JPH09313173A - Measurement of megakaryocyte cell projection-forming activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a substance having a megakaryocyte cell projectionforming ability, such as IL-6, in good repeatability and in high sensitivity. SOLUTION: This method for measuring the megakaryocyte cell projection forming activity of a specimen comprises (a) a process for culturing myeloma cells in a serum-free culture medium containing a mpl ligand in a concentration of 0.01-1ng/ml, (b) a process for separating the megakaryocyte prepared in the process (a), (c) a process for bringing the separated megakaryocyte into contact with the specimen and culturing the treated megakaryocyte in a serum- free medium not containing the mpl ligand, and (d) a process for detecting the cell projection-formed megakaryocyte.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention provides a method for producing megakaryocytes, particularly megakaryocytes capable of forming vesicle projections, but which does not substantially cause formation of cysts, and measurement of megakaryocyte projection formation activity. The present invention relates to a method, a method of selecting a substance having megakaryocyte projection process forming activity, a method of selecting a substance having megakaryocyte procession formation inhibitory activity, and a method of measuring the level of platelet production of bone marrow cells.

[0002]

BACKGROUND OF THE INVENTION Platelets play an important role in hemostasis. It is considered that platelets are derived from megakaryocytes differentiated from bone marrow cells, and the megakaryocytes grow and differentiate to cause endoplasmic reticulum formation and release platelets. Studies have been conducted on substances that promote the growth of megakaryocytes, and an mpl ligand has been discovered (1). However, due to the lack of an appropriate measurement system for the formation of ER processes, research has been delayed,
Only a few substances are known to promote the formation of endoplasmic reticulum, and no substance is actually used in medicine.

As a conventional method, a method (2) for separating megakaryocytes from bone marrow of various animals and using them is known.
However, since this method uses mature megakaryocytes that have already been subjected to various stimuli in the living body, more than half of the megakaryocytes will form endoplasmic reticulum projections even in the control without addition of any drug, It is unsuitable as a system for searching for a substance having megakaryocyte projection process-forming activity.

[0004] In order to search for such an ER process stimulating factor, a culture method capable of observing a series of processes in which megakaryocytes are induced from bone marrow cells to cause formation of ER processes is required. Instead, cells obtained by dispersing bone marrow in an appropriate medium are cultured in a medium containing 50 U / ml [ie, about 10 ng / ml (3)] of mpl ligand and serum. The method (4) using the prepared megakaryocytes is performed. That is,
In this method, mouse bone marrow cells were treated with 50 U / ml of mpl.
Induction of megakaryocytes by culturing in Iscove's modified Dulbecco's medium (IMDM) containing ligand, 10% fetal calf serum (FCS) for 1 to 3 weeks, and the induced megakaryocytes are separated, and IMDM containing 5% FCS Cells are cultured for 15 hours to 3 days, and cells that have undergone endoplasmic reticulum formation are observed. However, the value of megakaryocyte projection process forming activity measured by this method has an extremely large error and is extremely poor in reproducibility as a measurement method, and when used for screening a substance having megakaryocyte projection process forming activity. , A situation in which a substance that does not have megakaryoblastic process formation activity was erroneously detected as a substance having activity was also observed.
[(1) Hideaki Mizoguchi, History of Medicine, 174 , 695, 1
995 (2) Leven, R.A. M. , Journalo
f Cellular Physiology, 16
3 , 597, 1995 (3) Frederic, J. et al. d
e, Sauvage et al, NATURE, 36
9 , 533, 1994 (4) Nagahisa, H .;
et al, Blood, 87 , 1309, 1996.

[0005]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention In order to develop a drug for thrombocytopenia and thrombocytosis, to develop a substance that promotes or inhibits the formation of megakaryocyte endoplasmic reticulum, and
Development of a reproducible method to measure a large number of samples in order to diagnose how a substance that promotes or inhibits endoplasmic reticulum formation is involved in diseases that exhibit an increase or decrease in platelets Is required.

