JPH07274994A - Production of thrombomodulin - Google Patents

Abstract

PURPOSE:To obtain thrombomodulin having anti-thrombotic action, effective for treating and preventing cardiac infarction and thrombosis in a large production amount without adding a specific substance, by culturing a cell capable of producing thrombomodulin while applying shear stress to the cell. CONSTITUTION:A culture device capable of culturing a cell (e.g. human hemangioendothelium cell) capable of producing thrombomodulin while applying shear stress to the cell is formed as follows. A cell is bonded to a cover glass in a flow channel of a microflow chamber, the flow channel is changed by contriving cutting of silicone rubber, the channel close to a blood vessel in an organism is formed. A culture solution in a reservoir bottle is circulated in a tube by using a pump and the cell is cultured while applying shear stress having >=1.5 dynes/cm<2> and not destroying the cell to give the objective thrombomodulin in a high manifestation amount, having antithrombotic action, effective for treating and preventing cardiac infarction and thrombosis in a large production amount without adding a specific substance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing thrombomodulin capable of further increasing the production amount of thrombomodulin, which comprises culturing cells while applying shear stress.

[0002]

2. Description of the Related Art In recent years, research on the function of blood vessels has made remarkable progress. Among them, it is known that the role of vascular endothelial cells in blood vessels is very large, and as its functions, it regulates the permeability of substances from the vascular lumen to tissues, maintains the antithrombotic effect by the expression of anticoagulants, and There are adjustments of expansion and contraction. It is also pointed out that if these functions fail to work due to some influence, they will develop into various vascular diseases.

Thrombomodulin is one of the physiologically active substances produced by vascular endothelial cells. Thrombomodulin has an anticoagulant action and a thrombolytic action by promoting protein C activation by thrombin. Therefore, treatment and prevention of myocardial infarction, thrombosis, embolism, peripheral vascular occlusion, arteriosclerosis obliterans, intravascular blood coagulation syndrome (DIC), angina, transient ischemic attack, toxemia of pregnancy, etc. It is a substance that can be used for.

The gene encoding human thrombomodulin has already been cloned, and thrombomodulin has already been produced using recombinant cells into which the gene has been incorporated [JP-A-64-6219]. The production of thrombomodulin using cultured vascular endothelial cells is performed by adding various substances to the culture medium because the expression level thereof is very low. As substances that increase the production of thrombomodulin, phorbol esters represented by 4β-phorbol-12-myristate-13-acetate (PMA) [Journalof Biochemis
try, 108 , 839 (1990)], histamine [Biochemical Journal, 276 ,
739 (1991)], retinoic acid [Biochem.
ical Journal, 281 , 149 (199)
2)] and cyclic AMP analog [Journa
l of Cellular Physiology,
147 , 157 (1991)] and pentoxifylline.
OMBOSIS RESEARCH, 62 , 745 (1
991)] etc. have been reported.

[0005]

Thrombomodulin, which has an important antithrombotic effect in blood vessels, has a very low expression level in vascular endothelial cells, and it is very difficult to confirm its expression and activity. It is possible to add various substances and culture them in order to increase the expression level, but this is also a factor of cost increase, and there is a sufficient possibility that additional purification is required. Therefore, development of an efficient production method for obtaining a useful substance, thrombomodulin, was expected.

[0006]

Means for Solving the Problems The present inventor has conducted various studies in order to efficiently produce thrombomodulin produced by vascular endothelial cells. As a result, they have found that the expression level of thrombomodulin produced by vascular endothelial cells is increased by culturing the vascular endothelial cells in a culture device that applies shear stress, and have completed the present invention.

That is, the present invention is a method for producing thrombomodulin, which comprises culturing cells capable of producing thrombomodulin while applying shear stress. The present invention will be described in detail below. The cells are not particularly limited as long as they can produce thrombomodulin, but adhesive cells are preferable, and examples thereof include vascular endothelial cells,
Human vascular endothelial cells are particularly preferable. More preferred is human umbilical vein endothelial cell (HUVEC). This vascular endothelial cell is derived from human umbilical cells, 20 , 329 (1988) or Human Ce.
ll, 1, can be easily separated according to the method described in 188 (1988). It is also possible to obtain and use the separated secondary culture cells. The cells can be used alone, but it is also possible to use two or more types of cells at the same time.

