having activity, the activity was found in the culture supernatant of KHM-5M cell line derived from an undifferentiated thyroid cancer patient, HUT78 cell line derived from a patient with Cerisey's disease, etc. In addition to finding the presence of a substance having the following, from the culture supernatant, using the activity against the megakaryocyte-platelet system as an index, a target physiologically active substance is collected, purified, isolated, and its properties are clarified. It succeeded in doing so and came to complete the present invention.

An object of the present invention is to provide a novel physiologically active substance (platelet proliferating factor) which acts on the megakaryocyte-platelet system, promotes its differentiation and maturation, and promotes the production of platelets. To do.

Another object of the present invention is to provide a novel physiologically active substance (platelet proliferating factor) which acts on the megakaryocyte-platelet system and has an activity of promoting acetylcholinesterase production.

It is another object of the present invention to provide a pharmaceutical composition for treating thrombocytopenia, which comprises the physiologically active substance (platelet proliferating factor) as an active ingredient.

Further, the present invention is a pharmaceutical composition comprising the physiologically active substance as an active ingredient, which is used for treating or preventing diseases associated with thrombocytopenia or diseases associated with platelet dysfunction. It is intended to provide an effective pharmaceutical composition.

[0015]

The present invention which achieves the above-mentioned object is constituted by the following technical means (1) and (2). (1) the following properties; act on rodent megakaryocytic cells and have an activity to promote acetylcholinesterase production; have an activity to promote megakaryocyte differentiation and maturation in vitro; by SDS-PAGE The measured molecular weight is about 42 kD; the molecule has the following amino acid sequence Xaa Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile (Xaa indicates that the amino acid is not specified); A thrombocytosis factor characterized by:

(2) A pharmaceutical composition for treating thrombocytopenia, which comprises the thrombocytosis factor described in (1) above as an active ingredient.

[0017]

DETAILED DESCRIPTION OF THE INVENTION The contents of the present invention will be described in detail below. Novel bioactive substance (platelet increasing factor) of the present invention
Is characterized by acting on the rodent megakaryocyte-platelet system and promoting the production of acetylcholinesterase (hereinafter sometimes referred to as AchE).
Since the acetylcholinesterase is an enzyme produced with the differentiation and / or maturation of rodent megakaryocytic cells, the activity of promoting AchE production is the physiologically active substance of the present invention. It shows that it acts on the megakaryocyte-platelet system.

As described above, the physiologically active substance of the present invention has an activity on the megakaryocyte-platelet system. The activity on the megakaryocyte-platelet system here means the differentiation and maturation of megakaryocytes or their progenitor cells. It means that it has an activity to promote or to promote the production of platelets in the process of producing platelets from megakaryocytes.

Next, in order to measure the activity of the physiologically active substance of the present invention on the megakaryocyte-platelet system, for example, bone marrow cells or megakaryocyte cells are used, and the substance to be tested (sample) is used.
Is used as a suitable method for measuring the appearance of proteins and enzymes specific to megakaryocytes and platelets by acting on these cells.

Since rodent megakaryocyte cells produce acetylcholinesterase as they differentiate and mature, for example, the number of cells producing AchE can be measured by staining the cells or the produced AchE. Measuring activity with a spectrophotometer [Toshiro Nagasa
wa et al., Journal of Japan Society of Hematology, Volume 49, 1688-169.
5 page (1986)] and the like, the activity of the physiologically active substance on the megakaryocyte-platelet system can be measured. As will be described later, the physiologically active substance of the present invention, as a result of measuring its activity by the measurement method, acts on megakaryocyte cells and has an activity of promoting acetylcholinesterase production, and its differentiation, Promotes maturity,
It was found to have an activity of promoting the production of platelets.

Next, the physiologically active substance of the present invention is SDS-
PAGE (SDS-polyacrylamide gel electrophoresis)
The molecular weight measured by is about 42 kD. In general, the molecular weight of a physiologically active substance is determined by gel filtration or S
It is measured by a molecular weight measurement method such as DS-PAGE, and the measured value may slightly vary depending on the type of the measurement method, the type of carrier or molecular weight marker used for the measurement, the measurement conditions, etc. As described above, the active substance has a molecular weight as measured by SDS-PAGE.
It is about 42 kD. In this case, as described later, as a molecular weight marker, Pharmacia LMW kit
E (Pharmacia) 14.4kD, 20.1k
Measurements were made using D, 30 kD, 43 kD, 67 kD and 94 kD molecular weight markers.

