JP2000125863A - Gene transduction cell sheet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene transduction cell sheet into which an object gene can be readily transduced and can be prepared in a shortened time, particularly obtain a congenic cell sheet that can increase the take percentage to the living body and can produce the product of the introduced gene for a long period of time after the introduction. SOLUTION: This cell sheet is constituted with plural layers of mucosal epithelial cells into which the object gene is transduced. As the mucosal epithelial cells are more anaplastic, the object gene can be easily transduced. For example, in the case that the gene of an antimicrobial peptide is used as the object gene, the gene transduction cell sheet can be inhibited from its infection as a transplant thereby increasing the take percentage. In another case where human blood coagulation factor IX is used as the object gene, the formation of the blood coagulation factor IX can be retained in an organism for a long period of time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a transgenic cell sheet and a method for producing the same, and more particularly, to a transgenic cell sheet useful for gene therapy and for transplantation, and a method for producing the same.

[0002]

2. Description of the Related Art Living cells and tissues are sometimes transplanted or introduced into living bodies such as humans.

For example, a tissue as a wound dressing may be transplanted. In this case, the part where the tissue of the living body has been partially removed due to an operation, an accident, or the like is supplemented with another tissue (graft) to supplement the removed part or improve the appearance. Various implants have been used for such purposes. In particular, sheets of epidermal cells can be made from autologous epidermal cells. The epidermal cell sheet thus produced is highly evaluated as a rejection-free transplant useful in burns and the like. In addition, epithelial cells isolated from oral mucosal cells are undifferentiated cells than skin keratinocytes, and therefore have a shorter metabolic cycle than skin keratinocytes, and without keratinization This has the advantage that long-term maintenance is possible. From such properties, it is considered that oral mucosal cells are more useful as tissue for transplantation than epidermal keratinocytes.

[0004] On the other hand, in the field of gene therapy, cells are sometimes used as a carrier for a therapeutic gene, and a therapeutic gene is introduced into cells taken out of a living body and returned to the living body. Cells used for such gene carrier use include cells such as hematopoietic stem cells, vascular endothelial cells, and small intestinal epithelial cells. After being returned to the living body, these cells express the target gene in the living body.

[0005]

However, when transplanting a tissue into a living body, it is necessary to prepare a graft in a short time after the necessity of transplantation. In addition, grafts may not be able to survive successfully, because tissues that are not originally at the transplant site will survive in the living body. In particular, the transplant site has a weak resistance against bacteria and the like because the original tissue has been removed,
If a bacterial infection is caused, the graft cannot survive.

[0006] In addition, when carrying out gene transfer, it is necessary to collect cells as carriers for the target gene from the individual to be treated.
It may be difficult to collect from living organisms. For example, hematopoietic stem cells are undifferentiated cells, so that genes can be easily introduced and expressed, but must be collected from bone marrow. Further, even if a target gene is introduced, the gene product cannot always be produced in a living body for a long period of time. For this reason, even if an attempt is made to introduce a gene through, for example, epidermal cells, the growth rate is low, and the gene is hardly introduced. Even if the gene can be introduced, a gene product cannot be produced for a long time.

[0007] Accordingly, an object of the present invention is to provide a transgenic cell sheet that can easily introduce a target gene and can be prepared in a short time. In particular, an object of the present invention is to provide a transgenic cell sheet having improved engraftment efficiency to a living body and useful for transplantation. It is a further object of the present invention to provide a transgenic cell sheet useful for gene therapy that can produce a transgene product for a long period after transfection. Another object of the present invention is to provide a method for producing a transgenic cell sheet which can easily produce a useful transgenic cell sheet as described above.

[0008]

The gene-introduced cell sheet of the present invention is characterized by comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced. In addition, among the above-mentioned mucosal epithelial cells, the target gene is preferably introduced into 40% to 60% or 90% or more of the cells. Further, the gene-introduced cell sheet of the present invention is characterized in that the target gene is selected from the group consisting of a blood coagulation factor, insulin, a growth factor, a tumor antigen gene and a tumor suppressor gene.

