JP3133148B2 - Human cellular fibronectin, its production method and cell line - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to human cellular fibronectin, a method for producing the same, and a human hepatoblastoma-derived cell line HUH-6YM using the method. Fibronectin is useful as an additive for a medium in animal cell culture, and has uses as a diagnostic agent, cosmetics, and a pharmaceutical raw material.

[0002]

2. Description of the Related Art Generally, fibronectin is a bioactive protein having an action as a cell adhesion factor. Two types are known: plasma fibronectin present in plasma and cellular fibronectin mainly produced by fibroblasts and present in tissues. These actions of fibronectin are deeply involved not only in cell adhesion and spreading, but also in phenomena such as cell chemotaxis, phagocytosis, differentiation, cell morphology regulation, cell motility, canceration and cancer metastasis. It has been reported. Since fibronectin has various effects on cells in this way, it has been applied to pharmaceuticals such as therapeutic agents for tissue damage and cosmetics to improve the condition of skin tissue. I have. In addition, application of fibronectin to diagnostic agents utilizing the property of specifically binding to various substances such as heparin and collagen, and application as a diagnostic agent marker are also being promoted.

[0003] However, fibronectin is a proteinaceous factor derived from animal tissues or its plasma, etc. In order to prepare it in large quantities, it is necessary to secure its raw materials and impurities derived from the raw materials, such as viruses, for purified products. There is a problem such as mixing.

In order to obtain human cellular fibronectin, there has been known a method of extracting human fibronectin from human placenta or purifying it from a culture supernatant of fibroblasts. It was difficult to prepare in large quantities.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to prepare human cellular fibronectin in large quantities.

[0006]

Means for Solving the Problems The present inventors have developed HUH-6clone5 (Jap) previously isolated from human hepatoblastoma.
anne Cancer Research Res
source Bank, cell number JCRB0401)
Can be cultured in a serum-free medium supplemented with insulin alone as a protein factor, and produce a significant amount of plasma fibronectin in the culture supernatant, and a method for producing plasma fibronectin using the same was found. Patent filed. (JP-A-3-160989).

[0007] Then, the present inventors further developed HUH-6cl.
When cloning was repeated from one5 and examined, HUH-6Y, a human hepatoblastoma-derived mutant strain that can be cultured in a protein-free medium and can be subcultured without limit
M (hereinafter referred to as cell line HUH-6YM) was established and established as a cell line.

[0008] The cell line has been deposited with the Japan Institute of Fine Arts as FERM P-12890 as a cell line HUH-6YM.

[0009] Furthermore, surprisingly, the cell line HUH-
The fibronectin produced by 6YM was found to be cellular fibronectin, and the cellular fibronectin was found to be highly soluble in sucrose solutions. The present invention has been completed based on the above-mentioned findings. It comprises culturing human cellular fibronectin produced by cell line HUH-6YM and cell line HUH-6YM, separating cellular fibronectin from the resulting culture, The present invention relates to a method for producing cellular fibronectin, which is characterized by being purified and prepared at a high concentration, and a cell line HUH-6YM which is a novel human hepatoblastoma-derived cell line used in the method.

[0010]

According to the present invention, a method for producing human cellular fibronectin in large quantities is provided. The cellular fibronectin obtained by the present production method is a cell line HU
Since H-6YM can be obtained from a supernatant cultured in a protein-free medium, there is no danger of contamination with foreign proteins or viruses.

As a result of the present invention, it has been revealed that human cellular fibronectin can be mass-produced using a cell line obtained by cell selection and transformation.

[0012] Furthermore, the fact that fibronectin acts upon implantation of fertilized eggs, and that (Wartiovaara,
J. et al. , Develop. Biol. , 197
9; 247-257) Cellular fibronectin, unlike plasma fibronectin, is more highly expressed in fetal tissues than in adult tissues (Oyama, F. et.
al. , Biochemistry, 1989;
1428), and in particular, cellular fibronectin
It is thought to be an important factor for cell migration and chemotaxis.

In other words, it can be used as a medicament, cosmetic or diagnostic agent characteristic as a repair agent for cell migration upon tissue damage or a factor for maintaining epidermal cell homeostasis. Further, since it has basically the same biological activity as plasma fibronectin, it can be used for similar uses.

[0014]

DETAILED DESCRIPTION OF THE INVENTION The cell line HU used in the present invention
H-6YM is a mutant strain obtained as a result of cloning HUH-6clone5 at the cellular level.
It is clearly different from -6clone5, and a biological variation is observed. HUH-6 clone already reported
5 has 49 chromosomes (Sato, J. et.
al; Acta Med. Okaya 34 (2),
127-130 (1980)), the number of chromosomes in HUH-6YM is higher, and the cell line HUH-6YM is specific in that it can grow in a protein-free medium.

This cell line HUH-6YM was specifically obtained by the following cloning method. First, an e-RDF medium containing insulin (0.04 IU / ml) (Japanese Society of Agricultural Chemistry, Vol. 58, p575-583)
1984), a serial dilution of HUH-6 clone 5 was prepared, and seeded on a Petri dish having a diameter of 100 mm.
The cells were cultured under CO ₂ -containing air at 37 ° C. for 2 to 4 weeks.

Among the formed colonies, a colony considered to have been formed from a single cell was selected, transferred to a 24-well plate, and clones having a high production of cellular fibronectin were selected using the same method as above. By performing several-order cloning, cell line HUH-6YM was obtained. In addition, this cell line HUH-6YM was able to grow and subculture only in the e-RDF medium.

Further, cDNA probes for peptides called ED-A and ED-B regions, which are present only in cellular fibronectin, were prepared, and mU of HUH-6YM was prepared.
Examination of NA confirmed the presence of the ED-A and ED-B regions.

The cell line HUH-6 thus obtained is
The biological properties of YM are summarized below. It is a mutant derived from the human hepatoblastoma cell line HUH-6clone5. It grows on a basal medium consisting of amino acids, vitamins, sugars and inorganic salts and can be subcultured without limit. That is, the cell line does not require a protein factor as a growth factor. Produces significant amounts of human cellular fibronectin. Fibronectin mRNA containing the ED-A and ED-B regions is expressed.

Next, a method for producing human cellular fibronectin by culturing the cell line HUH-6YM will be described.

The cell line HUH-6YM is cultured in a petri dish as a seed culture. Repeat the passages to ensure the required number of cells in the next continuous culture. As the medium to be used,
A commercially available e-RDF medium is suitable, but not limited thereto, and any medium prepared according to its components can be used. As an additional component, a protein factor is not particularly required, but may be added as long as it does not adversely affect cells.

The culture conditions are not particularly limited, but the culture temperature is 36 to 37 ° C., and the gas phase condition is suitably air containing about 5% CO ₂ in seed culture using a petri dish or the like. . However, in continuous culture, CO
_There is no need for _2nd grade prepared air.

The method of continuous culture is not particularly limited, but it is possible to carry out by a general method using a roller bottle. In this case, the medium is changed at an appropriate time according to the number of cells in the roller bottle.

[0023] The cell line HUH-
The 6YM culture supernatant is used as a raw material for the next purification.

For purification of cellular fibronectin, a method generally used for protein purification can be used. One example is as follows.

First, an appropriate protease inhibitor is added to the culture supernatant, followed by centrifugation to remove impurities such as cell debris, and then concentration is performed by ultrafiltration. Raise the fibronectin to the appropriate concentration. This is to make the next purification more efficient. The concentrated supernatant was washed with PBS (-) (Phosphate Bu).
The gel is loaded on a gelatin affinity column equilibrated with the above-mentioned method (referred Saline). Then, PBS (-)
Is eluted with a buffer in which 6M urea is dissolved, and the eluted fraction is obtained. The purity of cellular fibronectin in this fraction is 95% or higher according to the assay by electrophoresis, but to further increase the purity, it is possible to increase the purity by using other methods such as a general ion exchange method. Although it can be purified, cellular fibronectin usually has a very low solubility in a buffer such as PBS (-), so that it is necessary to perform the separation operation in the presence of urea.

Thereafter, the urea is removed, and the buffer is exchanged with a phosphate buffer containing 5% sucrose to prepare a cellular fibronectin solution having a certain concentration or higher. The method of exchange with a buffer solution can be performed by a general method such as membrane dialysis or diafiltration using ultrafiltration.

The human cellular fibronectin thus obtained is obtained as a mixture of a dimer and a monomer. The structural features of cellular fibronectin include:
According to the analysis of SDS polyacrylamide gel electrophoresis under reducing and non-reducing conditions, it has a molecular weight larger than that of a plasma fibronectin preparation and reacts with a commercially available anti-cellular fibronectin antibody.

The properties of human cellular fibronectin produced using the cell line HUH-6YM are shown below. Molecular weight SDS polyacrylamide gel electrophoresis in reduced and non-reduced state showed dimer of 470 kd and monomer of 24
5 kd and 225 kd, with a dimer to monomer ratio of 2:
It is one. Isoelectric point: pH 5-6 Immunological specificity Commercially available anti-cellular fibronectin antibody (manufactured by Sigma, M
ONOCLONAL ANTI-CELLULAR F
IBRONECTIN, Product No. F61
40), and also with an anti-human fibronectin antibody (clone 30-8, manufactured by Takara Shuzo). As a result of the protease fragmentation, the protease fragment of human cellular fibronectin of the present invention was clearly different from the protease fragment of human plasma fibronectin. In addition, the human cellular fibronectin produced by the present invention has the previously reported biological effects of fibronectin such as cell adhesion promoting activity, gelatin bead aggregating ability and cell spreading activity.

As described above, human cell fibronectin can be produced very advantageously by continuously culturing a large number of cells in a cell-free medium using the cell line HUH-6YM.

[0030]

【Example】

[I. Production of substance] As seed culture, cell line HUH-6Y
M (FERM P-12890) was subcultured using e-RDF medium (manufactured by Kyokuto Pharmaceutical). Culture at 37 ° C, 5%
Performed for one month with air containing CO ₂ .