Next, the physiologically active substance of the present invention is characterized by having the following amino acid sequence in the molecule. The amino acid sequence is experimentally identified by a protein sequencer as described later. (Sequence) Xaa Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile (Xaa indicates that the amino acid is not specified)

The physiologically active substance of the present invention is preferably derived from the KHM-5M cell line derived from the undifferentiated thyroid cancer patient, the HUT78 cell line derived from the patient with Cerisey's disease, and the like. Its origin is not limited to this, for example, the culture supernatant of human-derived cells capable of producing the physiologically active substance of the present invention, those obtained from human body fluids such as urine, the physiologically active substance of the present invention It may be produced by genetic engineering using a gene. As will be described later, in the specific description of the present invention, the KHM-5M cell line and the HUT78 cell line are used, and those obtained from the culture supernatant of the KHM-5M cell line or the HUT78 cell line are used. It is disclosed as a suitable example.

Next, the method for producing the physiologically active substance of the present invention will be described. The physiologically active substance of the present invention can be efficiently produced by the following steps, for example. (1) Culturing a KHM-5M cell line or the like. (2) The culture supernatant of the KHM-5M cell line or the like is collected. (3) The culture supernatant is concentrated with an ultrafiltration membrane. (4) Purify according to the following. DEAE-Sepharose FF ion exchange chromatography 1st reverse-phase high performance liquid chromatography 2nd reverse-phase high performance liquid chromatography Gel filtration SDS-PAGE In the present invention, the production method by the step is mentioned as a suitable one. The method is not limited, and a manufacturing method in which other steps are added to these steps, if necessary, may be appropriately used.

Next, each of the above steps will be described. The step (1) of culturing the KHM-5M cell line or the like is appropriately carried out depending on the culture conditions under which the cells can grow. That is, the culture is carried out at 37 ° C. using a medium containing serum or growth factors such as fetal calf serum or insulin at concentrations suitable for growing the KHM-5M cell line or the like. As a medium, a commonly used DMEM (Dulbecc
o's Modified Eagle's Medi
um), IMDM (Iscove's Modify)
d Dulbecco's Medium) and RP
A preferable medium is a medium such as MI-1640. After culturing the cells under conditions suitable for their growth,
It is preferable to transfer to a condition suitable for recovery of the physiologically active substance of the present invention, for example, a completely synthetic medium containing no serum or the like and culture.

It is also possible to culture the cells using animal solids. That is, it is also possible to transplant the cells into an animal individual, or to culture using the body fluid of the animal in a diffusion chamber attached inside or outside the animal individual. Furthermore, the cells cultured by utilizing individual animals are taken out from the individual animals and dispersed,
It is also possible to culture in an appropriate growth medium. The animal used when culturing the cells using an individual animal can be appropriately used as long as the cells can proliferate, for example, thymectomized or anti-thymocyte antibody-treated mouse, rat, Suitable examples include hamsters, nude mice, nude rats and the like.

Next, the step (2) of recovering the culture supernatant of the KHM-5M cell line or the like is carried out, for example, according to the step (1) above.
The culture supernatant may be recovered from the medium in which the HM-5M cell line or the like has been cultured by an appropriate means. After culturing the KHM-5M cell line or the like, the medium is taken out from the culture vessel and, if necessary, centrifuged. The cells in the medium are separated from the culture supernatant by a means such as filtration, and the culture supernatant is collected. Thus, the culture supernatant contains KH containing the physiologically active substance of the present invention.
The culture supernatant of the M-5M cell line or the like may be a medium containing serum or the like, or an animal body fluid.

Next, the concentration of the culture supernatant with the ultrafiltration membrane of the above (3) is carried out by a conventional method using a commercially available ultrafiltration membrane, whereby the culture supernatant is concentrated to an appropriate level, and the liquid is concentrated. The material may be prepared, and the means therefor is not particularly limited. As the liquid concentrate containing the physiologically active substance of the present invention, the above-mentioned KHM-5M cell line, HUT78 cell line and the like are preferably used, but in addition, the physiologically active substance of the present invention can be produced. Tissue or cell culture supernatant,
A culture medium in which cells or microorganisms capable of producing the physiologically active substance of the present invention are cultured, or a concentrate such as a body fluid containing the physiologically active substance of the present invention can be appropriately used.

Next, the method for purifying the physiologically active substance of the present invention from the thus obtained concentrate of the culture supernatant is not particularly limited, and salting out, ultrafiltration and isoelectric point precipitation are carried out. ,
Many publications such as gel filtration, ion exchange chromatography, hydrophobic chromatography, antibody affinity chromatography, chromatofocusing, adsorption chromatography, reverse phase chromatography, etc.
[Biochemistry Experiment Account 1 Protein Science, edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (1976)], etc., an appropriate method may be selected and appropriately performed. Step (4) is DEAE-Sepha
rose FF ion exchange chromatography, ~
This is a suitable purification step that is a combination of reverse-phase high performance liquid chromatography, gel filtration, and SDS-PAGE. By utilizing this purification step, the physiologically active substance of the present invention can be efficiently purified.

In the step (4), the ion exchange chromatography is DEAE-Sepharos.
e FF ion-exchange chromatography is a reverse-phase high-performance liquid chromatography using Vydac Prot.
Suitable examples are ein C4 RP-HPLC and TSK G3000SWXL GPC for gel filtration. Although the physiologically active substance (platelet proliferating factor) of the present invention is purified in this manner, once it is collected and purified and its properties are elucidated, protein isolation using these properties as an index. It is possible to utilize an appropriate method used for purification.

Further, the physiologically active substance of the present invention can be produced by genetic engineering means. For example, from the KHM-5M cell line of the above-mentioned cell line, etc.
NA was isolated and cDNA was used in a conventional manner using the mRNA.
A library can be created. This cDNA
A DNA probe for screening a library can be designed, for example, based on the partial amino acid sequence revealed by the present invention. Or,
Enzymatically or chemically cleaving the physiologically active substance of the present invention,
After determining the amino acid sequence of the fragment, a DNA probe can be designed based on the amino acid sequence.

Next, after inserting the thus obtained cDNA encoding the physiologically active substance into an appropriate expression vector,
The physiologically active substance of the present invention can be produced by transforming a host with the expression vector and culturing the transformant. Usual hosts such as prokaryotic cells such as Escherichia coli, lower eukaryotic cells such as yeast, and higher eukaryotic cells such as mammalian cells can be used as the host.

The characteristics of the physiologically active substance (platelet proliferating factor) of the present invention produced by the above steps are shown below. (1) Molecular Weight The physiologically active substance of the present invention has a molecular weight of about 42 kD measured by SDS-PAGE. (2) Partial amino acid sequence The physiologically active substance of the present invention has the following amino acid sequence in the molecule. Xaa Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile (Xaa indicates that the amino acid is not specified)

(3) Platelet increasing activity It has an activity of acting on megakaryocyte cells of rodents and promoting the production of acetylcholinesterase. It has an activity of promoting the differentiation and maturation of megakaryocytes in vitro.

The KHM derived from the undifferentiated thyroid cancer patient who was screened by the present inventors and established as a cell line.
-5M cell line is KHM-5M, deposit number: FERM BP-4901, on November 29, 1994, at the Institute of Biotechnology, Institute of Biotechnology, which is a public microbial depository institution.
Has been deposited (international deposit under the Budapest Treaty). Incidentally, as shown in Example 2 described later, the above K
HM-5M has been deposited as a KHM-5M cell line decontaminated with mycoplasma according to the notification from the microorganism depository institution.

Next, the pharmaceutical composition for treating thrombocytopenia of the present invention will be described. The pharmaceutical composition of the present invention is characterized by containing the physiologically active substance (platelet increasing factor) of the present invention as an active ingredient. As the physiologically active substance (platelet proliferating factor), a substance having the amino acid sequence at the N-terminal or an appropriate site in the molecule can be used. The pharmaceutical composition of the present invention can sufficiently exhibit its effect even if the physiologically active substance (platelet proliferating factor) is treated only in a pharmaceutically required step such as freeze-drying or sterilization filtration. As a matter of course, it goes without saying that a pharmaceutically acceptable auxiliary component may be appropriately added to the physiologically active substance to prepare a pharmaceutical product by a conventional method.

The auxiliary components include bases, stabilizers, preservatives, preservatives, emulsifiers, suspending agents, solubilizers, solubilizers, lubricants, corrigents, coloring agents, fragrances, and soothing agents. Examples include agents, excipients, binders, thickeners, buffers, and the like. Specific examples include calcium carbonate, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, and cocoa butter. , Distilled water for injection, sodium chloride aqueous solution, Ringer's solution, glucose solution, human serum albumin (HSA) and the like.

When the pharmaceutical composition of the present invention is prepared using these auxiliary ingredients, for example, a list of pharmaceutical additives (Tokyo Pharmaceutical Industry Association Medical Law Committee and Osaka Pharmaceutical Industry Association Medical Law Research) As described in (Published by the Committee), the auxiliary component may be appropriately selected and used. Further, the amount of the auxiliary component used may be appropriately selected depending on the drug form of the pharmaceutical composition and the like within a pharmaceutically acceptable range.

The dose of the pharmaceutical composition of the present invention is appropriately determined according to the patient's condition, age, sex, body weight and the like. In addition, the administration method is appropriately selected from various administration methods such as oral administration, intramuscular administration, intraperitoneal administration, intradermal administration, subcutaneous administration, intravenous administration, intraarterial administration, and rectal administration according to the patient's condition. To be done.

The pharmaceutical composition comprises thrombocytopenia and thrombocytopenic purpura associated with chemotherapy and bone marrow transplantation, various diseases showing bleeding tendency which may be caused by thrombocytopenia, and functions of megakaryocytes and / or platelets. It is useful as a therapeutic or preventive drug for diseases accompanied by abnormalities.

[0041]

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited thereto. In the following description, a part of the conventional abbreviations used in this field is used.

Example 1 (1) Subculturing of human-derived KHM-5M cell line The human-derived KHM-5M cell line is an established cell line derived from an undifferentiated thyroid cancer patient as described above. The subculture of the cell line was performed by the following method. First, 1
RPMI-1 containing 0% inactivated fetal calf serum (FCS)
The human-derived KHM-5M cell line was inoculated into 100 ml of 640 medium, and cultured at 37 ° C. for 3 days in a culture flask (Falcon) having a bottom area of 175 cm 2.

After completion of the culture, the medium is removed and RPMI-
After washing twice with 1640 medium, about 2 to 5 ml of EDTA-containing trypsin solution was added to detach the cells and 10%.
RPMI-1640 medium containing FCS was added to prepare a cell suspension, which was cultured in a culture flask at 37 ° C. Thereafter, subculture was performed in the same manner.

(2) Culture of KHM-5M Cell Line and Preparation of Culture Supernatant The KHM-5M cell line was prepared with 100 ml of RPMI-1640 medium containing 10% inactivated fetal bovine serum to give a bottom area of 17
5 square centimeter culture flask at 37 ° C, 5%
It was cultured for 3 days under the condition of CO ₂ and humidity of 100%. After completion of the culture, the medium was removed, the cells were washed 3 times with IMDM medium, and further cultured for 3 days using 100 ml of IMDM medium. The culture supernatant was collected and centrifuged at 8000 rpm for 20 minutes, and the supernatant was used as the culture supernatant of the KHM-5M cell line.
It was stored frozen at 20 ° C.

(3) Purification of physiologically active substance (platelet proliferating factor) of the present invention 1) Concentration by ultrafiltration membrane About 20 L of the above culture supernatant was thawed at room temperature, and the ultrafiltration membrane (Asahi Medical Co., Ltd.) was used. Manufactured by PAN 1200 and Amicon, PM-10) and concentrated to 500 to 1000 ml, and the concentrate was frozen and stored.

2) DEAE-Sepharose FF
Ion exchange chromatography The above concentrated culture supernatant was dialyzed against 20 mM Tris-HCl buffer pH 7.4. This was adsorbed on a column of DEAE-Sepharose FF (Pharmacia) equilibrated with the same buffer, and eluted by gradually increasing the concentration of NaCl to 250 mM and 1000 mM. The following purification was performed on the fractions eluted with 250 mM NaCl.

3) Vydac Protein C4
RP-HPLC Next, the fraction eluted with the above 250 mM NaCl was
Vydac Protein C4 (manufactured by Vydac)
Fractionation was performed by reversed-phase high performance liquid chromatography using the column described in 1. That is, it was adsorbed on a column equilibrated with a 0.1% trifluoroacetic acid solution containing 40% acetonitrile at a flow rate of 8 ml / min, and eluted by linearly increasing the concentration of acetonitrile to 80% in 60 minutes.
Figure 1 shows the results of elution by the high performance liquid chromatography.
Shown in As a result of measuring the activity of the obtained fraction, the fraction No. The activity was recognized in 19-21.

4) 2nd Vydac Protein
C4 RP-HPLC The above active fraction was again subjected to the second Vydac Pro.
Fractionation was performed by reversed-phase high performance liquid chromatography using a column of tain C4 (manufactured by Vydac). That is, it is adsorbed on a column equilibrated with a 0.1% trifluoroacetic acid solution containing 40% acetonitrile at a flow rate of 1 ml / min, and the concentration of acetonitrile is 80% in 40 minutes.
Increased linearly until. The elution result of the second high performance liquid chromatography is shown in FIG. As a result of measuring the activity of the obtained fraction, the fraction No. The activity was observed in 7-9.

5) TSK G3000SWXL GPC Next, the above active fractions were respectively added to TSK G300.
Gel filtration was performed using a column of 0SWXL (manufactured by Tosoh Corporation). Gel filtration was performed on a column equilibrated with a 0.1% trifluoroacetic acid solution containing 40% acetonitrile at a flow rate of 0.25 ml / min. As a result of measuring the activity of the obtained fractions, the fraction No. The activity was recognized in 6-9. As an example, 2nd Vydac P
protein C4 RP-HPLC fraction N
o. The result of fractionating 8 with TSK G3000SWXL GPC is shown in FIG.

6) SDS-PAGE For each fraction of TSK G3000SWXL GPC above, SDS-PAGE was correlated with activity.
In E, a silver-stained band having a molecular weight of 42 kD was recognized. To confirm that this protein is the active substance, SDS-
PAGE was performed to extract from the gel. That is, 2nd Vydac Protein C4 RP
-HPLC fraction no. 7 for TSK G3000
Fraction No. fractioned by SWXLGPC. Non-reducing conditions (4% SDS, 20% glycerol, 0.
Add 125 M Tris-HCl buffer (pH 6.8) to 100
Immediately after the treatment at 3 ° C for 3 minutes, the solution was mixed with the sample at a ratio of 1: 1) to perform SDS, and SDS-PAGE mi was performed.
Ni gel (manufactured by TEFCO, 10 T%) was used for electrophoresis at constant current (concentrated gel: 10 mA, separation gel: 15 mA).

As the molecular weight marker, 14.4 kD, 20.1 kD, 3 of LMW kit E manufactured by Pharmacia Co.
0 kD, 43 kD, 67 kD and 94 kD were used. After electrophoresis, slice the gel into 2mm intervals and cut with 0.1% BSA.
The protein was eluted by placing it in IMDM medium (1 ml) containing The activity was measured by filtering with a pore size 0.2 μm filter (New Stella Disk, manufactured by Kurabo Industries). As a result, as shown in FIG. 4, the activity was recognized around the molecular weight of 42 kD (42-45 kD). From this, the silver-stained band observed at a molecular weight of 42 kD was considered to be the active substance.

(4) Activity (action on megakaryocyte cells)
1) AchE Assay (acetylcholinesterase assay) Preparation of cell suspension for activity measurement C57BL / 6Cr mouse (6 to 10 weeks old) (Japan SL
C. Bone marrow cells were collected from femurs and tibias (manufactured by K.K.) and suspended in 10% FCS / IMDM medium. After removing bone fragments etc. by filtration with a mesh diameter of 100 μm, sow it in a petri dish for cell culture, 37 ° C, 5%
It was left to stand for 90 minutes under the condition of CO ₂ . The cells attached to the dish are removed, and 1/10 amount of K is added to the non-adherent cell suspension.
AC2 solution (manufactured by Otsuka Assay Laboratories) was added and incubated at 37 ° C. for 1 hour with stirring every 10 minutes.

This non-adherent cell suspension was layered with 10 ml per 4 ml of Lympholite M solution, and centrifuged at 25 ° C. at 1,500 rpm for 30 minutes using a centrifuge. The non-phagocytic cells present at the interface between the Lympholite M solution and the medium were collected and centrifuged at 2,000 rpm for 5 minutes to collect the cells as a precipitate. The non-adherent and non-phagocytic cells thus obtained were diluted with 0.1% BSA / IMDM medium to
Washed twice.

Measurement of activity The sample for activity measurement was diluted with 0.1% BSA / IMDM. A 96-well microplate was prepared, and the above sample was added to the experimental group, and 0.1% BSA / IMDM medium was added to the control group at 100 μl / well. Next, 10% DFP (Diis
oppropyl Fluoro Phosphate)
0.8 × 1 in IMDM medium containing 10% of treated FCS
Suspend at ⁶ cells / ml and add 100 μl
Each well was added and the cells were cultured at 37 ° C. for 4 days.