The transgenic cell sheet of the present invention is used for gene therapy used as a gene carrier in gene therapy. The transgenic cell sheet of the present invention is used as a transplant.

[0010] The method for preparing a transgenic cell sheet of the present invention is a method for preparing a transgenic cell sheet comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced. And, to obtain a supporting cell compatible with the mucosal epithelial cells, to introduce an expression system containing the target gene and the marker gene into the mucosal epithelial cells, and culture in a serum-free serum-free selective culture medium ,
The mucosal epithelial cells into which the expression system has been introduced are selected, and the mucosal epithelial cells into which the expression system has been introduced are cultured together with the supporting cells in a growth medium containing serum to form a multi-layer mucosal epithelial cell. And obtaining the following.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The transgenic cell sheet of the present invention comprises a plurality of layers of mucosal epithelial cells into which a target gene has been introduced. As the mucosal epithelial cells of the present invention, oral mucosal cells, nasal mucosal cells, pharyngeal mucosal cells, vaginal mucosal cells, gastrointestinal mucosal cells, and the like can be used, and oral mucosal cells are particularly preferable from the viewpoint of easy collection. By being composed of such mucosal epithelial cells, it can be directly used as a graft or a gene carrier. In addition, mucosal cells are unlikely to be keratinized, unlike epidermal cells, and thus can be transplanted to a wide variety of sites in a living body.

These mucosal epithelial cells can be collected from a suitable living body. For example, oral mucosal cells can be collected from the oral cavity of a healthy subject together with the submucosal tissue. Since the collected mucosal tissue contains connective tissue in addition to mucosal cells, it is necessary to isolate mucosal cells. In the isolation of mucosal cells, an appropriate enzyme such as dispase or trypsin is used to remove connective tissue and dermis from the collected mucosal tissue. Also, debris and the like are removed using a nylon mesh or the like.

The target gene is introduced into the mucosal cells isolated as described above using a commonly used expression system. Expression systems used in such applications may be those known in the art, and include, for example, expression plasmids. The expression system that can be used here includes a marker gene, a gene introduction site into which the target gene is inserted, and a control site such as a promoter that controls the expression of the inserted target gene.

A promoter and an enhancer are arranged at the control site. Those commonly used can be used as they are, for example, Rous sarcoma virus (R
SV), human immunodeficiency virus (HIV), retrovirus such as Sendai virus (HVJ), adenovirus, adeno-associated virus, and the like.

[0015] The marker gene is a gene for confirming whether or not the target gene has been introduced into the expression plasmid, and a gene known in the art can be applied. Such marker genes include antibiotic resistance genes such as neomycin resistance gene and hygromycin resistance gene, β
-A chromogenic gene that develops a specific color such as galactosidase, luciferase and the like.

A target gene is inserted upstream of the promoter. The target gene used here can be selected according to the use of the transgenic mucosal cell sheet of the present invention.

When the gene-introduced mucosal cell sheet of the present invention is used as a graft, a factor for facilitating graft engraftment, for example, a factor for preventing bacterial infection during graft engraftment Growth of, for example, antibiotics, antimicrobial peptides (such as defensins), and factors that promote graft engraftment, such as platelet growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HFG) The gene of the factor can be the target gene. These genes are known in the literature and can be easily obtained from public institutions.

When the gene-introduced mucosal cell sheet of the present invention is used as a carrier for gene therapy,
The therapeutic gene can be the target gene. Target genes that can be targeted for such gene therapy include congenital and acquired deficiency factors such as blood coagulation factors, insulin, various growth factors, tumor suppressor genes, tumor antigen genes, and the like. The sequences of these target genes are already known in the literature and the like, and are available from various public institutions.

Two or more types of expression systems as described above may be used at the same time, and if possible, two or more types of target genes may be arranged in one expression system.

The above expression plasmid is transfected into virus-producing cells by a conventional method such as calcium phosphate, ribosome, electroporation and the like.
Various virus producing cells are known, and any of them can be used.

For gene transfer into mucosal epithelial cells, the virus-producing cells may be used as they are by simultaneous culture or the like, or the culture supernatant of the virus-producing cells may be used. It is preferable to use the culture supernatant of the virus-producing cells from the viewpoint of easily removing the possibility that the virus-producing cells are mixed into the cell sheet, and from the viewpoint of easy operation.

Gene transfer into mucosal epithelial cells using a viral supernatant can be performed by adding the viral supernatant to a culture system of mucosal epithelial cells seeded on a plate. In this case, 3 to 8 μg together with the virus supernatant was used.
/ Ml of polybrene is preferably added. The mucosal epithelial cells are infected with the virus in the virus supernatant within a few hours, whereby the target gene is introduced into the mucosal epithelial cells. After the infection, the infected cells are cultured in a selection medium described below, whereby mucosal epithelial cells into which the target gene has been introduced (hereinafter, introduced cells) are selected.

The selection medium used in the present invention contains a selection agent but does not contain serum. The selection agent corresponds to the marker gene introduced into the cell together with the target gene, and the marker gene is introduced, that is, only the target cell is killed by killing cells other than the cell into which the target gene has been introduced. Drugs that can be used to make a selection. Such a combination of a marker gene and a selection drug is well known in the art. For example, when a neomycin resistance gene is selected as a marker gene, a drug such as G418 is used. In the case of the transgenic cell sheet of the present invention, the amount of G418 used may be smaller than that of epidermal cells and the like, and may be 100 to 200 μg / ml, preferably 100 to 200 μg / ml.
150 μg / ml is sufficient.

In the present invention, it is essential that the selection medium does not contain serum. By not containing serum in the selection medium, the differentiation potential of the cells can be suppressed and the undifferentiated state can be maintained. Such a selection medium includes an epidermal cell selection medium (KGM). This K
GM is commercially available from Kurabo Industries as a serum-free liquid medium for growing normal human epidermal keratinocytes (HuMedia-KG2).

By culturing in such a selective medium, the ratio of transduced cells in the transgenic cell sheet can be adjusted. The percentage of transfected cells in the transgenic cell sheet affects the amount of transgene product produced from the sheet and the preparation period.

The transfected epithelial cell sheet of the present invention is characterized in that the ratio of transduced cells in the obtained cell sheet is 40 to 6
It can be 0%. Such a cell sheet can be prepared at an early stage with a short culturing period while the product of the transgene is properly produced. A transgenic cell sheet into which a gene has been transfected at a rate of 40% to 60% can be easily obtained by culturing in a selective medium for about 5 days.

The gene-introduced epithelial cell sheet of the present invention has a ratio of transduced cells of 9
It can be 0% or more. Such a cell sheet can sufficiently produce the product of the transgene.
The transfected cell sheet into which the gene has been transfected at a rate of 90% or more can be obtained by culturing for 10 to 14 days in a selective medium. In addition, the thing of the ratio of 100%
It can be obtained by further extending the period of culture in the selection medium.

The ratio of transfected cells in the transgenic cell sheet may be changed depending on the type of transgene. For example, when a gene such as an antimicrobial peptide is used as a transgene to increase engraftment efficiency, the ratio is as high as 90% or more.
A ratio of about 0 to 60% may be effective.

In the present invention, feeder cells are used in order to grow mucosal epithelial cells, which are transfected cells, in a layered manner with high efficiency. The supporting cells include, for example, fibroblasts, for example, mouse NIH3T3 cells, 3T3J2, and Swiss 3T3, and 3T3J2 cells are preferable from the viewpoint of the thickness of the obtained transfected cell layer and the growth rate of the transfected cells on the supporting cells. . The feeder cells lose their proliferative ability so as not to interfere with the growth of the introduced cells. The proliferation ability can be eliminated by a method well known in the art, for example, treatment with mitomycin C or irradiation. In addition, culture
This is performed using a culture vessel such as a normal plastic dish.

The transfected cells may be co-cultured with the feeder cells depending on the type of feeder cells, or may be seeded on feeder cells which have been seeded and grown in a layered manner. At this time, the introduced cells can be seeded at a cell density of 1 × 10 ⁵ / cm ² , in which case the supporting details are 1 × 10 ¹ / cm ²
It can be seeded at で 1 × 10 ³ / cm ³ or more. If the number of supporting cells is less than this, the role of supporting cells cannot be sufficiently fulfilled, and if the number of supporting cells is more than this, the growth of the introduced cells is hindered. Further, if the number of introduced cells is smaller or larger than this range, it cannot be efficiently propagated, which is not preferable.

