JP2009201443A - Arylsulfatase protein having modulating activity of cell form and function - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate for cell adhesion and a method for cell adhesion using the substrate, a substrate for cell morphology regulation and a method for regulating cell morphology using the substrate, and a substrate for cell culture and a method for culturing cells using the substrate. <P>SOLUTION: The substrate for cell adhesion, the substrate for cell morphology regulation and the substrate for cell culture each comprises an arylsulfatase protein. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell adhesion substrate containing an arylsulfatase protein.

  Cells of multicellular organisms are classified into adhesion-dependent cells that require cell scaffolds (most cells other than blood cells that make up living tissue) and adhesion-independent cells that do not require (floating cells such as blood cells). The Many adhesion-dependent cells can grow and proliferate only when they adhere to an extracellular matrix or adhere to each other. Such binding between cells is called cell adhesion. This cell adhesion is performed by intermolecular interaction of cell adhesion factors, and various molecules are known as such cell adhesion factors, such as collagen and fibronectin. Since each of these adhesive factors exhibits binding specificity, various functions such as morphogenesis, tissue organization, migration, differentiation, proliferation, and apoptosis during embryogenesis, as well as dynamic expression of adhesive factors in different cells. Is controlling. However, all cell adhesion factors are not yet covered, and it is possible that unknown substances are responsible for special cell functions related to cell adhesion.

Aryl sulfatase was discovered as an enzyme that hydrolyzes the aryl sulfate of an artificial substrate and dissociates the sulfate group from the aryl group (Non-patent Document 1). N-acetylgalactosamine 4-sulfate (Non-patent Document 1), Cerebroside 3-sulfate (Non-patent Document 2) and the like are listed as natural substrates for arylsulfatase, but the activity as an enzyme for these is extremely low. . Furthermore, human arylsulfatase function-deficient genetic diseases include mucopolysaccharidosis IV accompanied by growth retardation, hepatosplenomegaly, heart disease, etc. (Non-Patent Documents 3 and 4), and abnormal demyelination of central peripheral nerves. Dye white matter atrophy (Non-patent Documents 5 and 6) is known. However, there are no reports of capturing the protein as an extracellular matrix.
Yogalingam, G., Litjens, T., Bielicki, J., Crawley, AC, Muller, V., Anson, DS, Hopwood, JJ, 1996. Feline mucopolysaccharidosis type VI. Characterization of recombinant N-acetylgalactosamine 4-sulfatase and identification of a mutation causing the disease. J. Biol. Chem. 271, 27259-27265 Mehl, E., Jatzkewitz, H., 1968. Cerebroside 3-sulfate as a physiological substrate of arylsulfatase A. Biochim. Biophys. Acta. 151, 619-627 Evers, M. et al. Proc. Natl. Acad. Sci. USA. 93, 8214-8219 (1996) Harmatz, P. et al. J. Pediatr. 144, 574-580 (2004) Consiglio, A. et al. Nat. Med. 7, 310-316 (2001) Bond, CS et al. Structure 5, 277-289 (1997)

  An object of the present invention is to provide a cell adhesion substrate containing an arylsulfatase protein.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that arylsulfatase proteins have cell adhesion, morphology and function regulating activities, and have completed the present invention.

That is, the present invention is as follows.
(1) A cell adhesion substrate comprising an arylsulfatase protein.
The cell adhesion substrate may further contain at least one extracellular matrix.
The arylsulfatase protein contained in the cell adhesion substrate may be derived from human, mouse or rat.
Further, the cell to which the cell adhesion substrate adheres may be an endothelial cell.
(2) A cell shape regulation substrate comprising an arylsulfatase protein.
(3) A cell culture substrate comprising the cell adhesion substrate according to (1).
(4) A method of adhering cells using the cell adhesion substrate according to (1).
(5) A method for regulating cell morphology using the cell morphology regulation substrate described in (2).
(6) A method for culturing cells using the cell culture substrate according to (3).

  According to the present invention, a substrate for cell adhesion comprising an arylsulfatase protein is provided. The substrate for cell adhesion of the present invention is achieved by the arylsulfatase protein functioning as an extracellular matrix and regulating adhesion and complex morphogenesis of endothelial cells. This means that the cell adhesion substrate of the present invention is useful as a cell culture substrate.

