JP3859732B2 - Solid phase hepatocyte proliferating agent - Google Patents

Description

[0001]
[Industrial application fields]
TECHNICAL FIELD The present invention relates to a hepatocyte proliferating agent, and more specifically, hepatocyte proliferation in which the growth factor is immobilized by binding glycosaminoglycan, glucan or a derivative thereof to hepatocyte growth factor. It relates to the agent.
[0002]
[Prior art and problems to be solved by the invention]
In recent years, various cell growth factors have been cloned, and among them, a group of growth factors having a strong affinity for heparin has been found. This is collectively called heparin-binding growth factor, which includes fibroblast growth factor (hereinafter abbreviated as FGF), keratinocyte growth factor (KGF), pleiotrophin, granulocyte / macrophage colony formation stimulus. Various known or unidentified growth factors such as factor (GM-CSF), interleukins 3 and 7, vascular endothelial growth factor (VEGF) are included (Experimental Medicine, 9 (14), 1772-1776 (1991). )). These factors are known to bind to glycosaminoglycans represented by heparin and heparan sulfate. Among them, FGF is stored on the cell surface and extracellular matrix by binding to heparan sulfate proteoglycan, And it was confirmed that the biological activity of FGF can be regulated by heparin (Cell, 64, 841-848 (1991)).
[0003]
Carpenter et al. Found human hepatocyte growth factor (hHGF) from the plasma of patients with fulminant hepatitis that can extract hepatocytes from the living body and promote their growth in vitro (Patent Document 1: No. 63-22526), Kitamura et al., The amino acid sequence of hHGF protein, the gene (cDNA) sequence encoding the same (Japanese Patent Laid-Open No. 3-72883), and the production method and character of hHGF protein using this cDNA A converter was invented (JP-A-3-285893). The hHGF protein produced by this invention has been recognized to promote the growth of hepatocytes in vitro. In addition, not only hHGF but also HGF protein has been found to be a kind of the above-mentioned heparin-binding growth factor having a strong affinity for heparin.
[0004]
On the other hand, it is generally considered that not only humoral factors but also extracellular matrix (ECM) adjacent to cells play an important role in regulating cell growth and motility. The main components constituting the ECM are fibrous proteins such as collagen and elastin, adhesive proteins such as fibronectin and laminin, and complex carbohydrates such as proteoglycans having glycosaminoglycan sugar chains. Of these, it is not easy to prepare proteoglycans from biomaterials, and the analysis of the physiological significance of glycosaminoglycans in ECM and the interaction with growth factors has been delayed.
[0005]
Proteoglycan is a generic name for glycosaminoglycan and protein covalently bonded compounds, but in recent years, proteoglycan model compounds in which various glycosaminoglycans are covalently bonded to other proteins and phospholipids instead of natural core proteins (Neoproteoglycan; J. Biol. Chem., 264 (14), 8012-8018 (1989), Japanese Patent Laid-Open Nos. 4-80201 and 4-80202). This neoproteoglycan makes it possible for the first time to efficiently immobilize glycosaminoglycan sugar chains on a culture dish.
[0006]
In the prior art, it was possible to add various growth factors to the solution, including heparin-binding growth factors such as hHGF, and observe the action. Since the phase could not be obtained, the effect of the solid phase growth factor has not been analyzed. In addition, when a growth factor is administered in vivo, it has been technically difficult to keep the growth factor local.
[0007]
[Means for Solving the Problems]
The present inventor attempted to immobilize the growth factor using the binding property of HGF to glycosaminoglycan and to examine the action thereof. As a result, interestingly, HGF was immobilized on a glycosaminoglycan, and it was found that it could be a growth stimulus for hepatocytes, and the present invention was completed.
[0008]
That is, the gist of the present invention resides in a solid phase hepatocyte proliferating agent characterized by binding hepatocyte growth factor to glycosaminoglycan, glucan or a derivative thereof.
Hereinafter, the present invention will be described in detail.
As the hepatocyte growth factor (HGF) used in the present invention, any of those obtained by purification from biomaterials and those obtained by expressing the cDNA by a recombinant method can be used. Specific examples of human HGF (hHGF) include protein chemical separation and purification from the plasma of patients with fulminant hepatitis according to the method described in JP-A-63-22526, or JP-A-3-285893. Examples include those obtained by constructing an expression vector containing cDNA encoding hHGF according to the method described in the publication and expressing it in a host such as CHO cells. Such hHGF starts to show hepatocyte proliferating activity at a concentration of about 0.5 to 2 ng / ml, and reaches the maximum proliferation-inducing activity at a concentration of about 5 to 10 ng / ml.