In the present invention, a growth medium is used to grow the transfected cells on feeder cells. The growth medium used for this purpose can be selected from various media known in the art. By culturing in growth medium,
The transfected cells readily form multiple layers. On the other hand, the supporting cells die only by supporting the growth of the mucosal epithelial cells, and when the mucosal epithelial cells form a plurality of layers, they no longer exist in the culture system.

It is preferable to use an epithelial sheet forming medium (EFM) having a sheet forming ability as the growth medium. EF
M is a medium that is usually used to grow epidermal cells in layers, and is composed of a 3: 1 mixture of DMEM and Ham F-12 medium, 5% FBS, 5 μg / ml insulin, 5 μg / Ml transferrin, 2 ×
10 ⁻⁹ M triiodothyronine, 1 × 10 ⁻⁹ M cholera toxin, 0.4 μg / ml hydrocortisone,
0 U / ml penicillin, 0.1 μg / ml kanamycin, 0.25 μg / ml amphoteritin B and 10
ng / ml of human recombinant epidermal growth factor (E
GF).

The mucosal epithelial cells having a plurality of layers are separated from the culture vessel while maintaining the layer structure. This separation can be performed by a method known in the art, and can be easily performed by using a suitable enzyme such as dispase to inhibit the adhesion of the basal layer.

[0034] The obtained mucosal epithelial cell sheet is transplanted into a living body by a method known in the art. Since the gene-introduced cell sheet of the present invention is composed of multiple layers of mucosal epithelial cells, it can be directly transplanted to a transplant site, for example, as a transplant. In addition, since the gene-introduced mucosal epithelial cell sheet of the present invention is composed of mucosal epithelial cells, it can be transplanted onto the epidermis in contact with the outside world, but also in the oral cavity, under a humid environment such as under the skin. Also suitable for transplantation. In the case of gene therapy, if the gene product is released into the bloodstream, for example, it can be delivered to the whole body, while the gene product of the target gene can be produced at a fixed site.

The method of transplantation can be changed depending on the type of the introduced cell sheet and the expression site of the introduced gene, and can be carried out by applying a suitable known method as it is. For example, the transduced cell sheet can be transplanted under the skin, in the oral cavity, the epithelium of the small intestine, or the epithelium of the digestive tract, with or without a suitable transplantation carrier, and a flap is created on the back and placed underneath. You may.

The transduced mucosal epithelial cell sheet taken into the living body produces a substance corresponding to the introduced gene by the action of the expression plasmid. The transgenic mucosal epithelial cell sheet's ability to produce gene products is expected to last for at least 5 weeks, preferably as long as 8 weeks. The produced substance can be confirmed for its production by various known methods. Various methods are known in the art for confirming the product, but when an antibody against the product is known, it is preferable to carry out the ELISA method from the viewpoint of simplicity and sensitivity.

As described above, the transgenic cell sheet of the present invention can be easily prepared because it is composed of mucosal epithelial cells having high proliferative properties and easy transfection at the undifferentiated stage. Further, since it is a mucosal epithelial cell, it can be used as a mucosal cell under a humid environment and can be used as a substitute for an epidermal cell under dry conditions such as skin.

When a gene that promotes graft survival, such as a growth factor or a gene of an antimicrobial peptide for preventing infection, is selected as the target gene, the transplant can be used with higher efficiency. Can survive.
Furthermore, when a gene for treatment is selected as a target gene for the purpose of gene therapy, it can be easily prepared,
It can be used as a useful gene carrier having a long-term gene product production ability.

[0039]

EXAMPLES Examples representing the present invention will be described below. I. Preparation of Cells Mucosal tissue was obtained from healthy oral mucosa with the patient's consent. The submucosa was removed with scissors and chopped into small pieces. A small piece of mucosal tissue was treated with 1000 U / ml penicillin (Sigma,
St. Louis, MO), 1 mg / ml kanamycin and 2.5
In phosphate buffered saline (PBS) containing μg amphotericin B (Gibco, Gland Island, NY) for 30 minutes at 37 ° C.
Dipped twice. The tissue was then placed in Dulbecco's Modified Minimum Essential Medium (DMEM) containing 1000 PU / ml Dispase® (manufactured by Godo Shusei Co., Ltd.) for 16 hours 4
C. followed by treatment with 0.25% trypsin (Gibco, Gland Island, NY) for 30 minutes at room temperature to separate cells. Enzyme activity was determined using 10% fetal bovine serum (FBS) (Hyc
(lone, UT) by washing with DMEM.

Then, DMEM containing 10% FBS
After stirring for 30 minutes, the debris was filtered with a 50 μm nylon gauze to purify mucosal cells. The purified mucosal cells were centrifuged at 1500 rpm for 5 minutes, and the obtained cell pellet was resuspended in KGM (Kurabo). Purified mucosal cells were cultured at 37 ° C. in a 10% CO ₂ incubator. The culture was replaced with fresh culture every 2-3 days.

II. Plasmid Construction Moloney murine leukemia virus (MoMLV) mainly retroviral vector pLRNL is Rous sarcoma virus (RSV) neomycin resistance under the control of the promoter of (Neo ^r) contains a gene (Li et al., Virolog
y, (1989) 171: 331-341). Immediately upstream of this RSV promoter is a PstI site. In this PstI site, the entire coding region of β-galactosidase or c of blood coagulation factor IX
PstI fragment containing DNA (Matsushita et al., Thromb.
Res., (1993) 69: 387-393). This resulted in the construction of pLBZ or pLEXRNL (FIG. 1), respectively (Matsushita et al., Thromb. Res., 69: 387-393).
(1993)).

III. Virus production and transfection Virus-producing cells, PA317, were grown in DMEM containing 10% fetal calf serum (FCS) with high glucose. Plasmid pLBZ or pFIXRNL
As described above in the bidirectional packaging cell line PA317.
(Emi et al., J. Virol., (1991) 65: 1202-1207) and transfected with the calcium phosphate method for two weeks after which the cells were transfected with G418 (400 μg / m2).
1) selected for the expression of the neomycin resistance gene. Thereby, virus-producing cells, PA
317 / LAZ or PA317 / LIXRNL were obtained. The virus titer of each virus-producing cell relative to 208F cells was approximately 4 × 10 ⁴ / ml.

IV. Gene transfer efficiency into mucosal epithelial cells To study gene transfer efficiency into mucosal epithelial cells,
Mucosal epithelial cells were infected by different infection methods. The source of infection, 1 × 10 6 ^/ 35mm and virus supernatant of the virus-producing cells, the virus-producing cells as a feeder
A dish seeded with a dish was prepared. When a virus-producing cell was used as a supporting cell, the virus-producing cell was treated with mitomycin C to lose its growth ability.

Mucosal epithelial cells were inoculated to each of these infection sources. Mucosal epithelial cells were 1 × 10 ^{6 in} 35 mm dishes. The transfected cells contain 24
After hours, 5-bromo-4-chloro-3-indolyl B
-Specified by D-galactopyranoside (X-gal) staining. The evaluation was performed by comparing the number of positive cells in a 2 × 2 mm area.

As shown in FIG. 2, the gene could be similarly introduced into the mucosal epithelial cells using the virus-producing cells as the supporting cells or the virus supernatant of the virus-producing cells. For this reason, it was decided to carry out gene transfer using a virus supernatant which has good operability and can transfer genes without directly using virus-producing cells.

V. G418 concentration assay G4 used for selection of transfected cells
Eighteen optimal quantities were considered. Mucosal epithelial cells without neomycin resistance gene and other epithelial cell lines (PA
317) was prepared as described above, cultivated for 10 days, and then treated with trypsin to form a 3-mm in a 35 mm dish.
Seeded at a density of 6 × 10 ⁴ cells. When the cells became confluent, various amounts of G418 were added to the culture. Table 1 shows the results. "-" Indicates that cells are killed by the action of G418 (appropriately selectable),
“+” Indicates that the cells were not killed by the action of G418 (not properly sorted).