[0006]

Means for Solving the Problems The present inventor has conducted extensive studies in order to solve the above problems. First, the present inventor found that the cause of poor reproducibility in the above method (4) is
Since it is presumed that a serum-containing medium is used, the culture in a serum-free medium was examined. This is because serum contains an unknown substance, its composition is not constant, and there is a difference between lots, and generally, in culture using serum, the result may change depending on the lot difference of the serum used.

It is extremely easy to prepare megakaryocytes by culturing bone marrow cells in a serum-free medium containing 10 ng / ml of mpl ligand, and as shown in FIG. 1, a very large number of megakaryocytes were induced. It was confirmed that However, this megakaryocyte is interleukin-6 (IL), which is conventionally known as a substance having megakaryocyte procession forming activity.
Even if they are contacted with (abbreviated as -6), almost no vesicle projections are formed even if they are brought into contact with each other, and it has reached a difficult situation that it does not make any sense as a method for measuring megakaryoblastic projection formation activity.

Therefore, the present inventors presume that there may be some influence on the amount of mpl ligand added, mpl
An experiment was carried out in which the bone marrow cells were cultured in a serum-free medium in which the amount of ligand added was changed. As a result, surprisingly, as shown in FIG. 2, the smaller the amount of mpl ligand added to the medium, the greater the number of megakaryocytes that formed the ER process. On the other hand, however, it was also confirmed that the smaller the amount of mpl ligand added, the smaller the number of megakaryocytes induced from bone marrow cells. From bone marrow cells with a limit to the amount that can be easily obtained, it is necessary to induce an appropriate amount of megakaryocytes that can be used for the measurement of megakaryocyte procession formation activity, and the induced megakaryocytes are at least It is necessary that IL-6, which is known as a substance having megakaryocyte projection process-forming activity, be a megakaryocyte capable of exhibiting reticulum formation of megakaryocytes with good reproducibility and high sensitivity. These issues are mpl
It was confirmed that this can be overcome by setting the addition amount of the ligand within a specific range, and the present invention has been completed. The present invention has been completed based on the epoch-making ideas and experiments as described above.

That is, according to the present invention, bone marrow cells are added in an amount of 0.01 to
A method for producing megakaryocytes, which comprises culturing in a medium containing 1 ng / ml of mpl ligand. In addition, the present invention provides bone marrow cells with 0.01 to 1 ng / ml of mpl.
It is a method for producing megakaryocytes, which is capable of forming ER projections, which is characterized by culturing in a serum-free medium containing a ligand, but does not substantially cause ER projection formation as it is.

The bone marrow cells used in the method for producing megakaryocytes of the present invention are not particularly limited as long as they are cells that can be induced by megakaryocytes, and preferred examples are bone marrow cells of mammals including humans, for example, easily available mouse. As. It may be selected as bone marrow cells by removing mature blood cells (red blood cells, white blood cells, platelets), but isotonic liquid is usually used for the bone marrow of bones where hematopoiesis is actively performed like femurs. It is also easy to use the one dispersed in step 1.
Specifically, extruding bone marrow from the femur of a mouse, 1
Cells are dispersed by pipetting in isotonic solution such as calcium- and magnesium-free Hanks solution containing mM EDTA, or culture medium such as Iscove's modified Dulbecco's medium (IMDM), and the precipitated cells are separated by centrifugation. And a method of preparing the same by dispersing it to an appropriate concentration.

The mpl ligand used for inducing megakaryocytes from bone marrow cells is a factor that binds to mpl receptors expressed on the surface of megakaryocyte progenitor cells and megakaryocytes and promotes growth of megakaryocytes. , Mpl ligand gene-engineered recombinant mpl ligand (Frederic, J. de, Sauvage et al, NATURE, 369, 53
3, 1994), truncated without a C-terminal domain
Type mpl ligand (Tamaki, Wada et al, BIOCHEMICAL
AND BIOPHYSICAL RESERCH COMMUNICATIONS , 213, 1091,
1996), or native m purified from thrombocytopenic animals
pl ligand (Takashi, Kato et al, Journal of Bioc
hemistry, 118, 229 , 1995) and the like, and human TPO manufactured by Peprotech Co., Ltd. in the UK is commercially available as an mpl ligand, so that it is particularly preferable to use it. mpl
The concentration of the added ligand is usually 0.01 to 1 ng / ml, preferably 0.1 to 0.5 ng / ml, particularly preferably about 0.3 ng / ml.

