JPS61128974A - Artificial base membrane and its production - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Wood climbing I 1 day 1 10 people 10 people T 10 lit~dan 10! Window noise ↓
L market price h The basal surface of Izuki cells and mesothelial cells, or smooth muscle cells, transverse strangulated muscle cells, adipocytes, Schwann (Schw
ann) It is an intercellular interstitium whose main components are fulladen and glycoprotein, which surrounds the surface of free cells such as Ml follicles. Its functions include selective control of material movement into and out of cells;
Previous studies have shown that they play roles such as inducing cell differentiation and tissue development, supporting tissues through adhesion mechanisms with cells, and promoting and controlling cell proliferation.

At present, it is generally difficult to propagate epithelial cells such as epidermal cells, vascular endothelial cells, and hepatic parenchymal cells in vitro. Many epithelial cells exist in contact with the basal lamina in vivo, and are able to maintain their normal proliferation and function by being in contact with the basement membrane, which has the functions described above.

Therefore, the present invention provides a method that substitutes the function of the natural basement membrane.
That is, it is a therapeutically efficient drug that enables large-scale proliferation of epithelial cells in vitro, and can be used not only to simply cover injured areas, but also to help regenerate epithelial cells without developing keloids or hypertrophic scars. The purpose of the present invention is to provide an artificial basement membrane that is also useful as a wound dressing.

That is, the present inventors completed the present invention based on the fact that an artificial basement membrane having type 1 fulladen, glycoprotein, and acidic mucopolysaccharide and having physical strength can achieve the above object. To. Below, each material, manufacturing method, effects, etc. of the artificial basement membrane of the present invention will be explained in more detail.

Materials The essential material constituting the artificial basement membrane of the present invention is ■-type fulladen.

Among glycoproteins and acidic mucopolysaccharides, laminin and fibronectin are particularly suitable as glycoproteins, and heparan sulfate and chondroitin sulfate are particularly suitable as acidic mucopolysaccharides. Moreover, it is more effective to further contain a basement membrane extract and platelet-containing plasma in addition to the above-mentioned materials.

In the present invention, the basement membrane extract includes not only type II collagen but also unknown components that exist in the living body as an intercellular matrix.

Naturally, these materials should be selected in consideration of their adaptability to the human body and a certain degree of physical strength.

Examples of methods for obtaining each material are as follows.

1. Type ■Fladen is extracted and purified from human placenta and human amniotic membrane using a conventional method. That is, for example, the following documents may be referred to.

T., F., Krisna, etc., Biochemistry (T.,
F, Kresina et aL Bioch
emistry) 1979t18, 3089-309
7.R, varnish, pine crystal, etc., biochemistry (
R, S, MacWright et aL
Biochemistry) 1983w22, 49
40-4948 Also, Mouse Type Collagen manufactured by BRL Co., Ltd. and Collagen Type Colladen derived from bovine anterior lens capsule manufactured by Nitta Gelatin Co., Ltd. may also be exemplified as suitable materials, and both are readily available as commercial products.

28 laminin purified from human placenta or EHS S arcoma can be used. Especially EH3Sarcom
A is more suitable for obtaining laminin because it produces a large amount of basement membrane.

Methods for obtaining laminin from EHS S arcoma include methods such as
Inventory - Inlet Sea (Pamela Gehr)
on Robey et al.
Methods in Enzy-molog
yt) Vol, 82.831 838. is referenced. That is, ① tumors were grown subcutaneously in C57BL mice, 5
- Harvest after reaching 15g amount.

■ Tumor tissues (approximately 1 kg) were treated with a total of 6 to 81 chilled 3,4 M NaCl, 0.05 M Tris-H.
CI-pH7,4 (as a protease inhibitor, 0.0
Homogenize three times with 1 M EDTA, 50 Mg/ml p-hydroxymercurybenzony), 50 Mg/ml phenylmethanesulfonylfluoride).