[0047]

[Table 1]

As shown in Table 1, the mucosal epithelial cells
Selection with G418 was possible at lower concentrations than normal epithelial cell lines. Therefore, it was decided to use G418 at a concentration of 150 μg / ml when introducing genes into mucosal epithelial cells.

VI. Formation of Transduced Mucosal Epithelial Cell Sheet In the same manner as described above, the prepared mucosal epithelial cells were cultured for 10 days, then treated with trypsin, and transferred to a 35 mm dish.
Seeded at a density of −6 × 10 ⁴ cells. After overnight culture,
Mucosal epithelial cells were infected for 3 hours with 2 ml of virus supernatant containing 5 μg / ml polybrene (Sigma, St. Louis, MO). 24 hours after infection, G418 (15
0 μg / ml; K containing (Gibco, Gland Island, NY)
Transgenic mucosal epithelial cells were selected by culturing them for 10-14 days in GM medium. The mucosal epithelial cell population obtained by the selection was treated with trypsin-EDTA for 1 hour.
A cell suspension of × 10 ⁵ cells / ml was prepared.

3T3-J2 cells as feeder cells were treated with 4 μg / ml mitomycin C (Kyowa Hakko, Tokyo) in serum-free DMEM. Two hours later, PB
Rinse several times with S (-) to remove mitomycin C,
After trypsinization, a cell suspension of 1 × 10 ⁴ cells / ml was prepared.

A 1 × 10 ⁵ cell / ml mucosal cell suspension and a 1 × 10 ⁴ cell / ml feeder cell suspension were added to each well of a 6-well plate in an amount of 1 ml, and KGM was added.
Cultured in selective medium. Every 2-3 days, half of the medium was replaced with fresh EFM selective medium and maintained to form a layered epithelial cell sheet.

The mucosal epithelial cells seeded and cultured on the supporting cells proliferated on the supporting cells. Also, KGM
By the selective culture in the selective medium for 10 to 14 days, the mucosal cells at the undifferentiated stage could be maintained, and the ability to form a layer and an epithelial cell sheet was maintained (FIGS. 3A and 3B). ). By culturing on feeder cells for about 2 weeks,
Thick enough for transplant (3-5 layers, about 100 μm)
Became. Since the epithelial cell sheet can be formed by layering in this manner, the transgenic cell sheet is
It can be used as an implant or as a gene carrier for therapy. On the other hand, cells cultured in the EFM selection medium containing serum alone without using the KGM selection medium formed an irregular layer and were enucleated (FIG. 3 (C)).
This means that terminal differentiation has already taken place and the ability to form a layer to be uniformly layered has been lost.

VII. Transplantation of Transgenic Mucosal Epithelial Sheet Transgenic mucosal epithelial cells became confluent and stratified 10-14 days after the start of culture. This cell sheet was used as a graft. The epithelial cell sheet was detached with dispase (400 PU / ml) and washed twice with PBS. The cell sheet is bent barbital 0.04
Implantation was performed on nude mice (5-6 weeks old, male, BALB / c nc / nc) (Japan SLC Corporation, Hamamatsu) under general anesthesia by intraperitoneal administration of mg / g.

The transplantation was performed by the method of Barrandon et al.
N et al., J. Invest.Dermatol., (1998) 91, 315-318) and the method of Sugimura et al. (Sugimura et al., J. cranio-maxillofac Surg., (1997) 2
4,352-359). That is, after the back of the mouse was sufficiently disinfected with 10% Hibiten alcohol, 30 ×
A 30 mm rectangular flap was made to expose the muscular layer. A sterilized silicone membrane was placed over the entire exposed mouse muscle layer to isolate the flap from the muscle layer. Next, the prepared cultured mucosal epithelial cell sheet was placed on a sterilized silicone membrane as a transplant carrier, and transplanted onto the silicone membrane so that the cultured mucosal epithelial cells were in contact with the inside of the flap. Then, the flap was returned so as to cover the transplanted mucosal epithelial cell sheet and the silicone membrane, and sutured with a 5-nylon thread to complete the transplantation. Thus, the mucosal epithelial cells were fixed such that the surface (the basal layer) that had been in contact with the plate during the culture of the mucosal epithelial cells was in contact with the inside of the mouse flap.