The present invention is described in detail below.
1. The substrate for cell adhesion of this invention TECHNICAL FIELD This invention relates to the substrate for cell adhesion containing arylsulfatase protein. An aryl sulfatase protein (hereinafter sometimes referred to as “protein of the present invention”) is an enzyme having an activity of hydrolyzing an aryl sulfate of an artificial substrate and releasing the sulfate group from the aryl group. This enzyme has previously been thought to localize to lysosomes.

  The inventors made a recombinant protein for the above protein and proceeded with analysis of its properties, and as a result, found that the protein had a molecular weight of about 60,000. Furthermore, when the mouse liver was stained with an anti-arylsulfatase antibody, it was found that arylsulfatase was present on the surface of sinusoidal endothelial cells and that arylsulfatase coexists with heparan sulfate in the tissue, and this protein exists as an extracellular matrix. It revealed that. In addition, the protein has an affinity for heparin (FIG. 2) and collagen, and has the property of binding to an extracellular matrix composed mainly of acidic polysaccharide and collagen (FIG. 3) in vivo. It has been found that there is activity to regulate adhesion, morphology and function.

  Aryl sulfatase proteins are present in a wide range of organisms, and any protein derived from any organism can be used as the cell adhesion substrate of the present invention, but suitable proteins for the present invention are derived from humans, mice, rats, etc. Of the protein. Here, among mouse arylsulfatase (ARS) proteins, the amino acid sequence of mouse ARS A protein is SEQ ID NO: 2 (GeneBank Accession No. NP_033843), and the amino acid sequence of mouse ARS B protein is SEQ ID NO: 3 (GeneBank Accession No. NP_033842). Among the rat arylsulfatase proteins, the amino acid sequence of rat ARS A protein is SEQ ID NO: 4 (GeneBank Accession No. XP_235566), and the amino acid sequence of rat ARS B protein is SEQ ID NO: 5 (GeneBank Accession No. NP_254278). Among the human arylsulfatase proteins, the amino acid sequence of human ARS A protein is represented by SEQ ID NO: 6 (GeneBank Accession No. CAA36399), and the amino acid sequence of human ARS B protein is represented by SEQ ID NO: 7 (GeneBank Accession No. CAA51272) ( FIG. 1). The protein of the present invention includes a protein consisting of the amino acid sequence represented by SEQ ID NOs: 2 to 7 and a protein having a function equivalent to that of the protein, and may be naturally derived or may be derived from DNA by genetic engineering techniques. Artificially produced recombinant protein or chemically synthesized protein may be used. Furthermore, as long as it has a function equivalent to the protein of this invention, the protein which consists of a part of amino acid sequence shown by the said sequence number 2-7, or the protein by which the part was mutated may be sufficient. The “protein having an equivalent function” means a protein having “regulatory activity of cell adhesion, morphology and function” as described above. “Partially mutated protein” means, for example, one or more (1 to 50, 1 to 25, 1 to 10) amino acids in the amino acid sequence represented by SEQ ID NOs: 2 to 7 Is a protein consisting of an amino acid sequence deleted, substituted or added. In general, substitution between amino acids having similar properties (for example, substitution from one hydrophobic amino acid to another hydrophobic amino acid, substitution from one hydrophilic amino acid to another hydrophilic amino acid, substitution from one acidic amino acid to another When a substitution to an acidic amino acid or a substitution from one basic amino acid to another basic amino acid is introduced, the resulting mutant protein often has the same properties as the original protein. Techniques for producing recombinant proteins having such mutations using gene recombination techniques are well known to those skilled in the art, and such mutant proteins are also included within the scope of the present invention.

  The “substrate for cell adhesion” of the present invention is an extracellular matrix and refers to a substrate used for adhering cells, which contains the protein of the present invention having a cell adhesion function. “Adhering cells” means binding by direct contact between cells or cells. Then, except for non-adhesion-dependent cells of the blood cell lineage, cells generally bind to the extracellular matrix via an extracellular matrix receptor such as integrin and extend its morphology. Thereafter, the cells form foliate and dendritic limbs, and further morphological changes and cell movements are induced. In addition, the survival signal is activated by adhesion between the cells and the cells, and the cells can escape from cell death (apoptosis).

  The “cell adhesion substrate” of the present invention may be used in combination with other extracellular matrix. The extracellular matrix is a substance that fills the extracellular space of multicellular organisms, and at the same time, the skeletal role of animal cartilage and bone, the role of scaffolds in cell adhesion such as basement membrane and fibronectin, and heparan It refers to a substance that plays a role in holding and providing cell growth factors such as cell growth factor FGF that binds to sulfuric acid, and refers to glycoproteins such as collagen, proteoglycan, fibronectin and laminin, and cell adhesion molecules. Specific examples of other extracellular matrix include collagen; fibronectin; chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, proteoglycan such as dermatan sulfate proteoglycan; hyaluronic acid as a kind of glycosaminoglycan; laminin; tenascin; Elastin; fibrillin; vitronectin and the like, but not limited thereto.

  The cells to which the present invention is applied include, but are not limited to, endothelial cells, oligodendrocytes, and the like. Of the endothelial cells, microvascular endothelial cells, aortic endothelial cells, and umbilical vein endothelial cells are particularly preferable.

  In addition to the protein of the present invention, any substance used for normal cell culture may be added to the cell adhesion substrate of the present invention. If necessary, serum, cell growth factor, antibacterial agent, etc. may be added. You can also use it. The culture medium, serum and its use amount, cell growth factor and its use amount, antibacterial agent and its use amount may be appropriately determined according to the cell type, cell seeding amount, culture conditions, and cell recovery method after culture. If it is a trader, those conditions can be set.

  It can be confirmed as follows that the substrate for cell adhesion of the present invention adheres cells. That is, an appropriate cell is selected, and cultured for an appropriate time under culture conditions suitable for the cell. The cell adhesion substrate of the present invention is prepared to an appropriate concentration, dropped onto a glass slide, allowed to stand at room temperature for an appropriate time, and then blocked with a bovine serum albumin solution. Thereafter, cells prepared at an appropriate concentration are added to the substrate and cultured. After an appropriate time, the cultured cells are fixed with formaldehyde phosphate buffer and then stained with Coomassie brilliant blue. By using the stained specimen, observation with an optical microscope can confirm whether cell adhesion, cell extension, and cell protrusion are occurring. In addition, the number of cells adhered on the glass surface can be counted to quantitatively confirm the number of adhered cells.

From the above, the cell adhesion substrate of the present invention can also be used as a cell culture solution. This means that “3. It is described in the section “Substrate for cell culture of the present invention”.
To the cell adhesion substrate of the present invention, an extracellular matrix such as collagen and laminin, and growth factors such as FGF, EGF, and PDGF can be added, but not limited thereto.
2. The substrate for regulating cell morphology of the present invention The present invention also relates to a substrate for regulating cell morphology comprising an arylsulfatase protein.

  The cell shape regulation mechanism of the cell shape regulation substrate of the present invention is as follows. That is, when an endothelial cell is adhered onto an arylsulfatase using the cell shape-regulating substrate of the present invention, the ability to project a temporary foot is imparted to the cell. In addition, when collagen is further added to the cell shape regulating substrate of the present invention, the number of pseudopods of cells increases and the cell shape changes. From this, it can be said that the substrate for cell shape regulation of the present invention has a function of regulating cell morphology. This is also clear from the fact that when only collagen is added without using the cell shape-regulating substrate of the present invention, the cells only extend evenly. Specifically, it can be said that the cell shape-regulating substrate of the present invention is involved in the complex morphogenesis of endothelial cells in vivo as an extracellular matrix and regulates the morphogenesis. Thus, it can be seen that the present invention is also useful as a substrate for cell shape regulation.

  It can be confirmed by the following method that the substrate for regulating cell morphology of the present invention can regulate the morphology of cells. That is, an appropriate cell is selected, and cultured for an appropriate time under culture conditions suitable for the cell. The cell shape-regulating substrate of the present invention is prepared to an appropriate concentration, dropped onto a glass slide, allowed to stand at room temperature for an appropriate time, and then blocked with a bovine serum albumin solution. Thereafter, cells prepared at an appropriate concentration are added to the substrate and cultured. After an appropriate time, the cultured cells are fixed with formaldehyde phosphate buffer and then stained with Coomassie brilliant blue. By using the stained specimen, it is possible to confirm that the morphogenesis of the cells is regulated by observing the fine structure of the cells with an optical microscope. For example, taking the case of confirmation using endothelial cells as an example, it is shown that when cells are cultured only with collagen without using the cell shape control substrate of the present invention, the endothelial cells are well stretched and adhered. On the other hand, the shape of endothelial cells cultured using the substrate for cell shape regulation of the present invention is different from the shape of endothelial cells adhered on collagen. It can be determined that the morphology of the cells is regulated using the formation as an index.

The substrate for cell shape regulation of the present invention may include an extracellular matrix such as laminin and growth factors such as FGF, EGF, PDGF, etc., but is not limited thereto.
3. The cell culture medium of the present invention The present invention also relates to a cell culture medium as described above.

