JP3091113B2 - Hybrid gel secreting bioactive substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hybrid gel capable of secreting a physiologically active substance. More specifically, the present invention relates to a physiologically active substance useful for the treatment of various intractable diseases requiring long-term continuous administration of a physiologically active substance necessary for maintenance of a biological function, such as artificial skin. The present invention relates to a new hybrid gel capable of secreting active substances.

[0002]

2. Description of the Related Art The following three methods can be considered as methods for treating diseases caused by the loss or deterioration of the function inherent in living cells for some reason. That is, the lost or reduced function is compensated for by 1) drug administration, 2) by organ transplantation, tissue transplantation, cell transplantation, etc., 3) by gene therapy.

For example, in the case of insulin-dependent diabetes, in the case of insulin-dependent diabetes, β-cells (cells that produce insulin having a function of negatively regulating blood sugar level) in the pancreatic islets of Langerhans are destroyed. ing. Therefore, patients with insulin-dependent diabetes
Blood sugar levels are high, and as a result, the concentration of sugar in urine, from which the name of diabetes is derived, also increases. When blood sugar levels remain high, the functions of various cells in the living body are impaired, which causes serious complications of diabetes.

[0004] Therefore, in the treatment of insulin-dependent diabetes, a deficient insulin must be administered from the outside to control the blood sugar level. Insulin-dependent diabetics require several daily doses of insulin throughout their lifetime. This is not only a serious physical and mental distress for the patient, but also the inherent risk of life due to erroneous administration of the patient's own administration of the drug.

[0005] Therefore, as an alternative to the self-administration of insulin, a treatment method using transplantation of pancreas or islets of Langerhans has been implemented (Keiichi Kubota, Yasuo Dezuki,
Japanese Clinical Laboratory, 48: 1052, 1990). However, these methods have problems such as lack of donors, difficulties in controlling immune rejection of transplanted organs or tissues by patients, difficulties in transplantation operations requiring advanced techniques, and risks of surgery. There are many.

[0006] On the other hand, gene therapy, which has been undergoing clinical trials mainly in the United States since the 1990s, is a new medical technology that solves the above problems (NM SUMMERS. BIOTECHNO).
LOGY 12:42, 1994). Ideas on how to treat diabetes with this have already been proposed (RF SELDEN
etal, THE NEW ENGLAND JOURNAL OF MEDICINE 317
(17): 1067, 1987). In the case of this treatment method, an insulin gene is introduced into cultured cells, and the cells are transplanted into a patient's body so that insulin produced by the transgene is continuously secreted into the body. However, this method is currently difficult to control insulin secretion by transplanted insulin-producing cells, and can not collect cells once transplanted into the body,
It has many problems. Further, it is known that this gene therapy is a promising advanced medical technology not only for the above-mentioned genetic diseases such as insulin-dependent diabetes and severe immunodeficiency but also for intractable diseases such as cancer and AIDS. Various attempts have been proposed for that, and
In fact, this gene therapy method is being implemented, but many of these gene therapies use retrovirus-derived vectors to introduce genes into cells using viral cell infection. Is considered.

However, in the method using a retrovirus-derived vector, the efficiency of gene transfer depends on the affinity of the virus for cells, and the inactivated virus vector is transformed into a wild-type retrovirus in vivo. There is an inherent risk of mutating into a virus. Also, in general,
Conventional gene therapy has a problem that it is difficult to control the introduced gene from outside the body.

Accordingly, the present invention has been made to solve the problem while keeping in mind the effectiveness of the conventional gene therapy method. The purpose of the present invention is to provide a new technical means that allows a transgene to be controlled from outside the body. More specifically, an object of the present invention is to eliminate the drawbacks of the prior art by making it possible to transplant a bioactive substance-producing cell into which a gene has been introduced into a living skin as a hybrid gel (cell-incorporated gel).

[0009]

SUMMARY OF THE INVENTION The present invention provides a hybrid gel for secreting a physiologically active substance, comprising a physiologically active substance-producing cell and a gel derived from a living body. In such a hybrid gel, the physiologically active substance-producing cells must be encapsulated in a gel derived from a living organism, or overlaid on the gel, or overlaid on a gel containing the physiologically active substance-producing cells. Is a preferred embodiment.

[0010] The present invention also provides a physiologically active substance producing cell,
Provided is a bioactive substance-secreting hybrid gel comprising a gel derived from skin cells and a living body. In such a hybrid gel, skin cells are layered on a gel derived from a living body that encapsulates biologically active substance-producing cells,
The bioactive substance producing cells are layered on the gel in which the skin cells are encapsulated, the skin cells and the physiologically active substance producing cells are encapsulated in the gel, or the skin cells and the physiological active substance producing cells are encapsulated in the gel In a preferred embodiment, skin cells or physiologically active substance-producing cells are overlaid thereon.

[0011] In a preferred embodiment of the present invention, when the bioactive substance-producing cells are encapsulated in a gel, the cells are encapsulated in a gel together with a network or a porous membrane. Furthermore, in the present invention, the above-mentioned physiologically active substance-producing cells are skin cells (for example, skin fibroblasts, skin epidermal cells, etc.) carrying an expression vector incorporating a gene encoding a physiologically active substance such as insulin. The expression vector is a plasmid vector incorporating an insulin cDNA and a neomycin resistance gene, a plasmid vector pBMG neo neos or a plasmid vector incorporating a mutant insulin gene which expresses insulin which is convertible by the enzyme furin and has excellent stability. pR
The embodiment also includes that it is IS / proins / Ifur / IIfur / B10D.

[0012]

In the hybrid gel of the present invention, the cells encapsulated or layered in the gel produce bioactive substances required or lacking in the living body, which are continuously secreted into the living body. . In addition, bioactive substance-producing cells are encapsulated in a gel together with a network such as a mesh or a porous polymer film, thereby increasing the amount of the physiologically active substance produced, for example, as an external preparation for artificial skin or the like. It can be used effectively. Since a gene expressing a physiologically active substance is introduced into cells by, for example, a plasmid vector, there is no danger of mutation into a wild-type retrovirus by a retrovirus-derived vector as in conventional gene therapy. In addition, by implanting the transfected cells into the skin, the transgene can be easily controlled from outside the body.

More specifically, the following effects are obtained. 1) After transplantation, the drug can be stably administered for a long period of time without the patient's consciousness. This can greatly alleviate the physical and mental distress that has been given to patients by conventional repeated administration treatment. 2) Since transplantation to the skin and its removal are very simple operations, the amount of artificial skin can be adjusted at any time while monitoring the course of treatment. This facilitates finding optimal conditions for treatment.

3) Expression of a DNA sequence encoding a physiologically active substance using an appropriate promoter, and application of various promoter-specific inducing stimuli (various hormones, heavy metals, temperature, etc.) to the artificial skin graft site, The amount of drug secretion by cells in or on the gel can be controlled.
This allows fine adjustment of the amount of secretion. 4) Because the transplanted cells are encapsulated or layered in a gel,
Less susceptible to immune rejection in patients. Therefore, the amount of immunosuppressants generally used at the time of transplantation of tissue or the like can be reduced. This can greatly reduce the risk of side effects from immunosuppressants. Of course, in the case of gene therapy using the patient's own cells, immunological rejection is not a problem because of autotransplantation.

5) Since the operation can be performed by a simple operation that does not require hospitalization, there is no danger associated with the operation, and the operation is safe. Also,
Since the transplant is performed on the skin, the state of the transplant can always be observed from the outside. If necessary, it is possible to remove the transplanted artificial skin. In the present invention, various physiologically active substance-producing cells can be used in which an expression vector incorporating a gene therefor has been incorporated into the cell. For example,
Insulin-producing cells can be prepared by transfecting animal cells with a plasmid vector pBMG-neo-ins into which insulin cDNA and a neomycin resistance gene (selectable marker) have been incorporated, according to a standard method. Alternatively, a plasmid vector pRIS · which can convert proinsulin expressed by the insulin gene into insulin by the enzyme furin, and incorporates a mutant insulin gene that expresses insulin with excellent stability by amino acid substitution at the tenth amino acid in the insulin B chain. p
Roins / Ifur / IIfur / B10D may be transfected into animal cells.

The gel enclosing or layering the physiologically active substance-producing cells can be prepared and used according to a conventional method, for example, from collagen, fibrin, agarose or the like derived from a living body. For example, the following describes the case where a hybrid gel enclosing cells incorporating the insulin gene is used as an artificial skin for treating diabetes.

(1) A piece of skin is collected from the body surface of an experimental animal, and epidermal cells and fibroblasts, which are main constituent cells, are separated and cultured. (2) An expression vector incorporating the insulin gene is incorporated into these cells to obtain a cell line that secretes insulin. (3) A hybrid artificial skin having insulin secretion ability is constructed from these cell lines using collagen gel or the like.

(4) Transplanting an insulin secreting hybrid artificial skin. More specifically, the hybrid gel capable of secreting a physiologically active substance of the present invention is, for example, ASAGA
(H. ASAGA etal, EXPERIMENTAL CELLRESEARCH
193: 167, 1991).

That is, a 4-fold concentrated cell culture solution, serum, purified water, and, for example, collagen (0.5% solution)
Are mixed under ice cooling at a ratio of 2.5: 1: 2.5: 4 according to the required amount. To this, a 1N aqueous sodium hydroxide solution is dropped and mixed to adjust the pH to 7.4, and 2 ml each is dispensed into a 35 mm-diameter hydrophobic plastic petri dish, and immediately transferred to a 37 ° C. thermostat. Within minutes, the collagen solidifies and can form a gel. Physiologically active substance-producing cells can be encapsulated in a gel by mixing them with the above mixture just before solidifying collagen.

When a network or a porous membrane is allowed to coexist, these may be mixed with the above-mentioned mixed solution together with the physiologically active substance-producing cells. In addition, a commercially available product can be used for the culture solution, serum, and collagen. In order to facilitate transplantation to the skin, it is also effective to give the collagen gel an appropriate strength. The moderate strength of the gel is, for example,
BELL et al.'S method (E. BELL etal, PROCEEDINGS OF THE NAT
IONAL ACADEMY OF SCIENCES 76 (3): 1274
1979), by mixing an appropriate number of skin-derived fibroblasts in the gel. The gel's contracting action by the fibroblasts can result in the gel having a suitable strength. Skin-derived fibroblasts can be obtained, for example, by the primary transplant method (RI Freshrey, Cultu
re of Animal Cells, Alan R. Liss, Inc., New York, 1
According to 987), it can be obtained by culturing from a small amount of skin collected from patients.

In order to obtain a good state of engraftment of the gel on the skin to be transplanted, it is also effective to multiply culture the skin-derived epidermal cells on the gel before transplantation to the skin. The skin-derived epidermal cells layered on the gel can be obtained by a method such as GREEN (H. GREEN, etal, PROCEEDINGS OF THE NATIONAL).
According to ACADEMY OF SCIENCES 76: 5665, 1979), it can be obtained by culturing from the skin of the patient as well as fibroblasts.

It is needless to say that the present invention is also effective when a gel is implanted subcutaneously without superimposing epidermal cells. Specifically, various modes are possible. Hereinafter, the present invention will be described in more detail with reference to Examples. Of course, the present invention is not limited by the following examples.

[0023]

【Example】

Example 1 A hybrid gel (hereinafter sometimes simply referred to as a gel) of the present invention was prepared, and its effects in drug administration methods and application to diabetes treatment were evaluated in vitro in vitro experiments and in animal models of diabetes. The system was evaluated. In vitro test A gel encapsulating or layering proinsulin producing cells was cultured, and proinsulin secreted into the culture solution was measured. 1) Cells used The following three cell lines were used.

Fibroblasts derived from mouse embryos (NIH3T
3) Rat skin-derived fibroblasts (RSFins) incorporating insulin gene Insulin-derived rat skin-derived epidermal cells (RSKins) RSFins and RSKins cells are fibroblasts (RSF) and epidermis obtained by primary culture from rat skin Insulin gene (GI BELL et a) in cells (RFK)
1, NATURE 284, (6): 26, 1980). The integration of the insulin gene into fibroblasts and epidermal cells was performed using a plasmid-based expression vector pBMG-ne incorporating human insulin cDNA.
o · ins (Y. KAWAKAMI et al, DIABETES 41:95
6,1992) using CHEN and OKAYAMA (C. CHEN an
d OKAYAMA, MOLECURAR AND CELLULAR BIOLOGY 7
(8): 2745, 1987). Thereafter, the cells incorporating the vector were selectively expanded in a culture solution containing G418 at a concentration of 400 μg / ml.

Since these cells do not have an enzyme for converting to insulin, they secrete proinsulin, a precursor of insulin. However, proinsulin also has an insulin action (SN Davis et al., Journal of Clinic
al Endocrinologuy and Metabolism, 75 (5): 1282-128
8, 1992). 2) Culture solution For RSFins, 10% fetal calf serum (Hycron) was added to Dulbecco's modified Eagle's minimum medium (Gibco) and used (Culture solution A).

For RSKins, a mixture of Dulbecco's modified Eagle's minimum medium and MCDB152 medium (Far East) at a ratio of 7: 3 was mixed with hydrocortisone (0.4 μm).
g / ml), insulin (5 μg / ml), transferrin (5 μg / ml), triiodothyronine (2n
M), cholera toxin (0.1 nM), adenine (10
0 μM) and fetal calf serum (10%) were used (culture solution B).

After encapsulating the cells in the gel and layering, the hybrid gel was cultured using the culture solution A. 3) Test method 500,000 RSFins cells were encapsulated or layered in a gel to obtain gel A and gel B. On the other hand, 500,000 R
FKins cells are encapsulated or layered in gel, and gel C
And Gel D. NIH3T3 cells were simultaneously encapsulated in gel C and gel D to impart contractility. Table 1 summarizes the composition of this gel. These are 37
Incubated at ℃. One day later, the culture was continued by adding 2 ml of the culture solution, and then the medium was replaced with a fresh medium every two days. The exchanged culture solution was stored frozen, thawed if necessary, and the proinsulin concentration in the culture solution was measured. Proinsulin concentration was measured using an EIA kit (Sanko Junyaku)
It was measured as a value of immunoreactive insulin (Immunoreactive Insurin: hereinafter sometimes referred to as IRI).

[0028]

[Table 1]

4) Test results The test results are shown in Table 2. A stable amount of proinsulin was secreted into the medium over 25 days of culture from both cell lines encapsulated in the gel or layered on the gel.
Therefore, by implanting this gel into the skin, it becomes possible to administer proinsulin to the body.

[0030]

[Table 2]

Animal Test 1 A hybrid gel in which proinsulin-producing cells were encapsulated or layered was implanted into a diabetic model animal, and its therapeutic effect was evaluated by measuring the blood glucose level. 1) Experimental animals Balb / c nude mice (5-week-old males)
The diabetic condition was induced by intraperitoneal administration of streptozotocin (Sigma) at a dose of 3 mg / kg for 4 days and used at 7 weeks of age. 2) Cells used Three kinds of cell lines derived from rat skin were used.

RSF RSFins RSKins 3) Gel preparation method 500,000 RSFins cells were encapsulated in a collagen gel, and the same number of RSKins cells were layered thereon to obtain gel E. On the other hand, 500,000 RSF cells were encapsulated in a collagen gel, and the same number of RSKins cells were overlaid on this to obtain gel F. The composition of these gels is summarized in Table 3. These gels were used for transplantation after culturing at 37 ° C. for 6 days.
Proinsulin production of the gel at this point was 484 μl U / day / gel and 4
04 μl U / day / gel.

[0033]

[Table 3]

4) Test Method The skin on the backs of the two diabetic models (ID Nos. 3 and 4) was squared about 25 mm and about 200 mm, respectively.
It was removed over a square area. Here, the gels (E, F) cultured for 6 days after the preparation were prepared in the same area (each gel weight was 2).
(4 mg wet weight and 191 mg wet weight). After transplantation, about 20 μl of blood was collected from the tail every two days and the blood glucose level was measured. Two control animals (ID numbers 1 and 2) used a non-transplanted diabetes model. The blood glucose level was measured using a glucose CII test (Wako Pure Chemical Industries, Ltd.). 5) Test results The test results are shown in Table 4. In the gel transplantation group, the continuous increase in blood glucose level as seen in the control group was suppressed, and a tendency for the blood glucose level to decrease was observed.

[0035]

[Table 4]

Animal Experiment 2 A hybrid gel containing proinsulin-producing cells was implanted into a diabetic model animal, and the therapeutic effect was evaluated by measuring blood glucose level and body weight. 1) Experimental animals Balb / c nude mice (7-week-old males)
A diabetic condition was induced by continuous intraperitoneal administration of streptozotocin (Sigma) at 2 mg / kg for 2 days and used in the test. The gel was implanted two days after the end of streptozotocin administration. 2) Cells used Three kinds of cell lines derived from rat skin were used.

RSF RSFins RSK 3) Gel preparation method One million RSFins cells were encapsulated in a collagen gel, and the same number of RSK cells were overlaid on this to obtain a gel M.
On the other hand, 1,000,000 RSF cells were encapsulated in a collagen gel, and the same number of RSKins cells were layered on this to obtain gel N. The composition of these gels is summarized in Table 5. These gels were used for transplantation after culturing at 37 ° C. for 7 days. The amount of proinsulin produced by the gel at this point was 300.8 μlU / hr / gel and 1.5 μlU / hr / gel for gel M and gel N, respectively.

[0038]

[Table 5]

4) Test Method For three of the six diabetes models described above, the skin on the right flank was removed in a circular shape having a diameter of 8 to 10 mm, and the gel M which had been cultured for 7 days after preparation was used for each individual. One was transplanted. After transplantation, about 5 μl of blood was collected from the tail every two days and the blood glucose level was measured. Body weight was also measured about every two days. Gel N was similarly transplanted as a control animal for the remaining three of the six diabetic model animals. The measurement of the blood glucose level was performed using Glutest E (Sanwa Chemical). 5) Test results The test results are shown in Table 6. In the gel M transplantation group, there was a tendency to suppress the increase in blood glucose level as seen in the control group,
In particular, it was remarkable on day 13 after transplantation. In the gel M-transplanted group, the tendency to suppress weight loss as observed in the control group was always observed throughout the observation period.

[0040]

[Table 6]

Example 2 Another hybrid gel of the present invention was prepared, and its effect was evaluated using the following in vitro test and the system of a diabetic model animal. In-vitro experiment 1 A gel enclosing insulin-producing cells transduced with a mutant insulin gene, which can be converted with furin and has excellent stability, was cultured, and the IRI value secreted into the culture solution was measured. 1) Cells used Two kinds of cell lines derived from rat skin were used.

Rat skin-derived fibroblasts transfected with a mutant insulin gene convertible with furin (RSFins
fur) RSK RSF insfur cells are obtained by mutation of furin into fibroblasts (RSF) obtained by primary culture from rat skin (insulination by cleavage of proinsulin chain at two sites).
Mutant insulin gene (DJGROSKREUTZ et a
l, The Jounal of Biological Chemistry, 269 (8), 624
1, 1994). Such gene transfer into RSF is carried out by using a plasmid-based expression vector pRIS / proins /
Using Ifur / IIfur / B10D, the above CHEN-OKA
Performed according to YAMA's method. Thereafter, the cells incorporating the vector were selectively expanded in a culture solution containing G418 at a concentration of 600 μg / ml.

From these cells, 32 clones were isolated, and the cell having the highest IRI value was selected. This strain RSF
insfur is 24.5 μl per million cells
U / hr of immunoreactive insulin was secreted into the culture. Since these cells simultaneously express the mutant enzyme furin to mutant insulin, proinsulin, a precursor of insulin, is converted to insulin depending on the furin activity of the cells.

Using an anti-furin monoclonal antibody (Genentic) and anti-insulin rabbit serum (Austroral Biological), RSFinsf was used.
Immunohistological studies on ur cells were also performed. The results are as shown in Table 7, and it was also confirmed immunohistologically that the cells produced insulin and furin.

[0045]

[Table 7]

2) Culture solution For the RSF Insfur cells, the culture solution A of Example 1 was used.
And the culture solution B of Example 1 was used for RSK cells. After enclosing the cells in a gel and layering, the cells were cultured using a culture solution A. 3) Test method 3 million RSF insfur cells were encapsulated in a gel,
Further, 3 million RSK cells were overlaid thereon to obtain gel G. Table 8 summarizes the structure of this gel G. This gel G was cultured at 37 ° C. in a 6 ml culture solution. The culture solution was replaced with a new one every two days. 8 after culture
On the day, the gel was washed three times with a culture solution, replaced with a new culture solution, and cultured for 8 hours, and the IRI value in the culture solution was measured using an insulin EIA kit (Sanko Junyaku).

[0047]

[Table 8]

4) Test Results As a result of this test, Gel G showed 25.2 μm for 8 hours.
It was confirmed that IRI of IU was secreted. That is, since this gel G shows stable secretion of insulin over a long period of time,
Insulin can be administered to the body. In vitro experiment 2 A method for enhancing insulin secretion from a gel was investigated in a gel in which insulin-producing cells transduced by furin and transfected with a mutant insulin gene encoding insulin having excellent stability were encapsulated. 1) Cells Used Cells similar to those used in the above-described in vitro test 1 were used. 2) Culture solution The same culture solution as used in the above-described in vitro test 1 was used in the same manner. 3) Test method One million RSFinsfur cells were encapsulated in a gel,
Further, one million RSK cells were overlaid thereon to obtain gel H. On the other hand, when 1 million RSF Insfur cells were encapsulated in the gel, a polyglycolic acid (PGA) mesh (Japan Redley, Dexon Mesh, Style 2) cut into a circle having a diameter of 15 cm and 25 cm was present, and furthermore, One million RSK cells were overlaid to obtain gels I and J, respectively. The composition of these gels is as shown in Table 9.

[0049]

[Table 9]

These gels were cultured at 37 ° C. in 2 ml of culture medium. The culture solution was replaced with a new one every 1-2 days. On the 8th day after the culture, each gel was washed three times with the culture medium, replaced with a new culture medium, and cultured for 8 hours. A small amount (50 μl) of the culture medium was collected with time, and the IRI value in the culture medium was measured. It was measured using an insulin EIA kit (Sanko Junyaku). 4) Test results The results of this test are as shown in Table 10. The gels I and J in which cells are encapsulated with the mesh and layered contain only cells, and the amount of insulin secreted is significantly larger than that of the gel H in which the cells are encapsulated. Therefore, the mesh (reticular body) coexists in the gel. Was found to be effective in increasing the amount of insulin secreted by cells.

[0051]

[Table 10]

Animal Experiment The gel containing insulin-producing cells was transplanted into a diabetic model animal, and the body transplantation and blood glucose level of the animal were measured to examine the effect of the gel transplantation. 1) Experimental animal Balb / c nude mouse (7-week-old male) was treated with 200
mg / Kg of streptozotocin (Sigma) was intraperitoneally administered twice in total over 2 days to induce a hyperglycemic state. After confirming that the animals showed hyperglycemia, they were used in experiments. 2) Cells to be used RSF RSFinsfur RSK using the following three cell lines derived from rat skin 3) Gel (artificial skin) preparation method Three million RSFinsfur cells were encapsulated in a collagen gel, and the same number of RSK cells were added thereto. Overlay to obtain gel K. On the other hand, 3 million RSF cells were similarly encapsulated,
The same number of RSK cells were overlaid on this to obtain gel L. The composition of these gels K and L is as summarized in Table 11.

[0053]

[Table 11]

4) Test Method The skin on the back of the above-described diabetic model animal was removed in a circular area having a diameter of about 11 mm, and gel K which had been cultured for 8 days and shrunk to about 10 mm in diameter was removed from the animal skin. Transplanted to the department. Control animals were similarly implanted with Gel L containing cells into which no gene was introduced. After transplantation, about 2
The body weight was measured every day, and about 5 μl of blood was collected from the tail, and the blood glucose level was measured using Glutest E (Sanwa Chemical). 5) Test results The results of this test are as shown in Table 12. In the control group (gel L transplant group), a decrease in body weight and an increase in blood glucose level were observed, whereas in the group of gel K transplant animals in which the mutant insulin gene-introduced cells were encapsulated, an increase in body weight and a decrease in blood glucose level were observed As a result, improvement in diabetes symptoms was confirmed.

[0055]

[Table 12]

[0056]

As described above in detail, according to the present invention, it is possible to stably administer a drug for a long period of time, alleviate pain to a patient, and finely adjust the dose.
Without using a vector derived from a retrovirus which has a risk of mutation to a wild type as in the past, it is possible to control genes from outside the body and to perform gene prescription by skin transplantation.

────────────────────────────────────────────────── ─── Continued on the front page (72) Robert Andrew Cuthbertson, Inventor, USA 94114, San Francisco, Douglas Street 559, San Francisco (72) Inventor Katsutoshi Yoshizato ) References Tokuhyo Hei 5-501206 (JP, A) Tokuhyo Hei 5-506169 (JP, A) (58) Fields studied (Int. Cl. ⁷ , DB name) A61L 27/00 WPI (DIALOG) BIOSIS (DIALOG)

Claims (14)

(57) [Claims] [1] A gene encoding a physiologically active substance is constructed.
Bioactive substance-secreting artificial skin consisting of biologically active substance-producing cells carrying the expression vector and a gel derived from a living body
Skin . 2. The artificial skin according to claim 1, wherein the physiologically active substance-producing cells are encapsulated in a gel derived from a living body. 3. The artificial skin according to claim 1, wherein the physiologically active substance-producing cells are overlaid on a gel derived from a living body. 4. The artificial skin according to claim 1, wherein the bioactive substance-producing cells are overlaid on a biological gel containing the bioactive substance-producing cells. 5. A method for constructing a gene encoding a physiologically active substance.
A biologically active substance-producing cell having an incorporated expression vector ,
An artificial skin for secreting a physiologically active substance comprising skin cells and a gel derived from a living body. 6. bioactive producing cells Human Engineering skin according to claim 5 in which the skin cells are layered on the gel of the biological encapsulation
Skin . 7. The artificial substance according to claim 5, wherein biologically active substance-producing cells are overlaid on a gel derived from a living body in which skin cells are encapsulated.
Skin . 8. The artificial skin according to claim 5, wherein the skin cells and the physiologically active substance-producing cells are encapsulated in a gel derived from a living body.
Skin . 9. The artificial skin according to claim 5, wherein skin cells or physiologically active substance-producing cells are layered on a gel derived from a living body in which skin cells and physiologically active substance-producing cells are encapsulated. 10. The method according to claim 2, wherein the physiologically active cells are encapsulated in a gel derived from a living body together with a reticulate body or a porous membrane.
4, 6, 8 or 9 artificial skin . 11. The skin cell to be encapsulated in the biological gel is a skin fibroblast, and the skin cell layered on the biological gel is a skin epidermal cell.
Artificial skin . 12. physiologically active substance-producing cells, or claim 1 insulin are skin fibroblasts or skin epidermal cells harboring the expression vector incorporating a gene encoding the
11. The artificial skin according to any of 11 above . 13. The artificial skin according to claim 12 , wherein the expression vector is a plasmid vector pBMG-neo-ins. 14. An expression vector, according to claim 12 which is a plasmid vector pRIS · proins · Ifur · IIfur · B10D
Artificial skin .