After the completion of the culture, the plate was centrifuged at 1,500 rpm for 10 minutes to remove 150 μl of the supernatant from each well, and instead, 150 μl of PBS (−) adjusted to pH 7.2 was added. Next, 3.15 mg / ml
DTNB / 1% Na citrate solution in 1% Triton
Dilute 10 times with X-100 solution, add 50 μl / well of each, and stir with a plate mixer.
nm and 690 nm were measured as the absorbance at 0 min.

Next, 20 μl / well of 2.17 mg / ml iodinated acetylthiocholine solution was added and reacted at room temperature for 30 minutes or 60 minutes.
And the absorbance difference at 690 nm is measured (30 min or 60
Absorbance at min), ΔOD (30 min or 60
The difference between the absorbance at min and the absorbance at 0 min) was obtained.
In addition, DTNB is 5.5'-dithiobis- (2
-Nitrobenzoic acid). When the ΔOD is obviously higher than that of the control group, it is assumed that the acetylcholinesterase activity is induced,
It was determined that there is an action on platelet-type hematopoiesis.

Results Using the protein eluted from the gel around 42 kD obtained in 6) of (3) above as a sample, AchE Assay was carried out by the method described above. As a result, it was confirmed that the acetylcholinesterase activity was higher than that of the control group.

(5) Determination of amino acid sequence 1) Identification method The second reverse phase high performance liquid chromatography (V
active fraction (fraction N) obtained with ydac
o. 8) was fractionated by TSK G3000SWXL gel filtration chromatography (manufactured by Tosoh Corporation). Fraction No. in which activity was observed SDS was added to 7 so that the final concentration was 0.04%, and N ₂ gas was blown to concentrate it. The concentrated fraction was neutralized with 0.1 N NaOH and then 1: 1 with a sample treatment solution (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.125 M Tris-hydrochloric acid buffer solution (pH 6.8)). Mix at 100 ℃
Was processed for 3 minutes. The obtained sample is SDS-PA
GE mini (manufactured by TEFCO, 10 T%) was added to the gel, and a constant current (concentration gel 10 mA, separation gel 15 m) was added.
The electrophoresis was performed in A). Post-migration semi-dry blotting device (Bio-rad, TRANS-BLOT SD
The PVDF membrane was stained with Coomassie blue staining solution after transfer of 140 mA for 4 hr using SEMI-DRY TRANSCELL (FIG. 5), (Hirano Hisa, Protein structural analysis for gene cloning, p41-
p71).

This 42 kD band is cut out and model 4
The N-terminal amino acid sequence in the molecule was determined using a 76A protein sequencer (manufactured by Applied Biosystems). That is, PTH released by Edman decomposition
-Standard P which was previously separated by detecting amino acids by UV absorption
Amino acids were identified based on the retention time of TH-amino acids (manufactured by Applied Biosystems).

2) Results As a result, the physiologically active substance of the present invention (platelet increasing factor)
Was confirmed to have the following amino acid sequence at the N-terminus. Xaa Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile 1 5 10 15 (Xaa indicates that the amino acid is not specified)

Example 2 (Mechanism of Production of Physiologically Active Substances of the Present Invention) It was notified that the KHM-5M cell line deposited by the Institute of Biotechnology, Institute of Industrial Technology was infected with mycoplasma. Therefore, in order to clarify the production mechanism of the physiologically active substance of the present invention, the present inventors examined the effect of mycoplasma infection on the production of the physiologically active substance of the present invention (platelet proliferating factor).

(1) Method Other than the method shown below, the same method as in Example 1 was followed. 1) Passage maintenance of cell line and preparation of culture supernatant The cell line was RPMI-containing 10% fetal bovine serum (FBS).
The cells were subcultured and maintained in 1640 medium or IMDM medium. The sample for confirming the production of the present physiologically active substance is F cells
Culture was performed in the presence or absence of BS, and cells were removed by centrifugation, and the supernatant was used.

2) Mycoplasma infection Mycoplasma infected with a cell line is M from Dainippon Pharmaceutical.
Removed with C-210. MC-210 was added to RPMI-1640 medium or IMDM medium containing 10% fetal bovine serum (FBS) to a final concentration of 0.5 μg / ml, and subcultured for 7 to 14 days. After that, MC-2
A cell line from which mycoplasma had been removed was obtained by subculturing several times in a medium to which 10 was not added.