The medium containing mpl ligand may be either serum-free or serum-containing medium, but serum-free medium is preferred, in which case the prepared megakaryocytes are capable of forming endoplasmic reticulum, but as they are. Then, it becomes a megakaryocyte that does not substantially cause the formation of alveolar process. The type of medium is not particularly limited as long as it is a suitable basal medium for animal cells, but Iscove's modified Dulbecco's medium (IMDM) is preferable. When serum is added, it may be serum of mammals such as humans and cows, but fetal bovine serum is preferable. When using a serum-free medium, additives such as minerals, vitamins and proteins generally used for serum-free culture are added to the medium in an appropriate amount. Transferrin and insulin are preferably added, and the concentration thereof may be generally used 0.1-100 μg / ml. More preferably, 5 μg / ml transferrin and 10 μg / ml insulin are added. That is, specifically, 10 μg / ml bovine-derived insulin, 5 μg / ml
ml IMD containing holotype transferrin derived from bovine
M medium is exemplified. The number of culture days varies depending on the type of bone marrow cells and additives, but is 1 to 5% CO ₂ at 37 ° C.
Five days is good, and 2-3 days is more preferable.

Further, the present invention provides (a) bone marrow cells with 0.0
A step of culturing in a serum-free medium containing 1-1 ng / ml of mpl ligand, (b) a step of separating the megakaryocytes prepared by the step, (c) a contact of the separated megakaryocytes with a subject And a step of culturing in a serum-free medium containing no mpl ligand, and (d) detecting a megakaryocyte that has undergone formation of alveolar process. It is a measuring method.

The bone marrow cells (a) of the present invention are added in an amount of 0.01 to 1
The step of culturing in a serum-free medium containing ng / ml of mpl ligand is the same as described above. The step (b) of separating the megakaryocyte prepared in the step (a) is carried out by using a method of separating by using an antibody against a protein specifically expressed on the surface of the megakaryocyte or a difference in specific gravity of cells. Although megakaryocytes can be separated by an appropriate method such as a method of separating cells, preferably, a cell dispersion containing megakaryocytes is layered on a solution in which a concentration gradient of bovine serum albumin (BSA) is formed in balanced salt and allowed to spontaneously precipitate. The method of separating the megakaryocytes is exemplified. More preferably, the cell culture solution is layered on a calcium-magnesium-free Hank's solution containing 16%, 4% and 2% BSA in this order, and the cells are allowed to stand for 1 hour. You can separate it. The megakaryocytes isolated in this manner are megakaryocytes that are capable of forming vesicle projections but do not substantially form vesicle projections as they are.

The separated megakaryocytes are brought into contact with the subject and cultured in a serum-free medium containing no mpl ligand (c) The subject used in the step (c) is not particularly limited, but may be human or animal blood, Urine, cell supernatant,
Fermentation products, or purified products obtained from them, known physiologically active substances to be measured, and the like, do not pretreatment such as purification unless the assay method of the present invention is particularly inhibited Is preferred because it is simple. The contact with the subject is performed together with the culture in the step (c), that is, the method of adding the subject to the serum-free medium containing no mpl ligand used for the culture is the most simple and preferable, but other than that, Prior to substantially culturing, there is a method in which a test subject is contacted with a megakaryocyte in advance, the megakaryocyte is taken out, and the megakaryocyte is placed in a serum-free medium containing no mpl ligand and cultured. . In the case where the subject and the megakaryocyte are brought into contact with each other in advance, of course, an appropriate buffer solution or medium can be used in addition to the subject and the megakaryocyte, but it is preferable that the mpl ligand and the serum are not contained.