The shear stress is not particularly limited as long as it has such a strength that thrombomodulin can be increased, but is usually 0.1 dyne / cm ² or more, preferably 1.5 dyne / cm ² or more, and more preferably Is 15
For example, dynes / cm ² or more is exemplified. The upper limit is not so strong as to destroy cells or float without adhering, and may be selected from the viewpoint of dynamic economic efficiency, but usually 1000 dynes / cm ²
Hereafter, preferably 25 dynes / cm ² or less is exemplified. The value of the shear stress allows the endothelial cells to be cultured under the shear stress applied to the in vivo arterial endothelial cells and venous endothelial cells. The value of shear stress can be obtained by the following method.

Shear stress = viscosity × ((6 × flow rate) / (flow channel width × flow channel height ² )) That is, the viscosity of the culture solution, the flow rate of the culture solution, and the shape of the flow channel can be changed. Thus, the value of shear stress can be easily changed in various ways. When producing thrombomodulin, it is preferable that the shear stress applied to cells is in the above preferable range as an average value. It is also possible to load each cell uniformly, and when the flow path is deformed so that the width and the height do not match, the cell surface is adhered to the Since the shear stresses to be applied to the plates are different, a quadrangular prism preferably surrounded by a plane is exemplified. In this quadrangular prism, it is possible to apply a uniform shear stress to cells by either adhering the cells to one surface or by adhering the cells to the surface opposite to that surface and not adhering to the side surface. .

The culture medium used for culturing cells may be of a generally known composition. For example, in the case of vascular endothelial cells, a cell culture medium containing 0 to 20% of serum of an animal such as bovine is added. It is preferable to use, more preferably E
-GM UV medium (containing 2% fetal calf serum, manufactured by Kurabo) or M199 medium supplemented with 20% fetal calf serum is exemplified. Furthermore, ECGS is added to the culture solution.
(Endothelial cell growths
supplement), EGF (Epidermal)
growth factor; J. of Cell B
iology, 77 , 774-788 (1978)) or basic-FGF (Fibroblast g.
rowth factor; J. of Cell Bi
It is also preferable to improve cell growth by adding a cell growth factor such as chemistry, 77 , 774-788 (1978)). Further, the viscosity of the culture solution can be increased by adding dextran or the like, and high shear stress can be applied to the cultured cells.

As a culture device capable of applying shear stress, a rotating disc type [Biorology, 2
5 , 461 (1988)], parallel plate type [Biotec
hology and Bioengineerin
g, 27 , 1021 (1985)] and a microcarrier type [JP-A-4-179476], and the like, but a parallel plate type is preferably used.

The microflow chamber is a small parallel plate type shear stress culture device, and by using this device, a flow path (branch, S-shape, etc.) closer to a blood vessel in the living body, which could not be obtained by the culture device described above, is provided. It is possible to reproduce in vitro, and it is possible to obtain a result in which the expression of a substance reflects the blood vessel in the body. The outer shape, material, etc. of the microflow chamber can be variously changed, and the construction method thereof is not specified, but a preferable example is shown below. As shown in FIGS. 1 and 2, the microflow chamber is a flat plate on which animal cells can be adhered and cultured, preferably a sheet having a certain thickness cut so as to form a flow path between two glass cover glasses, Preferably, it can be constructed by sandwiching silicon rubber and fixing it. As shown in FIG. 2, the silicon rubber is preferably used by being adhesively fixed to one cover glass with an adhesive.
The cover glass preferably has a thickness in the range of 0.4 to 0.6 mm and a size of 40 mm in length × 100 mm in width. The cover glass is preferably used by coating the surface with gelatin, collagen or the like in order to improve the adhesiveness of cells. The thickness of the silicon rubber and the width of the flow channel are preferably 2 mm in thickness and 3 mm in the flow channel. At the inlet and outlet of the culture solution, a silicon tube of appropriate thickness that fits inside the flow path,
It is preferable that a silicon tube having an inner diameter of 1.5 mm and an outer diameter of 2.5 mm is adhered and fixed so that the culture solution can be smoothly taken in and out and the culture solution can be prevented from leaking to the outside.