VIII. Histological analysis Histological analysis was performed to confirm the engraftment of the transplanted transgenic mucosal epithelial cells. Engraftment of the transplanted transduced mucosal epithelial cell sheet was confirmed by staining X-Gal of β-galactosidase expressed by pLBZ (including the β-galactosidase gene). At 1, 3 and 5 weeks after transplantation, the flap containing the transduced mucosal epithelial cells was collected, and the cryoprotective medium (OTC, Tissue,
Tek, Miles). Frozen sections (5 μm) of the flaps embedded in cryoprotective medium were fixed in 2.5% glutaraldehyde solution in PBS for 15 minutes at 4 ° C. The sections were then washed twice in PBS and stained (1 mM M
gCl ₂ , 5 mM K ₃ Fe (CN) ₆ , 5 mM K
₄ (CN) ₆ and 400 μg / ml X-Gal in dimethylformamide solution) overnight at 37 ° C.
Stained. The results are shown in FIGS.

One week after transplantation, the transplant of the transduced mucosal epithelial cell sheet was stained almost blue (FIG. 4 (A), × 5). This means that one week after transplantation, the transplanted transduced mucosal epithelial cells have produced β-galactosidase protein and have sufficiently survived. The graft one week after the transplantation was completely attached, and many epidermal cells had infiltrated along the graft (FIG. 5 (A), × 250). The staining of such grafts became weaker after 3 weeks (FIG. 4 (B)),
After 5 weeks, almost no staining was observed (FIG. 4).
(C)). Three weeks after transplantation, the expression of β-galactosidase was reduced, and the grafts became gradually thicker, indicating signs of cell degradation such as lack of basement membrane (FIG. 5).
(B)). However, 5 weeks after transplantation, β-galactosidase expression was detectable (FIG. 5).
(C)).

IX. Production of blood coagulation factor IX In order to confirm the production of the gene product introduced into mucosal epithelial cells, the gene for blood coagulation factor IX was introduced, and the blood concentration of this factor was measured. It has been already proved that the expression product of blood coagulation factor IX used has activity. The production of blood coagulation factor IX by the transduced mucosal epithelial cells is a model for gene therapy of hemophilia.

The transfected mucosal epithelial cells transfected and transfected using pLEXRNL were transfected with G418 as described above.
The cells were selectively cultured for a week, seeded in a 35 mm culture dish, and co-cultured with feeder cells. After the culture, human blood coagulation factor IX released into the growth medium over time was measured for each dish. The measurement of blood coagulation factor IX is performed by the aforementioned E
Performed by the LISA method (Takahashi et al., J. Lab. Cli
n. Med., (1991), 118: 317-325). Table 2 shows the results.

[0059]

[Table 2]

As shown in Table 2, the maximum value of blood coagulation factor IX detected in the growth medium was 30% from the start of culture.
It was day. Under culture conditions, the transgene was shown to be expressed for at least 7 weeks.

Next, the in vivo expression of the transgene was analyzed. After introducing the above human blood coagulation factor IX into mucosal epithelial cells and selecting them, and co-culturing them with feeder cells for 2 weeks,
The transgenic mucosal epithelial sheet formed as described above is 3 ×
It was placed on a 3 cm silicon membrane and implanted on the back of two nude mice. Blood from each mouse was collected by puncture under the orbit and collected in 1/10 volume of 3.8% sodium citrate solution. The cells are removed by centrifugation and the plasma sample is stored at -80C. Blood coagulation IX
Factor levels were measured in the ELISA described above.

In the two mice transplanted with the transfected mucosal epithelial cells, 1.5-1.8 ng was obtained early after transplantation.
/ Ml of human blood coagulation factor IX was confirmed to have been produced (FIG. 6). Human blood coagulation factor IX in the blood
Detected for more than 3 weeks. This means that the gene-introduced mucosal epithelial cells stably produce gene products for a long time after transplantation, as under the culture conditions.