[0009]
In the present invention, glycosaminoglycan, glucan or a derivative thereof is used as a carrier for immobilizing the above HGF. Glycosaminoglycan is a general term for polysaccharides containing hexosamine as a constituent sugar, and specifically includes hyaluronic acid, chondroitin, taicuronic acid, colominic acid, chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, Examples include chondroitin sulfate D and E (chondroitin polysulfate), heparin, keratosulfuric acid (keratan sulfate), heparitin sulfate (heparan sulfate), acidic glycosaminoglycan such as polyacetylgalactosamine phosphate, chitin, galactosaminoglycan, etc. It is done. On the other hand, glucan is a general term for polysaccharides containing glucose as a constituent sugar, and specifically includes dextran, dextran sulfate, laminaran, lihenan, cellulose, amylose, amylopectin, etc. In the present invention, dextran or dextran sulfate is preferred. . These can be appropriately prepared using known methods.
[0010]
Further, the glycosaminoglycan or glucan derivative is not particularly limited, but in the present invention, it is preferably a protein that can be covalently bound to glycosaminoglycan or glucan, or a derivative with phospholipid. Proteins include in vivo proteins such as albumin, and phospholipids include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol, and sphingophospholipids such as sphingomyelin. Can be mentioned. These protein derivatives can be prepared by a method known as a method for producing a so-called neoproteoglycan (for example, the method described in J. Biol. Chem., 264 (14), 8012-8018 (1989)), and phosphorous Lipid derivatives can be prepared according to the methods described in JP-A-4-80201, JP-A-4-80202, and the like.
[0011]
In the present invention, HGF is bound to glycosaminoglycan, glucan or a derivative thereof. At this time, since HGF is originally heparin-binding, it can be easily bound by mixing the two. However, phosphate buffer-saline may be allowed to coexist as long as the properties are not impaired. HGF and glycosaminoglycan, glucan or a derivative thereof are preferably combined at a molar ratio of 1: 1 to 1: 1000. Furthermore, in the present invention, glycosaminoglycan, glucan or a derivative thereof is previously bound on a cell adhesion protein (hereinafter abbreviated as “culture substrate”) such as collagen, elastin, fibronectin, laminin, vitronectin, cadherin. It is preferable because it can be solid-phased more stably. In this case, glycosaminoglycan, glucan or a derivative thereof may be coated directly or indirectly on a culture substrate, and HGF may be added in the form of a solution thereon to be immobilized. For example, a sheet-like polymer base material may be used. Such a polymer substrate is not particularly limited as long as it is a material compatible with a living body. When a culture substrate is used, it may be used after further binding on a polymer substrate. The thus-immobilized HGF can induce the inherent physiological activity of the growth factor locally by exposure to cells or tissues.
[0012]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples as long as the gist thereof is not exceeded.
Example 1 Preparation of a carrier capable of inducing hepatocyte proliferation of hHGF solid-phased with neoproteoglycan: A multi-well plastic dish (Corning) having a 2.3 cm diameter well was first prepared with a bovine-derived type I collage. Genogen solution (10 μg / ml, KOHKEN) was used as a culture substrate, and then heparin-phosphatidylethanolamine (Hep-PE) was used as a synthetic glycosaminoglycan lipid derivative (GAG-PE). Coating was carried out at a concentration of 10 μg / ml to obtain a solid-phase support (Hep-PE was prepared according to the method described in Example 1 of JP-A-4-80201). As a control experiment, a well not coated with a synthetic glycosaminoglycan lipid derivative was also prepared.
[0013]
Immobilization of hHGF: Recombinant hHGF (prepared according to the method described in JP-A-3-285893), 10 mM phosphate buffer-physiology containing 1% bovine serum albumin (BSA) to a final concentration of 10 ng / ml It was prepared with saline solution, pH 7.4 (PBS (−)) and added to the carrier. After reacting at 4 ° C. for 6 hours, the solution was removed, and the carrier was washed 5 times with PBS (−). As a comparative experiment, a well in which the carrier was further washed twice with 1M NaCl was also prepared.