The serum-free medium containing no mpl ligand used in step (c) is not particularly limited as long as it is a suitable basal medium for animal cells, but Iscove's modified Dulbecco's medium (IMDM) is preferable, and serum-free medium is generally used. Add appropriate amounts of additives such as minerals, vitamins and proteins used for culture to the medium. Transferrin and insulin are preferably added, the concentration of which is generally 0.1-1.
00 μg / ml is sufficient. More preferably 5 μg / ml
Transferrin and 10 μg / ml insulin are added. The incubator is not particularly limited as long as it is usually used for animal cell culture, but it is usually used for cell culture.
Plates are preferred. In the plate, since it is easy to observe the number of megakaryocytes per hole is about 100 to 1000, it is preferable to set the dispersion liquid to such a level. The number of culture days varies depending on the type of bone marrow cells and additives, but is preferably 1 to 3 days under 5% CO ₂ and 37 ° C. conditions, and more preferably 1 to 2 days.

Detect megakaryocytes that have undergone vesicle formation
In the step (d), for example, the megakaryocytes after culturing in each well of a 96-well plate are observed under a microscope to measure the number of megakaryocytes that have undergone vesicle formation, and to form vesicle formation as a negative control. This is exemplified as a method of comparing the number of megakaryocytes with the number of megakaryocytes that have undergone vesicle process formation when a test subject is added, and measuring the oocyst process forming ability of a sample. It is general that the strength of the vesicle formation activity is expressed as the ratio with the number of vesicle formation megakaryocytes with the addition of the negative control taken as 100%. As a measurable range, for example, IL-6 is usually 10 n.
It can be measured at about g / ml or higher.

Therefore, a particularly preferred specific example of the present invention is as follows. For example, the cells dispersed from the bone marrow of the femur of an easily available mouse are dispersed, and the dispersed cells are used as a medium for megakaryocyte induction, for example, a suitable basal medium, preferably I
Approximately 0.3 ng / ml of human TPO (mpl ligand) manufactured by British Peprotech Co., Ltd. was added to MDM, and if necessary, inorganic substances, vitamins, proteins, etc. used in serum-free culture, especially 5 μg / ml transferrin and 10 μg /
5 in megakaryocyte induction medium supplemented with ml insulin
Incubate for ₂ to 3 days under the conditions of CO ₂ and 37 ° C. After culturing, the induced megakaryocytes are overlaid with a cell dispersion containing megakaryocytes in a solution in which a concentration gradient of bovine serum albumin (BSA) is formed in balanced salt, and naturally precipitated.
Separate megakaryocytes. The separated megakaryocytes thus obtained were placed in a cell culture 96 plate at 100 to 1000 per well.
Individually added, and cultured in a serum-free medium containing no test substance and containing mpl ligand for 1 to 2 days, and after culturing, the megakaryocytes in each hole were observed under a microscope to cause the formation of endoplasmic reticulum. The number of megakaryocytes was measured, and the number of megakaryocytes that had undergone formation of vesicles as a negative control was compared with the number of megakaryocytes that had formed vesicles when the test subject was added. Good.

The present invention also provides (a) bone marrow cells to 0.0
A step of culturing in a serum-free medium containing 1-1 ng / ml of mpl ligand, (b) a step of separating the megakaryocytes prepared by the step, (c) a contact of the separated megakaryocytes with a subject And the step of culturing in a serum-free medium containing no mpl ligand, and (d) the step of detecting megakaryocytes that have undergone vesicle formation, in which the megakaryocyte spore formation activity in the subject is included. Is a method of selecting a substance having

Further, the present invention provides (a) bone marrow cells with 0.0
The step of culturing in a serum-free medium containing 1-1 ng / ml of mpl ligand, (b) the step of separating the megakaryocytes prepared by the step, (c) the separated megakaryocytes for the formation of endoplasmic reticulum. In a subject characterized by including a step of contacting with a factor to cause and a subject and culturing in a serum-free medium containing no mpl ligand, and (d) a step of detecting megakaryocytes in which vesicle formation has occurred Is a method of selecting a substance having an activity of inhibiting megakaryocyte projection formation.

Bone marrow cells were treated with 0.01-1 ng / ml of m
Culturing in serum-free medium containing pl ligand (a) step, separating megakaryocytes prepared by the step (b) step, and detecting megakaryocytes that have undergone cyst formation
Step (d) is the same as described above. On the other hand, the isolated megakaryocyte is contacted with a factor that causes the formation of endoplasmic reticulum and a subject, and is cultured in a serum-free medium containing no mpl ligand (c). Specifically, IL-6 can be used. The concentration of IL-6 added to the medium is
Generally 1 to 1000 ng / ml, but 10 to 100
ng / ml is preferred. Other media, culture conditions and the like are the same as above.

The present invention also provides (a) bone marrow cells to 0.0
Culturing in serum-free or serum medium containing 1-1 ng / ml of mpl ligand, (b) separating megakaryocytes prepared by the step, (c) separating megakaryocytes from mpl ligand And a step of culturing in a serum-free medium that does not contain, and (d) detecting megakaryocytes that have undergone formation of endoplasmic reticulum.

As described above, since it is considered that platelets are produced by the formation of endoplasmic reticulum by megakaryocytes differentiated from immature bone marrow cells, megakaryocyte endoplasmic reticulum projections derived from bone marrow cells in vitro. By measuring the forming ability, it is possible to measure the level of platelet producing ability of bone marrow cells. Using this assay, it is possible to determine whether the cause of the platelet count differing from normal in certain individuals is a problem with bone marrow cells or another environment.

The present invention is described in more detail by way of examples, but the scope of the present invention is not limited to these examples.

[0025]

【Example】

Example 1 Number of megakaryocytes induced by mpl ligand from mouse bone marrow cells The femurs of eight 7-week-old ddY mice (male) were collected, and both ends thereof were cut to obtain 1 mM EDTA (manufactured by Sigma, USA). Bone marrow was extruded into a 100 mm plastic dish using a plastic syringe (22 G needle) containing 20 ml of calcium- and magnesium-free Hanks solution (Sigma 7.4; pH 7.4) added with. The cells were dispersed by pipetting, transferred to a 15 ml tube, and centrifuged at 100 g for 20 minutes. 10 precipitating cells
Disperse in ml of IMDM, centrifuge at 350 g for 5 minutes, again disperse the pelleted cells in 10 ml of IMDM,
It was centrifuged at 0 g for 5 minutes. Precipitating cells at 5 × 10 ⁶ / m
0, 0.01, 0.03, 0.1, 0.
3, 1.0, 3.0 or 10 ng / ml mpl ligand (human TPO, Peprotech, UK) 10 μ
g / ml bovine-derived insulin (manufactured by Gibco, USA),
It was dispersed in IMDM medium containing 5 μg / ml bovine-derived holotype transferrin (manufactured by Gibco). Bone marrow cell suspension of mpl ligand concentration is different each 5ml can be eight acquired respectively 25 cm ² culture flasks (USA,
Becton Dickinson), 37 ° C, 5% CO ₂
The cells were cultured for 3 days under the conditions of. After pipetting a liquid containing megakaryocytes induced by culture several times, 15 ml
It was transferred to a tube and left for 1 hour. 4 ml of the upper part was sucked out, 1 ml of the lower part was collected, and the cells were dispersed by pipetting several times. In eight 15 ml tubes, from the bottom, 1.5 ml of calcium and magnesium-free Hanks solution containing 16% BSA, 3 ml of calcium containing 4% BSA and magnesium-free Hanks solution, 1.5 ml of 2
1 ml of each cell dispersion was overlaid on a liquid containing calcium- and magnesium-free Hanks' solution containing% BSA, and left standing for 1 hour. The top 5 ml was aspirated, leaving the bottom 2 ml. 8 ml of IMDM was added to the remaining liquid, and the mixture was centrifuged at 60 g for 5 minutes. The number of megakaryocytes in the precipitate was measured by observing cells having a size of 20 μm or more as megakaryocytes by microscopic observation. FIG. 1 shows the number of megakaryocytes induced by each concentration of mpl ligand. The number of megakaryocytes induced by mpl ligand increased in a concentration-dependent manner, and when 10 ng / ml of mpl ligand was added, the number of megakaryocytes induced was about 8-fold, as compared to when no mpl ligand was added. Example 2 Endoplasmic reticulum formation ability of megakaryocytes induced by mpl ligand from mouse bone marrow cells Femoral bones of 8 7-week-old ddY mice (male) were collected, and both ends thereof were cut to obtain 1 mM EDTA (US, The bone marrow was extruded into a 100 mm plastic dish using a plastic syringe (22G needle) containing 20 ml of calcium- and magnesium-free Hank's solution (manufactured by Sigma) (manufactured by Sigma; pH 7.4). The cells were dispersed by pipetting, transferred to a 15 ml tube, and centrifuged at 100 g for 20 minutes. 10 precipitating cells
Disperse in ml of IMDM, centrifuge at 350 g for 5 minutes, again disperse the pelleted cells in 10 ml of IMDM,
It was centrifuged at 0 g for 5 minutes. Precipitating cells at 5 × 10 ⁶ / m
0, 0.01, 0.03, 0.1, 0.
3, 1.0, 3.0 or 10 ng / ml mpl ligand (human TPO, Peprotech, UK) 10 μ
g / ml bovine insulin (Gibco), 5 μg /
ml Bovine-derived holotype transferrin (Gibco)
Was dispersed in IMDM medium containing Eight types of 5 ml bone marrow cell dispersions with different mpl ligand concentrations were obtained, and each of them was placed in a 25 cm ² culture flask (Becton Dickinson, USA) under conditions of 37 ° C. and 5% CO ₂
Cultured for days. The solution containing megakaryocytes induced by the culture was pipetted several times, then transferred to a 15 ml tube and allowed to stand for 1 hour. 4 ml of the upper part was sucked out, 1 ml of the lower part was collected, and the cells were dispersed by pipetting several times. 1.5 ml from the bottom in 8 15 ml tubes
Calcium containing 16% BSA, magnesium-free Hanks solution, 3 ml of calcium containing 4% BSA, magnesium-free Hanks solution, 1.5 ml of calcium containing 2% BSA, magnesium-free Hanks solution , 1 ml of each cell dispersion was layered and left standing for 1 hour.
The top 5 ml was aspirated, leaving the bottom 2 ml. 8 ml of IMDM was added to the remaining liquid, and the mixture was centrifuged at 60 g for 5 minutes. The number of megakaryocytes in the precipitate was measured by observing cells with a size of 20 μm or more as megakaryocytes by microscopic observation, and 10 μg / ml of each was determined to be 3000 megakaryocytes / ml.
I containing bovine-derived insulin (Gibco), 5 μg / ml bovine-derived holotype transferrin (Gibco)
It was dispersed in an MDM medium and used as a megakaryocyte dispersion.