3) Preparation of Peptide Antibody Containing Amino Acid Sequence of N-Terminal Portion of the Present Physiologically Active Substance 19
A peptide consisting of amino acids was synthesized by the MAP method (Sawasdee Technology Co., Ltd.) and used as an antigen. This MAP
The peptide was emulsified with Freund's complete adjuvant and immunized intradermally in rabbits. This rabbit serum was used as anti-Sb serum. A portion of the anti-Sb serum was used after affinity purification with a column on which a partial peptide of the physiologically active substance was immobilized (AF anti-Sb antibody).

4) Detection of the present physiologically active substance The present physiologically active substance was detected by Western blotting using anti-Sb serum or AF anti-Sb antibody. SDS-PA
GE was carried out by partially modifying the method of Laemmli et al. Samples were diluted to the appropriate concentration and mixed 1: 1 with TEFCO sample buffer and treated at 100 ° C for 3 minutes. The molecular weight is estimated by TEFCO molecular weight marker (TEFCO
CO) was used as a standard protein. Electrophoresis is TEFC
Bio-Rad Electrophoresi on an OSDS-PAGEmini (10 T%, 1 mm) gel.
s Buffer system was used, and constant current was 18 mA.
After the electrophoresis was completed, the protein in the gel was washed with Bio-Rad Sem.
PVD using i-Dry Transblot device
Transferred to F membrane. Bjerrum and with SDS
Electrophoresis was carried out for 30 minutes at a constant voltage of 10-20 V in a Schafer-Nielsen transfer buffer system to allow transfer. The standard protein lane was protein stained with CBB R250. In the lane of the sample, anti-Sb serum or AF anti-Sb antibody was used as the primary antibody, and anti-rabbit IgG antibody conjugated with alkaline phosphatase was used as the secondary antibody, and immunoenzymatic staining was performed.

5) Detection of Mycoplasma Mycoplasma PC was used for detection of Mycoplasma.
R Primer Set (STRATAGENE) was used. Genomic DNA from cells is QIA
Prepared with amp BLOOD KIT (QIAGEN).

6) Preparation of Mycoplasma-infected Cell Line As the mycoplasm to be infected, the culture supernatants of infected HUT78 and other cell lines which are expected to be infected with mycoplasma were used. These mycoplasmas were added to the previously decontaminated HUT78 cell line and subcultured several times to obtain infected cell lines.

(2) Examination and Results 1) Effect of Mycoplasma Infection on Production of the Bioactive Factor in KHM-5M Cell Line The KHM-5M cell line used in Example 1 was undifferentiated as described above. It is a cell line derived from a thyroid cancer patient, and Mycoplasma infection is Mycoplasma in the KHM-5M cell line.
It was confirmed by plasma PCR Primer Set. However, when the mycoplasma infected with the KHM-5M cell line was removed using MC-210, the physiologically active substance of the present invention could not be detected. This suggests that mycoplasma infection may contribute to the production of this bioactive factor.

2) Production of the present physiologically active factor in various cell lines and mycoplasma infection Therefore, it was examined whether or not the mycoplasma infection contributes to the present physiologically active substance producing cell lines other than the KHM-5M cell line. Among the cell line culture supernatants examined, the only cell line in which this physiologically active substance could be detected was HUT78. This HUT78 is a T cell line derived from peripheral blood of a patient with Sézary Syndrome (Cesery's disease). Mycoplasma infection caused by Mycoplasma P in this cell line
It was confirmed by CR Primer Set. Furthermore, KH
As in the case of the M-5M cell line, when the infected mycoplasma was removed using MC-210, the physiologically active substance in the culture supernatant could not be detected. HU
T78 is available as a commercial product (ATC
C TIB 161) is the HUT78 used in this Example 2.
Was somehow mycoplasma contaminated during subculture.

3) Production of the present physiologically active factor by infecting mycoplasma From the above results, in the cell lines producing the physiologically active substance of the present invention, both KHM-5M derived from an undifferentiated thyroid cancer patient and HUT78 derived from a Cezery's disease patient It became clear that it was infected with mycoplasma. Moreover, in all cases, when mycoplasma was removed, a specific band could not be detected by Western blotting. From these results, it was estimated that mycoplasma infection is involved in the production of the physiologically active substance. On the other hand, since other cell lines infected with Mycoplasma do not produce this physiologically active substance, it was considered that the type of Mycoplasma or the type of cell line contributed. Therefore, H from which mycoplasma was removed
The UT78 cell line was infected with mycoplasma infected with HUT78 or mycoplasma infected with another cell line, and whether or not the present physiologically active substance was produced was analyzed by Western blot.