  Suitable cells for the cell culture medium of the present invention include, but are not limited to, endothelial cells, dendrocytes, and the like. Preferred examples of the endothelial cells include microvascular endothelial cells, aortic endothelial cells, umbilical vein endothelial cells, and skin epidermal cells.

As a cell culture method using the cell culture solution of the present cells, normal culture conditions can be applied, and therefore culture conditions suitable for the cells to be used may be appropriately selected.
As the cell culture solution of this cell, a commercially available medium to which arylsulfatase protein is added can be used, and among these, Medium 200S medium (Kurabo), K110 medium and the like are preferable.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.

In Example 1, in order to examine the property of purified arylsulfatase, SDS polyacrylamide electrophoresis was performed on the purified recombinant mouse arylsulfatase.
(1) Production of recombinant arylsulfatase cDNA encoding a mouse arylsulfatase gene was amplified by PCR. The following are the primers:
Forward primer: 5'-CGGGATCCACCATGGCCCTGGGGACC-3 '(SEQ ID NO: 8)
Reverse primer: 5'-GCTCTAGAGGACTGGGAGCCTGG-3 '(SEQ ID NO: 9)
The sequence of was used. A nucleotide sequence (SEQ ID NO: 1) encoding a mouse arylsulfatase (mouse ARS A) gene (GeneBank Accession No. BC098075) was introduced into pcDNA3.1 / myc-HisA expression vector (Invitrogen).

  Recombinant mouse arylsulfatase was expressed using the FreeStyle ™ 293 Expression system (Invitrogen). Specifically, FreeStyle 293-F cells are transfected with a vector into which the mouse ARS A gene (SEQ ID NO: 1) is inserted. Thereafter, the transfected cells were cultured, and the mouse arylsulfatase protein secreted into the culture medium was separated by acetone fractionation, and then affinity purification was performed using a HiTrap Chelating HP (Amersham BioSciences) column. Purified 500 ngm recombinant mouse arylsulfatase was separated by 10% SDS polyacrylamide gel electrophoresis. The gel after electrophoresis was stained with Coomassie Brilliant Blue R-250.

  As a result, the purified recombinant mouse arylsulfatase was detected as a single band of about 60 kDa. This was used as a starting material in subsequent experiments. Recombinant mouse arylsulfatase is associated with a His antigen site derived from the designed vector.

(2) Measurement of binding between heparin and arylsulfatase The recombinant mouse arylsulfatase (3 μg) purified in (1) was dissolved in 200 μl of physiological sodium chloride concentration Tris buffer (pH 7.4). This solution was mixed with 100 μl of heparin-conjugated sepharose resin (Amersham Biosciences). The mixed solution was centrifuged, and the precipitated supernatant was used as an unbound fraction. The heparin sepharose resin is washed twice with 500 μl of physiological sodium chloride concentration Tris buffer. The heparin sepharose resin was then washed with 200 μl of 2M sodium chloride concentration Tris buffer to elute the bound protein. Thereafter, the heparin sepharose resin was washed with a Tris buffer containing 2% SDS and 1% mercaptotoethanol, and the protein that was not eluted with 2M sodium chloride was recovered. Next, the unbound fraction and the 2M sodium chloride / SDS-eluted fraction were separated by 10% SDS polyacrylamide gel electrophoresis. Components in the gel after electrophoresis were electrically transferred to a PVDF membrane, and recombinant mouse arylsulfatase on the transferred membrane was detected with a peroxidase-labeled anti-His antibody. The chemiluminescence reagent Super Signal West Dura Extended Duration Substrate (PIERCE) was used for the detection, and the X-ray film was exposed to light and detected.

  As a result, it was shown that when recombinant arylsulfatase was mixed with heparin sepharose resin, arylsulfatase bound to heparin sepharose resin and was no longer detected from the supernatant (“Supernatant” in FIG. 2). The resin to which arylsulfatase was bound did not dissociate even when washed with 2M NaCl ("2MNaCl" in Fig. 2). However, when treated with 2% SDS, the bond was dissociated and the arylsulfatase was removed in the supernatant. Could be recovered (“2% SDS” in FIG. 2).