As described above, the present gene-introduced cell sheet can be easily prepared, and even if the sheet is transplanted to the back in a sheet form, human blood coagulation factor IX can be produced in blood for a long time.

[0064]

According to the gene-introduced mucosal epithelial cell sheet of the present invention, since it is composed of more undifferentiated mucosal epithelial cells, the target gene can be easily introduced and can be prepared in a short time. Can be. Thereby, it is possible to provide a useful gene therapy carrier that produces a gene product of a transgene for a long period of time, and to provide a graft with high engraftment efficiency in which graft engraftment is promoted. it can. In addition, according to the method for producing a gene-introduced mucosal epithelial cell sheet of the present invention, a useful carrier for gene therapy and a graft with high engraftment efficiency as described above can be easily produced.

[Brief description of the drawings]

FIG. 1 shows the construction of a plasmid containing human blood coagulation factor IX used for gene transfer.

FIG. 2 is a graph showing the efficiency of infection of oral mucosal epithelial cells.

FIG. 3 (A) is a view of transduced mucosal epithelial cells selectively cultured in a serum-containing selection medium, and FIG.
(C) is a diagram of the transduced mucosal epithelial cells selectively cultured in a serum-free selection medium.

FIG. 4 (A) is a 10-fold enlarged view showing the stained state of the transduced mucosal epithelial sheet one week after transplantation, and FIG.
A 10-fold enlarged view showing the stained state of the transgenic mucosal epithelial sheet three weeks after transplantation, and (C) is a 10-fold enlarged view showing the stained state of the transgenic mucosal epithelial sheet 5 weeks after transplantation. .

FIG. 5 (A) is an enlarged view of 250 times showing the stained state of the transduced mucosal epithelial sheet one week after transplantation, (B)
Is a 250-fold enlarged view showing the stained state of the transduced mucosal epithelial sheet 3 weeks after transplantation, and (C) is the stained state of the transduced mucosal epithelial sheet 5 weeks after transplantation.
It is a double enlarged view.

FIG. 6 is a graph showing changes in human blood coagulation factor IX concentration in blood in a mouse to which a transgenic mucosal epithelial cell sheet has been transplanted.

[Procedure amendment]

[Submission date] November 16, 1998 (1998.11.
16)

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 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Nobuhiko Emi 65, Tsurumai-cho, Showa-ku, Nagoya-shi Nagoya University Medical School (72) Inventor Hirokazu Mizuno 65, Tsurumai-cho, Showa-ku, Nagoya-shi Nagoya University Medical School F-term (reference) 4B024 AA01 BA80 CA04 DA02 EA02 FA02 FA10 GA11 HA01 4B065 AA93X AA97X AA99Y AB01 BA02 BC41 CA24 CA31 CA44

Claims (7)

[Claims] 1. A transgenic cell sheet comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced. 2. 40% to 60% of the mucosal epithelial cells,
The transgenic cell sheet according to claim 1, wherein the target gene has been introduced. 3. The transgenic cell sheet according to claim 1, wherein the target gene is introduced into 90% or more of the mucosal epithelial cells. 4. The transgenic cell sheet according to claim 1, wherein the target gene is selected from the group consisting of a blood coagulation factor, insulin, a growth factor, a tumor antigen gene, and a tumor suppressor gene. 5. The transgenic cell sheet according to claim 1, which is used as a gene carrier in gene therapy. 6. The method according to claim 1, which is used as an implant.
The transgenic cell sheet according to item 1. 7. A method for producing a transgenic cell sheet comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced, comprising: a mucosal epithelial cell for producing a cell sheet; Cells, an expression system containing the target gene and the marker gene is introduced into the mucosal epithelial cells, and cultured in a serum-free selective culture medium containing no serum, and the mucosal epithelium to which the expression system has been introduced. Selecting a cell, culturing the mucosal epithelial cell into which the expression system has been introduced, together with the supporting cells in a growth medium containing serum, and obtaining a mucosal epithelial cell having a plurality of layers formed therein. Method of manufacturing.