In this microflow chamber, shear stress can be applied to cells by adhering and culturing animal cells on a cover glass in the channel and flowing a culture solution in the channel. By investigating vascular endothelial cells loaded with shear stress, it is possible to analyze vascular functions in vivo and the role played by vascular endothelial cells. In addition, when observing the interaction between two different types of cells, or when observing substance permeability of vascular endothelial cells or infiltration of blood cells such as leukocytes into vascular endothelial cells, the culture layer is made of a porous membrane or the like. It is also possible to divide the culture into two layers and culture. Furthermore, their action in the presence of shear stress can be observed under a microscope in real time.

In this microflow chamber, various channels (branches, S-shapes, etc.) closer to blood vessels in the living body can be formed by devising cutting of silicon rubber and changing the channels. By creating such a complicated channel and culturing vascular endothelial cells under shear stress,
It is possible to observe the state of flow (reverse flow, vortex, etc.) in various channels actually existing in the living body, and to investigate the response of vascular endothelial cells to the flow. Further, by exchanging only the chamber portion from the culture device capable of perfusing the medium, cell culture under different conditions can be instantaneously performed. Furthermore, by connecting two or more chambers in series or in parallel, it is possible to simultaneously perform cell culture under various kinds of conditions.

During the culture using the microflow chamber, the effect can be observed in detail by adding the test substance to the culture medium. Furthermore, in the removed state, the culture medium is exchanged with a culture medium containing a desired test substance, and if necessary, with a culture vessel containing fresh cells, it is possible to screen a large number of samples. Therefore, substances such as cell culture supernatants, tissue extracts, etc.
It is also possible to screen from various synthetic substances or fungal metabolites.

As the culture apparatus capable of perfusing the medium, any apparatus capable of aseptically circulating the culture solution at a constant rate may be used.
For example, there is a system shown in FIG. In this system, the culture solution in the medium reservoir bottle can be circulated in the tube using a pump and flowed into the flow path of the parallel plate type culture device such as a microflow chamber. In the production of thrombomodulin, culture of vascular endothelial cells is not specified, but for example, the method shown below is exemplified. The vascular endothelial cells are adhered to the cell culture surface in a cobblestone shape, and the culture device is set. The culture temperature may be any temperature at which thrombomodulin is produced well and cells can be cultured, but is preferably 37 ° C., and further preferably in an incubator filled with 5% carbon dioxide gas. Although the number of cells is not particularly limited, it is preferably in a preferable level for thrombomodulin production, and may be in the level of ordinary culture, preferably 1 × 10 ⁴ or more, particularly preferably 1 ×.
It should be 10 ⁵ or more. Although the culture time is not specified, it may be any time as long as the production of thrombomodulin is good and the survival state of the cells is good, and preferably 6 hours or more and 48 hours or less.

The expression of thrombomodulin can be detected by various methods. For example, it can be detected at the protein level by a fluorescent antibody method using an anti-thrombomodulin antibody or FACScan. Furthermore, using mRNA extracted from cells, at the mRNA level by the Northern hybridization method using a probe for thrombomodulin, or the RT-PCR method using a primer designed to amplify a part of the thrombomodulin gene, etc. Can be analyzed.
Further, the activity of thrombomodulin can be detected as that which promotes the activation of protein C by thrombin. Usually, this activity was carried out by adding a sample to 20 mM Tris-HCl buffer (pH 7.4) containing 0.15 M sodium chloride, 2.5 mM calcium chloride, 1 mg / ml serum albumin in the presence of thrombin and protein C, The amount of activated protein C produced is quantified and evaluated. It is also possible to detect the activity of thrombomodulin by measuring the activity such as the anticoagulant activity and the platelet aggregation inhibitory activity described in JP-A-64-6219.

After culturing, thrombomodulin produced from vascular endothelial cells can be isolated by a commonly known method. For example, DIP prepared by binding diisopropylphosphorothrombin to bromocyanated agarose.
-Thrombin-agarose column chromatography can be used for purification. Also, an ion exchange column,
It can also be purified by combining an anti-thrombomodulin antibody column, a gel filtration column and the like.

[0019]

EXAMPLES In order to describe the present invention in more detail, the examples will be described, but the scope of the present invention is not limited to these examples.