In Example 2, to examine the distribution of arylsulfatase in vivo, mouse liver stained with an antiarylsulfatase antibody was observed.
(1) Preparation of fluorescence observation specimen A specimen for immunofluorescence microscope observation was prepared by the following method. Rat or mouse liver tissue was fixed in 4% paraformaldehyde-phosphate buffer containing calcium chloride and magnesium chloride. After washing with a phosphate buffer, the tissue was cut into 40 μm-thick sections using a micro slicer DTK-1500 (DSK Co). The sections were washed with a phosphate buffer, immersed in a phosphate buffer with 0.05% Triton X-100 for 30 minutes, and then blocked with 5% donkey serum. Sections were reacted with anti-rat arylsulfatase antibody and anti-heparan sulfate proteoglycan antibody. Thereafter, it was reacted with a Cy3-labeled donkey anti-rabbit or rat antibody. Furthermore, nuclei were stained with TO-PRO-3 (Molecular Probe, Eugen, OR). Stained specimens were mounted with 90% glycerol-Tris buffer with 0.5 mM p-phenylenediamine and observed with a confocal laser microscope (MRC-1024, BioRad, Hercules, CA, USA).

  The results are shown in FIGS. A and B are stained images of anti-arylsulfatase A (A) and anti-heparan sulfate (B) on the same section of mouse liver. D and E are stained images of anti-arylsulfatase B (D) and anti-heparan sulfate (E) on the same section of rat liver. C is an image of A and B, and F is an image obtained by superimposing D and E images. The localization of arylsulfatase A, B and heparan sulfate was consistent. The nuclei were stained blue. The scale in the figure is 20 μm.

(2) Preparation of specimen for electron microscope observation Electron microscope observation was performed as follows. That is, tissue fixation and section preparation were prepared in the same manner as the fluorescence observation specimen of (1). The sections were reacted with a rabbit anti-arylsulfatase antibody and then reacted with a horseradish peroxidase-labeled donkey anti-rabbit antibody. The specimen was then reacted with 0.05% 3,3′-diaminobenzidine (DAB) containing 0.05% hydrogen peroxide to visualize the reaction site. Thereafter, the specimen was fixed with 1% osmic acid and then dehydrated using ethanol. The specimen was embedded in Epon 812, an ultrathin section was prepared, stained with lead acetate, and observed with a transmission electron microscope JEM-1010 (JEOL, Tokyo, Japan).

  The results are shown in FIGS. G shows the localization of arylsulfatase A, and H shows the localization of arylsulfatase B. In the figure, En represents endothelial cells, He represents liver parenchymal cells, and Lu represents sinusoidal lumen. Arylsulfatase was localized on the surface of endothelial cells as indicated by arrows. The scale in the figure indicates 1 μm.

(3) Method for producing antibody The method for producing the antibody used in the above (1) and (2) is described below. That is, the antigen contains the following sequence for anti-mouse arylsulfatase A:
QYDAAMTFGPSQIAKGEDPA (SEQ ID NO: 10)
Synthetic peptides were used. This sequence corresponds to the amino acid sequence of 464-483 of mouse arylsulfatase A. For anti-rat arylsulfatase B, the following sequence: VASPLLKQKGVKSRELMHIT (SEQ ID NO: 11)
Synthetic peptides were used. This sequence corresponds to the amino acid sequence of 327-346 of rat arylsulfatase B. Rabbits were immunized with these antigens to obtain anti-arylsulfatase antibodies.

In Example 3, in order to examine the cell adhesion activity of arylsulfatase, the cell adhesion properties of arylsulfatase and other matrix proteins were compared.
Three types of cells were used in the experiment: human microvascular endothelial cells (purchased from Kurabo Industries), human aortic vascular endothelial cells (purchased from Kurabo Industries), and human skin epidermis cell-derived cell line E1L8. Endothelial cells were cultured using Medium 200S medium (Kurabo), and skin epidermal cells were cultured using K110 medium (Kyokuto Pharmaceutical). As a substrate for culture, collagen or recombinant mouse arylsulfatase was placed on a slide glass at a concentration of 10 μg / mL, allowed to stand at room temperature for 2 hours, and then blocked with a 1% bovine serum albumin solution. The cells were then cultured at a density of ² × 10 ⁴ / cm ² on each coated substrate. After 1 hour, the cells were fixed with 2% formaldehyde phosphate buffer and then stained with Coomassie brilliant blue. The stained specimen was observed with an optical microscope and photographed. After photographing, the number of cells adhered on a 1 mm × 0.7 mm glass surface was counted, and the number of adhered cells was compared (Table 1).