■ Immediately centrifuge the homogenized sample (9000
, 30ml, 4°C) to remove the soluble fraction.

■ The residue was incubated at 4°C overnight three times, i-i.
, sl of 0.5M NaCl, 0.05M Tri
Stir and extract in s-HCI, pH 7.4 (inhibitor above).

■ The extract contains a large amount of 2M urea, 0.05M
Dialyze against Tr+s-HCl, pH 8.6.

■ D E A E -cel 1uloseri a-p
Pour it on the thornfish. Samples that do not adsorb to the column are subjected to ultrafiltration (AIIlicon, Diafloyfiltration).
Concentrate to 50 mN volume on an erXM 100 A).

■ Equilibrated Agarose A-1,5m column (3,
5X140cm) through IM CaC1,,0,05
Elute with M Tris-HCl, pH 7.4.

■ 0.4M NaC1,0.05M Tris-H
Dialyzed against CI, pH 7.4, if necessary then 0
．． Dialyzed against 2M ammonium bicarbonate oH7-9, 4'C
Alternatively, commercially available mouse laminin manufactured by EY Laboratories may be used.

3. Heparan sulfate and funidroitin sulfate Mucopolysaccharides isolated from human or animal tissues by conventional methods are
It is obtained by fractionation and purification using c (cetylpyridium chloride), an ion exchange resin, or the like.

- As an example, the method for obtaining it from rat skin is shown below.

◎ Grind the male bovine skin ↓ Degrease with acetone ↓ Acetic acid buffer (0.1M pH 5.5 + semM
Suspend dried tissue at a ratio of g/20d in cysteine hydrochloride + 5mM EDTA) ↓ Add papain (2mg/g dry tissue) and store at 60°C for 24 hours.
hr digestion ↓ The solution is dialyzed in running water for 24 hr ↓ Dialysis with DW at 4℃ for 24 hr ↓ Trypsin digestion (pH 7, S, -2 hr) ↓ Addition of TCA (final 10%) ↓ The generated precipitate is removed by centrifugation ↓ Acid soluble part Dialyzed against DW at 4°C. Fractionated using CPC (cetylpyrinium chloride) A sodium chloride solution from CPC (K&K Labo) was added to the mucopolysaccharide concentrate, stirred, and allowed to stand at 37°C for 1 hr to precipitate.

↓ Add Celite (20mg/a+g polysaccharide) and stir ↓ Centrifuge (270Orpm* 30ml) ↓ Wash the precipitate with 0.03M sodium chloride solution containing 0.1% CPC and centrifuge (2700r4, 20') ↓ Wash 3 to 6 times with 0.4M NaCl solution containing 1% CPC (remove supernatant by centrifugation) to completely elute hyaluronic acid ↓ Chondroitin sulfate and heparan sulfate are eluted with 1.2M NaCl solution containing 0.1% CPC ■ Aqueous solution of fractionated and purified acidic mucopolysaccharide (5-10%) using ion exchange resin owex 1
(X2,200-400mech, C1-type) column (
0.9x44ca+) ↓ Wash well with water ↓ Elute with NaCl 0.5M → Hyaluronic acid 1.25M → Heparan sulfate 1.5M → Chondroitin sulfate 2.0M → Heparin, acidic mucopolysaccharides (heparan sulfate, chondroitin) Sulfuric acid, etc.) can be used by repurifying commercially available materials.

4. Basement membrane extract It can be isolated from human placenta, renal glomeruli, renal tubules, eye retina, brain capillaries, etc. by conventional methods, but human placenta from white rice is often used because it is generally easily available.

For isolation of human placental ground substance, for example, Japanese Patent Application Laid-Open No. 55-1
20517, a method of removing cellular components from capillaries isolated from human placenta by treating them with a nonionic alcohol-based detergent and ultrasound, or by removing cellular components from a villus fraction. Gang et al., P.N.I.B.M.

(Nicholas F, Gangelal P,
S, E, B, M, +) 1972tVo1.14
0. 188-193 extraction methods can be used.