100 μl of each of the megakaryocyte dispersions was dispensed into a 96-well plate for culture (Sumitomo Bakelite Co., Ltd., Japan) and cultured at 37 ° C. under 5% CO ₂ . Two
On the day, the megakaryocytes were observed under a microscope, and the number of megakaryocytes that had undergone the formation of endoplasmic reticulum was measured. FIG. 2 shows the endoplasmic reticulum formation ability of megakaryocytes induced by each concentration of mpl ligand.
In megakaryocytes induced by mpl ligand, the ability to form endoplasmic reticulum formation was suppressed in a concentration-dependent manner, and 10n
Megakaryocytes induced by g / ml mpl ligand are
Almost no alveolar process was formed. From the results of Examples 1 and 2, it was found that the higher the concentration of mpl ligand is, the more the number of megakaryocytes induced from bone marrow cells increases, but the ability of the induced megakaryocytes to form endoplasmic reticulum processes decreases. Example 3 Increase in number of vesicle-forming megakaryocytes by IL-6 96-well plate for culture (Sumitomo Bakelite Co., Ltd., Japan)
100 μl of the megakaryocyte dispersion prepared in Example 2 was dispensed into each of the cells, and no IL-6 was added and 100 ng / ml of IL-.
6 (Upstate Biotechnology Inc., USA) was added and the cells were cultured at 37 ° C. under 5% CO ₂ . On the 2nd day, megakaryocytes were observed under a microscope, and there was no addition of IL-6 having megakaryocyte procession formation activity and 100 ng / ml of IL-.
The number of megakaryocytes that caused the formation of endoplasmic reticulum when 6 was added was compared. FIG. 3 shows the increase in the number of megakaryocytes that caused the formation of endoplasmic reticulum due to IL-6 when using megakaryocytes induced by each concentration of mpl ligand. The number of megakaryocytes in which 100 ng / ml of IL-6 caused the formation of endoplasmic reticulum was 0.
It was most often with megakaryocytes induced by 3 ng / ml mpl. Therefore, when measuring endoplasmic reticulum formation activity using megakaryocytes derived from bone marrow cells by mpl ligand, there is an optimum mpl ligand concentration, which is much higher than the concentration of 10 ng / ml which is conventionally used. It turned out to be very low. That is, it was found that a concentration of 0.01 to 1 ng / ml of mpl ligand was suitable for measuring the endoplasmic reticulum formation activity. Example 4 Method for measuring megakaryocytic process formation activity Based on the results of Examples 1 to 3, a method for measuring megakaryocyte process formation activity was established as follows, and the activity of IL-6 was measured.