The continuous culture for obtaining the culture supernatant is 175
1 × 10 ⁹ cells were seeded per one 0 cm ² roller bottle (manufactured by Falcon) and cultured at 37 ° C. in the atmosphere. Until three weeks after seeding, the entire amount of the medium was changed every 500 ml per roller bottle every two to three days, and thereafter, the obtained culture supernatant was collected. Continuous culture is about 3
I went for months. (Fig. 1)

The culture supernatant was treated with 5 mM sodium ethylenediaminetetraacetate (EDT) as a protease inhibitor.
After addition of A), the mixture was continuously centrifuged at 1500 g at a flow rate of 2 g.
Impurities such as cell fragments were removed at 0 L / min (domestic H600-S), and the supernatant was collected.

Next, a 10-fold concentrated solution of the above supernatant was prepared using an ultrafiltration membrane having a molecular weight cut off of 100 k (manufactured by Filtron, model OS100CO5). This is PBS
The column was loaded with a gelatin sepharose 4B column (manufactured by Pharmacia) equilibrated in (-), and washed with PBS (-).
Equilibrated. Subsequently, elution was performed with a buffer in which 6 M urea was dissolved in PBS (-).

The purity of the cellular fibronectin in the eluate was 95% or more as a result of an assay by SDS-polyacrylamide gel electrophoresis under a reducing condition.

The eluate was collected and subjected to an ultrafiltration membrane having a molecular weight cut off of 100 k (manufactured by Filtron, model OS-100CO).
Using 1), the eluate was first concentrated, and then the solvent was exchanged with a 0.1 M sodium phosphate buffer containing 5% sucrose by a diafiltration method.

[0036] A human cellular fibronectin standard was obtained by the above method.

[0037]

[II. Properties of the substance]

 (1) Molecular weight

The molecular weight of the above-mentioned human cellular fibronectin preparation was compared with that of a standard protein and human plasma fibronectin preparation (manufactured by Iwaki Glass) under reduced and non-reduced conditions by SDS polyacrylamide gel electrophoresis.

As a result, the molecular weight of the human cellular fibronectin preparation of the present invention was 470 kd for the dimer and 2 for the monomer.
It was estimated to be 45 kd and 225 kd and was not clearly found to have a higher molecular weight than human plasma fibronectin. (Figure 2) (2) Immunological reactivity

An anti-cellular fibronectin (Product No. F-6, manufactured by Sigma) which does not react with plasma fibronectin but reacts only with cellular fibronectin.
140), the reactivity was confirmed by immunostaining after Western blotting. As a result, this sample reacted with this specific antibody. It also reacted with a human fibronectin antibody (manufactured by Takara Shuzo, clone 30-8). (3) Fragmentation by protease

Using thermolysin, the difference in the molecular weight of the fragments produced by the protease of human plasma fibronectin and the cellular fibronectin of the present invention was examined. Further, the immunological reactivity of the obtained fragment was examined.

As a result, the cellular fibronectin of the present invention clearly differs in fragment size by protease from human plasma fibronectin, and further by the immunological reactivity of the fragment with the monoclonal antibody, ED-A and ED-B. It was considered to be cellular fibronectin containing the region.

As a result of analysis of the above molecular weight and immunological reactivity, fragmentation by protease and mRNA of cells, fibronectin obtained in the present invention was found to be ED-A and ED-A.
It was determined to be human cellular fibronectin containing the B region.

(4) Water solubility As previously reported, cellular fibronectin has a pH of 6 to 8.
Is reported to be very poorly soluble in

In the present invention, it has been found that cellular fibronectin is easily soluble in a buffer containing sucrose. The solubility for each sucrose concentration is shown in Table 1 below.

[0046]

[Table 1]

[Brief description of the drawings]

FIG. 1. Cell line HUH-6YM during continuous culture
The figure which shows the change with respect to the culture period of the fibronectin density | concentration produced in a culture supernatant.

FIG. 2 shows that human cellular fibronectin of the present invention is expressed by SDS.
The result which applied to polyacrylamide gel electrophoresis is shown.
As a control, a human plasma fibronectin preparation was used.

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 21/00-21/02 C07K 14/78 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Cell lines HUH-6YM, FERMP-
A method for producing human cellular fibronectin, comprising culturing 12890 and isolating human cellular fibronectin having the following properties from the obtained culture. SDS polyacrylamide gel electrophoresis in a non-reducing state shows a dimer of 470 kd. Isoelectric point: pH 5-6 Immunological specificity Reacts with commercially available anti-cellular fibronectin antibodies and anti-fibronectin antibodies. It has various biological effects such as fibronectin's cell adhesion promoting activity, gelatin bead aggregating ability and cell spreading activity which have been reported to date. Cell lines HUH-6YM, FERM P-1289
0 can be cultured and produced from the resulting culture. 2. A cell line HUH-6Y having the following properties:
M, FERM P-12890. It is a mutant derived from the human hepatoblastoma cell line HUH-6clone5. It grows on a basal medium consisting of amino acids, vitamins, sugars and inorganic salts and can be subcultured without limit. That is, the cell line does not require a protein factor as a growth factor. HUH-6YM produces significant amounts of human cellular fibronectin. Fibronectin mRNA containing the ED-A and ED-B regions is expressed.