That is, an example of a method for separating the basement membrane of the placenta is as shown below.

From a normal placenta immediately after full-term delivery of a human pregnancy, decidua, uterine membrane,
! Remove the band and use only the villous fibers. Next, add 2 pieces of this villous fiber.
After cutting into ~3 cm strips and washing several times in saline to remove blood, metal
mesh) (first No., 80° then No.
, 200 metal mesh
) with physiological saline). Furthermore, this filtrate is No. 325 Metal Metal! (metal m
The terminal villi remaining on the mesh were collected and suspended in physiological saline at 1*500 rpm.
Centrifuge for 5 minutes. After centrifugation, the precipitate is resuspended in physiological saline and centrifuged at 1°, 500 rpm for 15 minutes five times until the supernatant becomes clear. The terminal villus fraction obtained was suspended in physiological saline and sonicated at maximum output (
The basement membrane and cell components are separated under conditions of 150 W, 100 V). Next, the suspended sample is transferred to a centrifuge tube, and centrifugation is repeated 5 to 6 times at 5+000 rpm for 15 minutes to obtain basement membrane components as a precipitate.

The basement membrane extract separated in this way contains unknown basement membrane factors in addition to existing factors such as tyl)e IV Col Iagens laminin, fibronectin, and heparan sulfate, and it is possible to create a clinically useful artificial basement membrane. important to configure. In particular, regarding unknown intercellular matrix components, the method of perfusing animal organs with a protein denaturing agent, which the present inventors have already announced at the 57th Annual Meeting of the Japanese Biochemical Society (October 7, 1980), is effective. According to this method, a component containing an unknown basement membrane component is obtained. This extract can be used as it is or, if necessary, after being subjected to del filtration or the like.

Next, using cubic atelocollagen as a substrate, type ■colladen, glycoprotein, acidic mucopolysaccharide, and basement membrane extract are overlaid or sprayed on top of it to form a gel, and then platelet-containing plasma is uniformly applied and freeze-dried. can get. A specific example of the manufacturing method is as follows.

Example 1 4℃ neutral atelocollagen solution (o, oi~0.5%)
Place it in a quartz plate or glass plate to a thickness of 0.1 to 2 m+a, and layer type II collagen, glycoprotein, acidic mucopolysaccharide, and basement membrane extract on the liquid surface, or mix the four. I'll put it on. At this time, be very careful not to make the whole part uniform. Next, this multilayered material was kept warm at 37°C to make it into a delta, platelet-containing plasma (PRP) was evenly adhered to cover the whole, and freeze-dried.
Obtain a sheet-like artificial basement membrane. Incidentally, the ``type fulladene glycoprotein'' and the mucopolysaccharide may be used in equal amounts, or the mixing ratio may be changed appropriately depending on the purpose.

Example 2 Sponno-like atelocolladen with a thickness of 0.1 to 2 mm was sprayed with ■-type fulladen, glycoprotein, mucopolysaccharide, and basement membrane extract in sequence, kept warm at 37°C, and then PRP was applied in the same manner as in the previous example. Attach and freeze-dry.

Example 3 Type 1 fulladen in atelocollagen solution (0.01-0.5%).

A mixture of Tong protein, acidic mucopolysaccharide, and basement membrane extract is mixed, applied onto delta-like atelocollagen (0.1 to 2 cm thick), gelled at 37°C as in the previous example, and freeze-dried. . PRP is then uniformly deposited and freeze-dried again to obtain an artificial basement membrane.

Atelocollagen is used for the purpose of increasing the physical strength of the artificial basement membrane, and as a coating substrate, biocompatible or safe pond materials, such as segmented materials with good air permeability, can be used as the coating substrate. It is also possible to use polyurethane, silicone, gucron, etc., or cellophane film.

The artificial basement membrane obtained as described above can be treated with ultraviolet rays,
It is sterilized by irradiation with gamma rays and can be stored until use.