Femurs of eight 7-week-old ddY mice (male) were collected, both ends thereof were cut, and calcium- and magnesium-free Hanks solution (manufactured by Sigma) containing 1 mM EDTA (manufactured by Sigma, USA). A plastic syringe (22G needle) containing 20 ml of pH 7.4, 100 m
m The bone marrow was extruded into a plastic dish. The cells were dispersed by pipetting, transferred to a 15 ml tube, and centrifuged at 100 g for 20 minutes. The precipitating cells were dispersed in 10 ml of IMDM and centrifuged at 350 g for 5 minutes, and the precipitating cells were again dispersed in 10 ml of IMDM and centrifuged at 350 g for 5 minutes. Precipitate cells 5x1
IMD containing 0.3 ng / ml of mpl ligand (Peprotech), 10 μg / ml bovine insulin (Gibco), 5 μg / ml bovine holotype transferrin (Gibco) so as to be 0 ⁶ / ml.
M medium). About 40 ml of the bone marrow cell dispersion can be obtained, and 5 ml of each is dispensed into eight 25 cm ² culture flasks (manufactured by Becton Dickinson, USA).
Cultivation was carried out for 2 days under the conditions of ° C and 5% CO ₂ . After pipetting a solution containing megakaryocytes induced by culture several times, 10 ml was dispensed into four 15 ml tubes,
Let stand for 1 hour. The upper 8.5 ml was sucked out, the lower 1.5 ml was recovered, and the cells were dispersed by pipetting several times. In two 15 ml tubes, from the bottom 1.5
ml of calcium containing 16% BSA, magnesium-free Hank's solution, 3 ml of calcium containing 4% BSA,
Hanks liquid without magnesium, 1.5 ml of 2% BSA
3 ml of the cell dispersion liquid was overlaid on a liquid containing calcium- and magnesium-free Hanks' liquid containing the above and left for 1 hour. 7 ml of the upper part was sucked out, leaving 2 ml of the lower part. 8 ml of IMDM was added to the remaining liquid, and the mixture was centrifuged at 60 g for 5 minutes. The number of megakaryocytes in the precipitate was measured by observing cells with a size of 20 μm or more as megakaryocytes by microscopic observation, and
IMD containing 10 μg / ml bovine insulin (Gibco) and 5 μg / ml bovine holotype transferrin (Gibco) to give 000 megakaryocytes / ml
It was dispersed in M medium and used as a megakaryocyte dispersion.

100 μl of the megakaryocyte dispersion was dispensed into a 96-well plate for culture (Sumitomo Bakelite Co., Ltd., Japan), and 10 μl of the test subject was added. Further, as a negative control, a non-addition test group to which no subject was added was also set up. After culturing this plate for 2 days, the megakaryocytes in each hole of the plate were observed under a microscope to measure the number of megakaryocytes that had undergone vesicle formation, and the number of megakaryocytes that had undergone vesicle formation was used as a negative control. , The number of megakaryocytes that had undergone vesicle formation when the test subject was added was compared, and the vesicle formation ability of the sample was measured. The intensity of activity is 100% of that of the negative control
The ratio is expressed as

As a specific example of the measuring method of the present invention, an experiment was conducted in which the subject was IL-6. That is, the subject IL
When -6 was added so that the final concentration at the time of measurement would be 100 ng / ml, the strength of the activity of ER formation was 1
68%. Moreover, the final concentration of IL-6 is 10 ng.
/ Ml and 1 ng / ml were added so that their activity intensities were 133% and 102%, respectively, and it was confirmed that the measurement was sufficiently possible at a concentration of at least 10 ng / ml or more. .