First, the infection of these cell lines with Mycoplasma was confirmed by Mycoplasma PCR Prime.
Confirmed using rSet. FIG. 6 shows the result. In the figure, 1 is HUT78 decontaminated from mycoplasma,
2 is mycoplasma-infected HUT78, 3 is HUT78 reinfected with mycoplasma derived from another cell line, and 4 is HU
HUT78 reinfected with T78-derived mycoplasma,
5 is a 100 bp Ladder marker (Pharma
Cia). In both the HUT78 cell line-derived mycoplasma and the HUT78 cell line infected with other cell line-derived mycoplasma, a band specific to mycoplasma was observed by PCR. But,
Since the bands had different sizes, it was presumed to be different types of mycoplasma. Mycoplasma derived from this HUT78 cell line is Mycoplasma fer
It showed a migration pattern similar to that of mentans. Next, when the production of the physiologically active substance in these culture supernatants was analyzed by Western blot, a specific band was detected in the culture supernatant of cells infected with mycoplasma derived from the HUT78 cell line, It could not be detected in the supernatant of cells infected with mycoplasma derived from other cell lines. FIG. 7 shows the result. In the figure, 1 is HUT78 decontaminating mycoplasma, 2 is HUT78 infected with mycoplasma, 3 is HUT78 reinfected with mycoplasma derived from another cell line, 4 is HUT78 reinfected with mycoplasma derived from HUT78, and 5 is a molecular weight marker ( Manufactured by TEFCO), respectively. From this,
It was presumed that specific mycoplasma infection contributed to the production of this physiologically active substance.

[0072]

The physiologically active substance of the present invention is a factor having a thrombocytosis activity which acts on the megakaryocyte-platelet system,
It has the activity of promoting the differentiation and maturation of megakaryocyte cells and promoting the production of platelets. Further, the pharmaceutical composition of the present invention containing the physiologically active substance as an active ingredient, thrombocytopenia and thrombocytopenic purpura associated with chemotherapy or bone marrow transplantation, various bleeding tendencies considered to be caused by thrombocytopenia It is useful as a therapeutic or preventive drug for diseases.

[0073]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Characteristic symbol: peptide Location: 1. . 16 Method of characterization: E sequence Xaa Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile 1 5 10 15

[Brief description of the drawings]

FIG. 1 shows a monitoring chart of reversed-phase high performance liquid chromatography of Example 1 of the present invention.

FIG. 2 shows a second high-performance liquid chromatography monitoring chart of the reversed phase system of Example 1 of the present invention.

FIG. 3 is a TSK G3000SWX of Example 1 of the present invention.
The elution pattern of the gel filtration by L is shown.

FIG. 4 shows the results of measuring the activity by extracting the protein after SDS-PAGE in Example 1 of the present invention.

FIG. 5: PVD after SDS-PAGE of Example 1 of the present invention
The pattern which transferred to F membrane and was stained with protein is shown.

FIG. 6: Mycoplasma PCR Primer
The result of having detected Mycoplasma of each cell line by Set is shown.

FIG. 7 shows the results of detecting the production of the physiologically active substance of the present invention in the culture supernatant of each cell line by Western blotting.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technology display location (C12P 21/00 C12R 1:91) (72) Inventor Masahito Higuchi 1-135 Komamon, Gotemba, Shizuoka Prefecture Chugai Pharmaceutical Co., Ltd. Inside the company

Claims (2)

[Claims] 1. The following properties; (1) it has an activity of acting on rodent megakaryocyte cells and promoting the production of acetylcholinesterase; (2) promoting the differentiation and maturation of megakaryocytes in vitro. (3) The molecular weight measured by SDS-PAGE is about 42 k.
(4) having the following amino acid sequence Xaa Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys Glu Lys Asp Ile (Xaa indicates that the amino acid is not specified) in the molecule. And a thrombocytosis factor. 2. A pharmaceutical composition for treating thrombocytopenia, which comprises the thrombocytosis factor according to claim 1 as an active ingredient.