[0014]
Cell preparation: According to the method of Seglen (Methods in Cell Biology, vol 13, p29, Academic Press, New York (1976)), from a Wistar male rat (body weight 200 g), 0.05% collagenase ( Hepatocytes were isolated using Type I, Sigma). The liver parenchymal cells were sowed in a multiwell plastic dish (Corning) coated with the above carrier and hHGF at a concentration of 2.5 × 10 ⁴ cells / 0.25 ml / cm ² and 5% carbon dioxide gas. Monolayer culture was performed at 37 ° C. in an air-containing gas phase (Tanaka et al., J. Biochem. 84, 937-946 (1978)). As a culture medium, 5% fetal calf serum (FCS, Filtron), Williams E (WE) medium (Fro Laboratories, Inc.) supplemented with 10 ⁻⁸ M insulin, 10 ⁻⁸ M dexamethasone and 60 μg / ml gentamicin. (Hereinafter abbreviated as “basic medium”).
[0015]
Measurement of cell proliferation inducing activity: 2 hours after the start of the culture, the medium was replaced with a new basic medium, and 18 hours later, the medium was replaced with a WE medium containing no fetal calf serum (containing 10 ⁻⁷ M insulin and 60 μg / ml gentamicin). 28 hours after the start of the culture, 1.25 μCi of [methyl ³ H] thymidine (Amersham) was added per well, and 10 μg / ml of aphidicolin (Sigma) was added as necessary. After 24 hours, cells were fixed with 1 ml of cold 10% trichloroacetic acid for 1-2 hours, washed 3 times with 95% ethanol, and lysed with 1M NaOH. A scintillator was added to the neutralized cell lysate, and after mixing well, the radioactivity incorporated into the cells was measured with a liquid scintillation counter. Replicated DNA synthesis values were calculated by subtracting the count in the presence of aphidicolin from each radioactivity count. As a control group, the radioactivity was obtained when GAG-PE was not added to each sample, and the result was shown as a ratio to the value (FIG. 1). As a comparative experiment, a similar experiment was performed using epidermal growth factor (EGF) having no affinity for glycosaminoglycan instead of hHGF. The results are shown in FIG. The vertical axis represents the DNA synthesis inducing activity of each sample as a ratio (%) to the DNA synthesis inducing activity of the control group that did not use GAG-PE. It can be seen that when Hep-PE is used as a culture substrate, hHGF added prior to cell infiltration is effectively solid-phased and can stimulate liver parenchymal cell DNA synthesis even after medium replacement. That is, an increase of 66% was recognized compared with the control (a).
Further, even when hHGF was added as a solution to hepatocytes on Hep-PE as a conventional solution, about 20% of DNA synthesis was enhanced as compared with the control (b). When immobilized hHGF was washed with 1M NaCl, the DNA synthesis enhancement decreased from 66% to 33% (c). This is interpreted as a part of the solid phase of hHGF was removed by washing with 1M NaCl. For EGF without heparin binding ability, no difference was observed depending on the presence or absence of GAG-PE (d and e). This suggests that hHGF binds on GAG-PE by binding to glycosaminoglycan as expected and is immobilized on a culture substrate.
[0016]
From the above results, by combining hepatocyte growth factor and glycosaminoglycan, glucan or derivatives thereof, the growth factor can be effectively immobilized, and the immobilized growth factor can exert physiological action. I understand that.
[0017]
【The invention's effect】
Conventionally, when using a hepatocyte growth factor, there is no known method for immobilizing the protein in such a state that the activity of the protein is retained or using the immobilized growth factor. As shown in the present invention, it is possible to immobilize hepatocyte growth factor while retaining activity by using an appropriate glycosaminoglycan, glucan or a derivative thereof. The growth factor has become available in vivo or in vitro. By devising a solid phase-immobilized carrier for hepatocyte growth factor based on the present invention, it can be expected to be widely used in the medical field, and can also be expected to have a sustained release effect and an accumulation effect when administered to a living body.
[Brief description of the drawings]
FIG. 1 is a drawing showing immobilization of hHGF with a synthetic glycosaminoglycan lipid derivative (Hep-PE).

Claims (2)

  A hepatocyte growth agent comprising a solid phase-enhanced hepatocyte growth factor, in which a hepatin-phosphatidylethanolamine-coated carrier is immobilized on collagen.   The hepatocyte proliferating agent according to claim 1, which has an ability to induce hepatocyte proliferation.

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