[0020]

Example 1 Changes in thrombomodulin expression level in human umbilical vein endothelial cells (HUVEC) (protein level) (1) Culture of HUVEC HUVEC (Endocell-UV, Kurabo Industries)
Used the third passage cell. Cell culture equipment is Biotec
hology and Bioengineerin
g, 27 , 1021 (1985), was used. A glass cover slip (46 × 26 × 0.9-1.2 mm, made by Matsunami Co., Ltd.) was allowed to stand in a 90 mmφ plastic dish (made by Nunc Co.) and 1% gelatin (derived from bovine skin, made by Sigma Co.) was used. 5 ml of physiological saline containing it was poured, and the mixture was left overnight at 4 ° C. to obtain a gelatin-coated cover slip. The culture solution (E-GM UV,
The gelatin-coated cover slips washed with Kurabo Industries Co., Ltd. were left to stand, and HUVECs were planted at 1.0 × 10 ⁴ pieces / cm ² . Set in a parallel plate type shear stress incubator and filled with 5% carbon dioxide gas 3
Perfusion culture was carried out for 24 hours so that a shear stress of 1.5 dynes / cm ² was applied in an incubator at 7 ° C. (2) Detection of thrombomodulin by fluorescent antibody method The cells subjected to shear stress were taken out together with the coverslip from the device, and phosphate buffered saline containing 1.1% formalin (PBS [137 mM sodium chloride, 2.7 mM potassium chloride, 4 mM). 3 mM disodium hydrogen phosphate and 1.4 mM potassium dihydrogen phosphate]) at 4 ° C. for 20 minutes, and then in PBS containing 1% bovine serum albumin (BSA) for 30 minutes, and then 0.5%. P including BSA
It was washed twice with BS (hereinafter referred to as 0.5% BSA-PBS). The obtained immobilized HUVEC was stained by the fluorescent antibody method using an anti-thrombomodulin antibody as follows. The anti-thrombomodulin antibody was prepared in mice according to the method described in the Monoclonal Antibody Experiment Manual (S. Tomiyama, S. Ando, T.) (1987). To the cells on the cover glass,
Anti-thrombomodulin monoclonal antibody solution (85μ
0.5% BSA-PBS was added so that the concentration would be g / ml), and the mixture was reacted at room temperature for 1 hour. 0.5% BS
After washing three times with A-PBS, FITC-labeled anti-mouse IgG goat antibody (manufactured by Silenus) diluted 15-fold with 0.5% BSA-PBS was added and reacted at room temperature for 1 hour. After washing twice with 0.5% BSA-PBS, it was washed with PBS, immersed in PBS containing 1.1% formalin for 5 minutes, and further washed with 0.5% BSA-PBS. The coverslip was placed on a slide glass for observation, sealed, and then observed under a microscope.

As a result, in the cells cultured under shear stress for 24 hours, a marked staining with the antibody was observed as compared with the cells which were statically cultured at the same time. That is, almost no staining was observed with the antibody in the statically cultivated cells, but the cells cultivated under shear stress were stained with green fluorescence as a whole, and yellow fluorescence was observed in some places. In other words, it was considered that the amount of thrombomodulin produced by cells was clearly increased by the load of shear stress. (3) Quantitative analysis by FACScan The FACScan used was manufactured by BECTON-DICKINSON.

The cells subjected to the shear stress culture according to the method (1) are taken out from the apparatus together with the coverslip, and then 5 m in length.
It was washed twice with 1 PBS. The cells were allowed to stand in 5 ml of 0.1% disodium ethylenediaminetetraacetate (EDTA, manufactured by Dojindo Laboratories) for 10 minutes, and then thoroughly stirred with a Pasteur pipette to detach the cells from the glass surface. The cell suspension was passed through a nylon filter (75 μm mesh) and transferred to a 15 ml centrifuge tube. 0.5 ml of 1%
After adding BSA-PBS, 4 ° C, 1,200 rpm
Centrifuge at 6 minutes for 6 minutes to collect 5 ml of 0.1% B
Suspended in SA-PBS. Count the number of cells, put 2 × 10 ⁵ cells in a tube for exclusive use of FACS, and add 0.1% BSA.
-The volume of the liquid was adjusted to 300 μl using PBS. 0.1 mg
/ Ml anti-thrombomodulin antibody (15 μl)
Let stand for 0 minutes. 2 ml of 0.1% BSA-PBS was added and the mixture was centrifuged at 4 ° C. and 1,200 rpm for 6 minutes, and the supernatant was removed to 250 μl. 3 μl of FITC-labeled mouse Ig (H + L) (rb) (manufactured by Seikagaku Corporation) was added, stirred, and allowed to stand for 30 minutes in a light-shielded state. 2 ml of 0.1
% BSA-PBS was added and stirred, and the precipitate obtained by centrifugation was stirred in 200 μl of 0.1% BSA-PBS, and then measurement was started with a FACScan device.

As a result, the cells subjected to the shear stress culture fluoresced about 50% more strongly than the statically cultured cells (FIG. 4). From this, it was considered that the expression level of thrombomodulin in HUVEC was increased by the shear stress loading.

[0024]

Example 2 Change in Thrombomodulin Expression Level in HUVEC (mRNA Level) (1) Culturing of HUVEC HUVEC was cultured in the same manner as in (1) of Example 1. However, the shear stress is 15 dynes / cm ² ,
The culture time was 6 hours. (2) Extraction of total RNA from cells HUVEC cultured in shear stress (about 3 × 10 ⁵ cells)
Remove the coverslip containing
Solution D [4M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.
5% N-lauroyl sarcosine sodium and 0.1
Adherent cells were detached using M 2-mercaptoethanol]. Immediately, using a 1 ml syringe equipped with a 23 G needle, a solution containing cells was put in and taken out about 10 times,
The cells were completely destroyed. This solution was transferred to a 1.5 ml-volume microtube, 50 μl of 2M sodium acetate (pH 4.0) was added, and the solution was mixed by inversion upside down.
Furthermore, 0.5 ml of water-saturated phenol (phenol (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved at a temperature of 60 ° C. and dissolved, and an equal volume of DEPC-treated water (0.2 ml of diethyl pyrocarbonate (100 ml of water) (Manufactured by Yakuhin Kogyo Co., Ltd.), stirred vigorously and left overnight, and sterilized under high pressure for 20 minutes at 120 ° C.). Chloroform-isoamyl alcohol mixed solution (49: 1) was added and the mixture was turned upside down and then vigorously stirred for 10 seconds using a vortex mixer. After leaving in ice for 15 minutes, 4 ℃, 15,000r
Centrifuged at pm for 20 minutes. Transfer the water layer to a new tube and add an equal amount of isopropyl alcohol to it.
It was left to stand at ℃ for about 1 hour. Furthermore, 4 ℃, 15,000r
The precipitate obtained by centrifugation at pm for 20 minutes was dissolved in 0.3 ml of solution D. After adding an equal amount of isopropyl alcohol again and leaving it at -20 ° C for about 1 hour, 4
The precipitate was collected by centrifugation at 15,000 rpm at 10 ° C for 10 minutes. After washing with 80% ethanol, the precipitate dried under vacuum was dissolved in 50 μl of water to give a total RNA solution. (3) Analysis of mRNA level by RT-PCR method RT-PC shown below using about 0.4 μg of total RNA
The mRNA level was analyzed by the R method. 0.5m
200 U of MM prepared in a microtube of 1 volume
LV reverse transcriptase
(Manufactured by GIBCO BRL) 10 mM dithiothreitol, 1.0 mM each of 2'-deoxyadenosine-5 '.
-Triphosphate, 2'-deoxycytidine-5'-triphosphate, 2'-deoxyguanosine-5'-triphosphate and thymidine-5'-triphosphate (both manufactured by Takara Shuzo), 0.5
μg oligo (dT) _12-18 (Pharmacia)
And 20 μl of cDNA firsts containing RNase inhibitor (manufactured by Toyobo Co., Ltd.)
rand buffer [50 mM tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris) -hydrochloric acid buffer (pH 8.3), 75 mM potassium chloride and 3 m]
M magnesium chloride] extracted with HUVEC from about 0.
4 μg of total RNA was added, 100 μl of mineral oil (manufactured by Sigma) was overlaid, and a PCR device (DNA The
rmal Cycler, manufactured by Takara Shuzo)
Single-stranded cDNA was synthesized by incubating at 60 ° C for 60 minutes, at 99 ° C for 5 minutes and at 4 ° C for 5 minutes.