As a result, it was shown that microvascular endothelial cells and aortic endothelial cells adhere to arylsulfatase and collagen, whereas skin epidermal cells adhere only to collagen.
Cells were cultured on an arylsulfatase coat and bovine serum albumin coat using the cells and culture conditions listed in Table 1, fixed after 1 hour, stained with Coomassie Brilliant Blue, and then observed with a microscope (objective). Lens x10) A photograph is shown in FIG. In FIG. 4, A and B are microvascular endothelial cells, C and D are aortic endothelial cells, and E and F are skin epidermal cells. A, C and E are cultures on arylsulfatase, B, D and F are cultures on serum albumin. The scale in the figure is 100 μm.

  As a result, in a cell adhesion assay using human-derived endothelial cells, arylsulfatase was shown to have higher cell adhesion than control serum albumin. Endothelial cells used were microvascular endothelial cells, aortic endothelial cells, and umbilical vein endothelial cells, all of which were adhered onto arylsulfatase. On the other hand, no adhesion was observed in human skin epithelial cells, and it was revealed that the adhesion of arylsulfatase is cell-specific (Table 1, FIG. 4).

  In Example 4, the morphology of endothelial cells on collagen and arylsulfatase was observed in order to compare the cell adhesion properties of arylsulfatase and collagen. The culture conditions, fixation, and staining conditions were the same as in Example 3. The culture time was 40 minutes, and the microstructure of the cells was observed with an objective lens x40. The result is shown in FIG. In FIG. 5, A shows the case where aortic endothelial cells are cultured on collagen, and B shows the culture on arylsulfatase.

  As a result, it was shown that the endothelial cells were well stretched and adhered on the collagen. On the other hand, the morphology of the endothelial cells adhered on the arylsulfatase was different from the morphology of the endothelial cells adhered on the collagen, and the cells were imparted with the ability to project a temporary foot. It became clear that arylsulfatase has a function to regulate cell morphology.

  In Example 5, in order to examine the effect of arylsulfatase on the morphological change of endothelial cells, endothelial cell adhesion was observed using a mixture of collagen and arylsulfatase as a substrate. That is, the glass surface was coated with a collagen solution (10 μg / mL) or an arylsulfatase mixture (5 μg: 5 μg / mL), and then blocked with 1% bovine serum albumin. The result is shown in FIG. In FIG. 6, A shows culture on a collagen and arylsulfatase mixture, and B shows culture on collagen alone as a control. As the cells, aortic endothelial cells were used. Normal human aortic vascular endothelial cells were cultured on this substrate, fixed with formaldehyde after 1 hour, stained with Coomassie brilliant blue, and observed with a light microscope. Endothelial cells adhered on collagen had few cell processes, but when arylsulfatase was added, cell morphology changed and cell processes were observed.

  As a result, it was shown that cells in the control collagen were evenly expanded, but when arylsulfatase was mixed, the cell's pseudopods increased and the cell morphology changed (FIG. 6). As a result, it has been clarified that the extracellular matrix arylsulfatase regulates the complex morphogenesis of vascular endothelial cells in vivo.

FIG. 1 shows the amino acid sequences of mouse, rat, and human arylsulfatases A and B using single letter abbreviations for amino acids. FIG. 2 is a photograph showing purification of recombinant mouse arylsulfatase and binding to heparin. FIG. 3 is a photograph showing the localization of arylsulfatase and heparin sulfate in liver tissue. FIG. 4 is a photograph of a rat and mouse liver tissue microscope showing adhesion of microvascular endothelial cells, aortic vascular endothelial cells, and skin epidermal cells to arylsulfatase. FIG. 5 is a photograph showing the morphology of endothelial cells on collagen and arylsulfatase. FIG. 6 is a photograph showing the effect of arylsulfatase on morphological changes in endothelial cells.

Claims (9)

  A cell adhesion substrate comprising an arylsulfatase protein.   The cell adhesion substrate according to claim 1, further comprising at least one extracellular matrix.   The cell adhesion substrate according to claim 1 or 2, wherein the arylsulfatase protein is derived from human, mouse or rat.   The cell adhesion substrate according to any one of claims 1 to 3, wherein the cells to be adhered are endothelial cells.   A substrate for cell shape regulation comprising an arylsulfatase protein.   A cell culture substrate comprising the cell adhesion substrate according to claim 1.   The method to adhere | attach a cell using the substrate for cell adhesion of any one of Claims 1-4.   A method for regulating cell morphology using the cell morphology regulating substrate according to claim 5.   A method for culturing cells using the cell culture substrate according to claim 6.

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