[Brief description of drawings]

FIG. 1 is a diagram showing the number of megakaryocytes induced by each concentration of mpl ligand.

FIG. 2 is a diagram showing the ability of megakaryocytes to form endoplasmic reticulum formation induced by various concentrations of mpl ligand.

FIG. 3 is a diagram showing the increased number of megakaryocytes that have undergone the formation of endoplasmic reticulum by IL-6 when using megakaryocytes induced by each concentration of mpl ligand.

Claims (8)

[Claims] 1. A method for producing megakaryocytes, which comprises culturing bone marrow cells in a medium containing 0.01 to 1 ng / ml of mpl ligand. 2. Bone marrow cells are cultivated in a serum-free medium containing 0.01 to 1 ng / ml of an mpl ligand, and vesicle formation is possible, but the vesicle projections are substantially formed as they are. A method for producing megakaryocytes that does not cause formation. 3. (a) 0.01-1 ng / m 2 of bone marrow cells
culturing in a serum-free medium containing 1 mpl ligand, (b) separating the megakaryocytes prepared by the step, (c) contacting the separated megakaryocytes with a subject, and mpl A method for measuring megakaryocyte projection activity of a subject, comprising the step of culturing in a serum-free medium containing no ligand, and (d) detecting megakaryocytes that have undergone formation of cell projections. 4. (a) The bone marrow cells are added in an amount of 0.01 to 1 ng / m 2.
culturing in a serum-free medium containing 1 mpl ligand, (b) separating the megakaryocytes prepared by the step, (c) contacting the separated megakaryocytes with a subject, and mpl A step of culturing in a serum-free medium containing no ligand, (d) a step of detecting megakaryocytes that have undergone vesicle formation, and a substance having megakaryocyte projection forming activity in a subject, which comprises How to choose. 5. (a) The bone marrow cells are added in an amount of 0.01 to 1 ng / m 2.
1) a step of culturing in a serum-free medium containing mpl ligand, (b) a step of separating the megakaryocytes prepared in the step, (c) a step of culturing the separated megakaryocytes and a factor which causes the formation of oocysts Megakaryocyte vesicles in a subject, which comprises a step of contacting with a body and culturing in a serum-free medium containing no mpl ligand, and (d) a step of detecting megakaryocytes that have undergone vesicle formation A method for selecting a substance having a protrusion formation inhibiting activity. 6. (a) The bone marrow cells are added in an amount of 0.01 to 1 ng / m 2.
culturing in a serum-free or serum medium containing 1 mpl ligand, (b) separating the megakaryocytes prepared in the step, (c) separating the megakaryocytes without mpl ligand A method for measuring the level of platelet production of said bone marrow cells, comprising the step of culturing in a serum medium and (d) detecting megakaryocytes that have undergone formation of endoplasmic reticulum. 7. The method according to any one of claims 3 to 6.
(a) The number of days of culturing in the step is 2 or 3 days, the method for measuring megakaryocyte projection process forming activity according to any one of claims 3 to 6, the method for selecting a substance having megakaryocyte projection process forming activity, A method for selecting a substance having a megakaryoblastic process formation inhibitory activity, or a method for measuring a platelet production level. 8. The method according to any one of claims 3 to 6.
(c) The number of days of culture in the step is 1 or 2 days, the method for measuring megakaryocyte projection process activity according to any one of claims 3 to 6, the method for selecting a substance having megakaryocyte process projection activity, A method for selecting a substance having a megakaryoblastic process formation inhibitory activity, or a method for measuring a platelet production level.