Next, 2.5 U of AmpliTaq DN
A polymerase (manufactured by Takara Shuzo), 370 kBq [α
- ³² P] dCTP (111TBq / mmol, NEN Co.) a set of human thrombomodulin primers each 0.1nmol consisting and sequence shown below (SEQ ID NO:
AmpliTaq buffer [10 mM containing the sense primer shown in 1 and the antisense primer shown in SEQ ID NO: 2, manufactured by Nippon Bio Service Co., Ltd.]
Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride and 0.001%
Gelatin] 80 μl was added, and an amplification reaction was performed as follows using a PCR device. After keeping the temperature at 95 ° C. for 2 minutes, an amplification reaction (hereinafter referred to as a PCR amplification reaction) having 1 cycle of 95 ° C., 1 minute, 60 ° C., 2 minutes and 72 ° C., 3 minutes was performed for 8 cycles. Next, a set of 0.1 nmol / μl of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers each having the sequence shown below (for the sense primer shown in SEQ ID NO: 3 and the sequence shown in SEQ ID NO: 4) 1 μl of each of the indicated antisense primers (manufactured by Japan Bioservices Co., Ltd.) was added, and subsequently PCR amplification reaction for 14 cycles was carried out, followed by incubation at 72 ° C. for 7 minutes and then incubation at 4 ° C. for 5 minutes.

Molecular Cloning,
A Laboratory Manual, Seco
According to the method described in nd Edition (1989),
A slab type 8.0% non-denaturing polyacrylamide gel of 13.5 x 13.5 cm was prepared. 1 μl of a dye solution [0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol] was added to 4 μl of the amplified DNA solution by PCR, and the solution was added to the sample lane of polyacrylamide gel, and 1 × TBE bu was added.
tter [90 mM Tris-borate buffer, 2 mM E
Electrophoresis was performed at 160 V for 2 hours using DTA (pH 8.0). The gel after electrophoresis was immersed in a 7% acetic acid solution for 5 minutes, and then the gel was dried at 80 ° C. for 60 minutes using a gel dryer (manufactured by BIO-RAD). The dried gel was fixed in a cassette, and an imaging plate for BAS-2000 imaging analyzer (Fuji Photo Film Co., Ltd.) was set. The image captured on the imaging plate after 30 minutes of exposure was captured by BAS-2000.
It was read by an imaging analyzer and the radioactivity was measured by a computer. For quantification, the radioactivity of the band obtained with the thrombomodulin primer was determined by GAPDH.
It was done by comparing the values divided by that.

The level of thrombomodulin mRNA in the cells cultured under shear stress for 6 hours was obviously increased by 240% as compared with the cells cultured statically (FIG. 5). From this, it was revealed that the increase in thrombomodulin expression level due to shear stress in HUVEC was also increased at the mRNA level.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, by culturing cells while applying shear stress and increasing the expression level of thrombomodulin produced by the cells, thrombomodulin production can be increased without adding a special substance. It is possible.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 21 Sequence type: Nucleic acid Sequence type: DNA Sequence CTCATAGGCA TCTCCATCGC G 21

[0030]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 21 Sequence type: Nucleic acid Sequence type: DNA sequence CCGCGCACTT GTACTCCATC T 21

[0031]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 22 Sequence type: Nucleic acid Sequence type: DNA Sequence TCTGGAAAGC TGTGGCGTGA TG 22

[0032]

[Sequence list]

 SEQ ID NO: 4 Sequence length: 22 Sequence type: Nucleic acid Sequence type: DNA Sequence TCAGTGTAGC CCAAGATGCC CT 22

[Brief description of drawings]

FIG. 1 is a diagram showing the structure of a shear stress culture device (microflow chamber).

FIG. 2 is a view showing a structure of a micro flow chamber and a cell adhesion surface.

FIG. 3 is a diagram showing a perfusion culture system for shear stress culture.

FIG. 4 is a diagram showing the results of quantitative analysis of thrombomodulin expression by FACScan.

FIG. 5 shows the level of thrombomodulin mRNA at RT-
It is the figure which showed the schematic diagram at the time of performing electrophoresis by polyacrylamide gel, after amplifying by PCR method, and the figure which showed the analysis result.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 15/09 ZNA (C12P 21/02 C12R 1:91) (C12N 5/06 C12R 1:91) 9281-4B C12N 15/00 ZNA A (C12N 5/00 E C12R 1:91)

Claims (3)

[Claims] 1. A method for producing thrombomodulin, which comprises culturing cells capable of producing thrombomodulin while applying shear stress. 2. The method according to claim 1, wherein the cells are human vascular endothelial cells. 3. The method according to claim 1, wherein the shear stress is 1.5 dynes / cm ² or more and the shear stress is not more than the shear stress at which cells are not destroyed.