In addition, if stronger strength is required for the Colladen substrate, it is desirable to irradiate it with ultraviolet rays, gamma rays, etc. while it is in a gel state.

Effects The artificial basement membrane of the present invention is different from existing artificial skin that has a mere covering effect, and is characterized in that it converts into a natural basement membrane when transplanted into a living body. Therefore, by covering the injured site with this artificial basement membrane, the regeneration of the epithelial cells of the tissue concerned is facilitated, and wound healing is promoted. Since it can regulate cell differentiation and suppress the development of keloids and hypertrophic scars, it is particularly effective as a dressing for the surgical side of extensive burns and hypertrophic scars.

It is also effective as a culture substrate for epithelial cells, which are difficult to proliferate in vitro. Furthermore, application to cell-integrated artificial skin is also possible.

'? JL Katsuragi (Production of artificial basement membrane) Atelocollagen derived from bovine skin (Koken; Pepsin digestion■
A 0.4% pH 7.4 aqueous solution of collagen type) was kept at 4°C and made into a 0.4% aqueous solution with a thickness of 0.4% pH 7.4. To make it Smm〃Lass play) (
50×50aus). ■Type fulladen and heparan sulfate are on the surface of this atelocollagen aqueous solution.

Laminin and basement membrane extract were sequentially layered and kept at 37°C to completely gel. Next, platelet-containing plasma (PRP) was uniformly adhered to this and freeze-dried. The obtained artificial basement membrane was irradiated with ultraviolet light.

Example 2 (Transplantation of Artificial Basement Membrane) The artificial basement membrane of the present invention was transplanted to an individual animal to examine its effect on wound healing. ) was cut into a size of 50 mm x 5 m II and used as a standard.

As experimental animals, guinea pigs (female? Hartley
Immediately before transplantation, the animal's back was shaved to a length of 60A+InX 30 mm using an electric knife, and the remaining hair was removed by vacuum suction. Anesthetized with ether, and the back 70/30
Improper tool/washed with water.

Next, a 50 mm x S mm area of the meat tissue membrane was cut out from the hair-removed area and dabbed with sterilized gel soaked in physiological saline to remove bleeding from the wound. The specimen was placed tightly over the wound, covered with sterile gel, and fixed with a bandage. When the progress of the wound treatment was observed every day, it remained unchanged from normal tissue, and on the 8th day,
The skin was completely epithelialized and healed well on the 200th day.
There was no visible difference from the surrounding area. When the transplanted area was cut out and histologically examined, it was confirmed that the skin was regenerated favorably in the following order: dermis, basement membrane, epithelial cells, and stratum corneum.

Patent Applicant Advance Development Research Institute Co., Ltd. Procedural Amendment (Method) % Formula % 1. Indication of the case Patent Application No. 248719 of 1982 2. Name of the invention Artificial basement membrane and its manufacturing method 3. Person making the amendment Case Relationship with Patent applicant (Shipping date: March 26, 1985) 5. Full text of the specification to be amended 6. Contents of amendment The academic literature etc. of the language were described more accurately, including the Japanese name.

(However, the content remains unchanged) The entire text of the amended specification is as attached.

Claims (4)

[Claims] (1) An artificial basement membrane characterized by having type IV collagen, glycoprotein, and acidic mucopolysaccharide. (2) The artificial basement membrane according to claim 1, further comprising a basement membrane extract. (3) The glycoprotein is laminin and/or fibronectin, the acidic mucopolysaccharide is heparan sulfate and/or chondroitin sulfate, and the basement membrane extract is a human placental villus extract. (
The artificial basement membrane according to item 1) or item (2). (4) A layered product obtained by layering or spraying type IV collagen, glycoprotein, acidic mucopolysaccharide, and basement membrane extract on a substrate made of atelocollagen is freeze-dried, and then platelet-containing plasma is applied and freeze-dried. A method for producing an artificial basement membrane, characterized by: