WO2006038462A1

WO2006038462A1 - Novel steroid-producing cell

Info

Publication number: WO2006038462A1
Application number: PCT/JP2005/017349
Authority: WO
Inventors: Shigeki Gondo; Toshihiko Yanase; Taijirou Okabe; Hajime Nawata
Original assignee: Kyushu University, National University Corporation
Priority date: 2004-10-01
Filing date: 2005-09-21
Publication date: 2006-04-13
Also published as: JPWO2006038462A1

Abstract

[PROBLEMS] To provide a novel steroid-producing cell which is usable in treating abnormal steroid hormone secretion such as steroid hormone deficiency via transplantation or the like. [MEANS FOR SOLVING PROBLEMS] A steroid-producing cell characterized by having been differentiated from a bone marrow cell or a multipotent stem cell by transferring a factor participating in steroid hormone synthesis thereto is provided. Moreover, a method of producing this steroid hormone-producing cell, utilization thereof, a medicinal composition with the use of a hormone secreted by the cell and a method of treating various diseases by using the cell are provided.

Description

Specification

New steroidogenic cells

Technical field

[0001] The present invention relates to the production of steroid-producing cells, and more specifically, to a novel steroid-producing cell produced thereby and a production method thereof. The invention also relates to the use of the cells so produced.

Background art

[0002] In general, steroid hormones are known as “steroidal agents” used in bronchial asthma and rheumatism in addition to external and internal use for atopic dermatitis. This steroidal agent has a strong immunosuppressive action, and it should be used with caution from the viewpoint of the power and side effects used in the treatment of a wide range of diseases.

[0003] On the other hand, steroid hormones produced in vivo are hormones that play a very important role in living organism maintenance. Insufficient production of steroid hormones in vivo reduces maintenance of regulation of glucose, regulation of Na'K balance, promotion of protein synthesis, control of inflammatory response 'immune response, sexual development, reproductive function, etc. When this steroid hormone is missing or decreased for various reasons, a condition called “steroid hormone deficiency” is caused.

[0004] One of these steroid hormone deficiencies is adrenal cortex dysfunction. This adrenal cortical dysfunction is caused by a lack of adrenal steroid hormones, and specific symptoms include generalized malaise due to lack of secretion of the stress-responsive hormone glucocorticoid cortisol. It may become stronger and medical findings may include hypotension, hypoglycemia, hyponatremia, hyperkemia, and pigmentation. In the worst case, acute adrenal insufficiency may result in death due to an absolute shortage of adrenal steroid hormones or a sudden increase in the amount required due to stress such as infection. Adrenal cortex dysfunction is often accompanied by poor secretion of the mineralocorticoid hormone aldosterone, further promoting hyponatremia and hyperkemia.

[0005] Other steroid hormone deficiencies include gonadal dysfunction. Gonadal dysfunction All have a decreased production of gonadal steroid hormones due to a variety of etiologies, such as genetic factors, tumors, inflammation, surgery, radiation, etc. Refers to the disease state. Basically it is not a life-threatening problem, but when it occurs in young people in their teens and twenties, secondary sexual characteristics are impaired and the lack of masculinity and femininity also causes complex problems. It can happen. At the same time, it can be a problem that affects future fertility. In addition, there is a problem that osteoporosis and arteriosclerosis are likely to occur due to a long-term absence of gonadal hormones that are caused only by the effects on the reproductive system.

[0006] Steroid hormone supplementation therapy has been established as a current treatment method for these steroid hormone deficiencies, and provides many benefits as described in Non-Patent Document 1, for example. However, especially in the case of adrenal insufficiency, it is necessary to continue supplementation throughout the lifetime. Glucocorticoids are generally supplemented, but mineral corticoids are also added if that alone does not correct the electrolyte dysfunction. Depending on the patient, it may be a fixed-dose oral administration, and it may be difficult to respond when the steroid requirement is rapidly increased due to infection, etc. If steroid excess continues, various side effects may occur.

[0007] Accordingly, new therapies such as gene therapy and steroidogenic cell transplantation are eagerly desired, but definitive treatment methods are still being developed at present.

[0008] Non-patent document 1: Laureti, S., Falorni, A., and Santeusanio, F. Improveme nt of treatment of primary adrenal insufficiency by administration of cortisone acetate in three daily doses. 〃J Endocrinol Invest. 20 03 ( 11): 1071 ~ 1075

Disclosure of the invention

Problems to be solved by the invention

[0009] The present invention has been made in view of such problems, and an object of the present invention is to provide a novel steroid-producing cell that can be used for the treatment of abnormal steroid hormone secretion such as steroid hormone deficiency. Specifically, steroid-producing cells that can secrete physiologically diverse steroid hormones, their production methods, uses, pharmaceutical compositions using the secreted hormones, and various diseases using the cells A cure for the disease For that purpose.

Means for solving the problem

Recently, many studies have suggested that bone marrow cell transplantation may contribute to the regeneration of hematopoietic / mesenchymal cell groups in various organs. In other words, it is thought that bone marrow cells contain pluripotent progenitor cells that can be divided into various organs. Bone marrow stem cells are known to grow into hematopoietic and mesenchymal cell groups, but before the present invention, they were not known to be separated into steroidogenic cells. However, the present inventors paid attention to the fact that bone marrow cells can be separated into muscles and fats derived from the mesenchymal system as well as the adrenal cortex 'gonadal gland, and when steroidogenic cells are separated from bone marrow cells, Based on this new knowledge, and based on this and sincere examination and experimentation, we have succeeded in producing steroid-producing cells with several properties as described below.

That is, according to the first main aspect of the present invention, there is provided a steroid-producing cell characterized by introducing a factor involved in steroid hormone synthesis into bone marrow cells and allowing the bone marrow cells to differentiate. Provided.

[0012] According to such a configuration, a novel steroid hormone-producing cell that can be used for treatment of abnormal steroid hormone secretion such as steroid hormone deficiency can be obtained by transplantation or the like.

Here, the bone marrow cells include, but are not limited to, bone marrow stem cells, bone marrow mesenchymal stem cells, hematopoietic stem cells, and pluripotent progenitor cells.

[0014] Further, according to the second main aspect of the present invention, a steroid characterized by introducing a factor involved in steroid hormone synthesis into a pluripotent stem cell and allowing the pluripotent stem cell to differentiate. A production cell is provided. Here, the pluripotent stem cells are preferably somatic pluripotent stem cells including mesenchymal stem cells and hematopoietic stem cells.

[0015] The pluripotent stem cells are preferably derived from bone marrow cells. However, the pluripotent stem cells of the present invention are not limited to those derived from bone marrow, but may be those derived from yarn and tissue where pluripotent stem cells are present.

[0016] The factor involved in the steroid synthesis is not limited to this, but is a transcriptional regulator of steroid hormone synthase. Transcription of this steroid hormone synthase The factor is preferably Steroidogenic factor 1 (SF—l).

[0017] According to one embodiment of the present invention, the steroid hormones produced in the steroidogenic cells are predanenolone, progesterone, oxycorticosterone, conoleticosterone. emissions (corticoste rone), 18-hydroxy-Kono retinyl corticosterone (18- hydroxycorticosterone), Anoredosute Ron, aldosterone Roh, 1 α Hitoro ³ r shea pregnenolone, 17 a- hyaroxypregnelone)

17-Hydroxyprogesterone (17 0; -11 (1): 0): 0865 6]: 0116), 11 deoxyconoreth, 11-deoxycortisol, Cortisol, DHEA (dehydroepia ndrosterone, Andros anion ( one or more of the steroid hormones consisting of androstenedione), estrone, androstenediol, testosterone, and estradiol.

[0018] The steroid-producing cells secrete steroid hormones over a predetermined period. The predetermined period is preferably at least 2 weeks. This can reduce the frequency of treatment and reduce the burden on the patient.

[0019] Furthermore, according to one embodiment of the present invention, the steroidogenic cells are adrenocorticotropic hormone (ACTH) responsive. Also here ACTH responsiveness is dose dependent. Thus, for example, when a steroidogenic bone marrow cell obtained in the present invention is transplanted to a patient with adrenal insufficiency, if the steroid in the adrenal cortex in the living body is insufficient, ACTH is secreted, and the adrenal gland is secreted from the cell. Cortical steroids are secreted and, conversely, if excessive, ACTH can be suppressed and, as a result, corticosteroid secretion can also be suppressed.

[0020] According to one embodiment of the present invention, the steroid-producing cells are cultured in a retinoic acid-containing medium or a medium containing retinoic acid and hCG (human chorionic stimulating hormone) to obtain a gonadal steroid hormone. To produce.

[0021] According to a third main aspect of the present invention, (a) a step of preparing bone marrow cells, and (b) introducing a factor involved in steroid hormone synthesis in the bone marrow cells, There is provided a method for producing steroid-producing cells, comprising the step of separating the cells into steroid-producing cells. This makes it possible to produce a variety of effective steroid hormones It becomes possible to produce steroidogenic cells that can be secreted.

[0022] According to one embodiment of the present invention, the method further comprises a step of culturing in a retinoic acid-containing medium, or a retinoic acid and hCG (human chorionic stimulating hormone) -containing medium. A method for producing a steroidogenic cell characterized by inducing differentiation.

[0023] According to a fourth main aspect of the present invention, there is provided a pharmaceutical composition comprising the physiologically diverse steroid hormone secreted by the steroidogenic cell force and a pharmaceutically acceptable carrier. . According to the pharmaceutical composition thus obtained, it becomes possible to effectively treat steroid hormone secretion abnormalities such as steroid hormone deficiency and various self-exemptions.

[0024] Further features and remarkable effects of the present invention will become apparent to those skilled in the art from the description of the embodiments of the present invention described below.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

[0026] As described above, according to the present invention, there is provided a steroidogenic cell obtained by introducing a factor involved in a steroid synthase into bone marrow cells or pluripotent stem cells and allowing the cells to differentiate.

[0027] # Summary and 亩 ^^ Summary

Recently, many studies suggest that bone marrow cell transplantation may contribute to the regeneration of hematopoietic and mesenchymal cells in various organs. In other words, it is thought that bone marrow cells contain pluripotent progenitor cells that can be divided into various organs. Bone marrow stem cells are known to grow into hematopoietic and mesenchymal cells, but before the present invention, they were known to be separated into steroidogenic cells. However, the present inventors paid attention to the fact that bone marrow cells can be divided into muscles and fats derived from the mesenchymal system as well as the adrenal cortex 'gonadal gland, and when steroidogenic cells are separated from bone marrow cells, As a result of obtaining new knowledge and conducting repeated sincerity studies and experiments based on this knowledge, we succeeded in generating steroid-producing cells having several desirable properties described below.

[0028] Here, bone marrow cells and pluripotent stem cells can be separated into steroidogenic cells. It is not limited to a good person or a mammal such as a mouse. The cell collection method may be a known method.

[0029] Introduction of transcription regulatory insulator for steroid synthase

In order to differentiate bone marrow cells and pluripotent stem cells into steroidogenic cells, it is considered necessary to synthesize steroid hormones by introducing factors involved in steroid synthesis.

[0030] Here, SF-1, a tissue-specific transcription factor of steroid hormone synthase, also known as ad renal 4 binding protein (Ad4BP), belongs to the nuclear receptor superfamily and is reported to be involved in many steroidogenic genes. (1). SF-1 is thought to be particularly important for steroidogenesis and steroidogenic tissue growth, as a complete absence of adrenal glands and gonadal glands was observed in this SF-1 knockout mouse.

[0031] On the other hand, when SF-1 was continuously expressed in embryonic stem cells, these embryonic stem cells acquired the ability to produce steroids, and P450scc was induced in a cAMP- and retinoic acid-dependent manner. There is also an experiment report that it produced. However, this ability to produce steroids is limited to the level of progesterone secretion, and the addition of 20 a-hydroxycholesterol, which is a foreign substrate and passes through the outer mitochondrial membrane, requires no physiological secretion. However, these results strongly suggest that SF-1 is the key to steroidogenic cell sorting.

[0032] In order to verify whether steroidogenic cells are generated by introduction of SF-1 into bone marrow cells, the present inventors have created an adenovirus (Ux-bSF-1) having ushi SF-1 However, it has been clarified that when a long-term cultured bone marrow cell derived from a mouse is infected with Adx-bSF-1, a significant amount of various steroids are produced.

[0033] However, in the present invention, the transcriptional regulatory factor is not limited to Steroidogenic factor 1 (SF-1), but may be a factor necessary for synthesizing steroid hormones in the cell. Yeah.

[0034] In the present invention, as a method for introducing and forcibly expressing a target gene in bone marrow cells, according to one embodiment of the present invention, for example, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lenti Virus vectors, HIV vectors, etc. This is done using a kuta. However, it can be carried out by using known techniques such as electoral positioning and protein direct introduction using only such methods using vectors derived from viruses.

[0035] Further, the method of separating bone marrow cells and the like into steroid-producing cells is considered to be a method in which an exogenous factor that acts only by introduction of an endogenous factor involved in steroid hormone synthesis is also possible. . That is, by culturing the bone marrow cells in a culture solution and medium containing exogenous factors, it is possible to separate them into target steroid-producing cells.

[0036] New Steroid 'Bovine. Cell, Its Function and Use

According to the novel steroid-producing cells of the present invention, the following pregnenolone, progesterone, deoxycorticosterone, corticosterone, 18 hydroxyconoleticosterone (18- hyd roxycorticosterone), Anoredosuteron (aldosterone), 17 α-hydroxy pregnane Nenoro down (1 / ■ α- hydroxypregnelone Roh, 1 "α shed ³ r Nfu opening Gesuaron (1 1 a-hydroxy progesterone ;, 11 Doya From Sikorchizonole (11—deoxycortisol), Cortezonole (cortisol), DHEA (dehydroepiandrosterone), Andros anion, androstene dione, Estrone, Androstenediol, testosterone, Estradiol (estradiol) At least one of the steroid hormones produced.

[0037] According to the analysis of the present inventors, the production of steroid hormones by steroid-producing cells according to the present invention increases the amount of steroid hormone synthase in bone marrow cells by introducing a synthase transcription regulatory factor. It seems to be the cause. That is, in the steroidogenic cells according to the present invention, it was found from experiments using RT-PCR that the mRNA expression of the steroid hormone synthase is enhanced by forcibly expressing the transcriptional regulatory factor. Furthermore, immunostaining experiments using anti-cytochrome P450 _SCC antibodies showed that the expression of the steroid hormone synthase is enhanced at the protein level.

[0038] Further, it is considered that the steroidogenic bone marrow cells obtained in the present invention are required to produce stable steroid hormones for a certain period or longer (at least 2 weeks, preferably 3 weeks or more). However, according to this example, steroid hormone production continued for at least 112 days. According to RT-PCR analysis, this long-lasting ability to produce steroids is due to the introduced adenovirus-derived urine SF-1 rather than by induction of endogenous mouse SF-1. It was confirmed. That is, even if the expression level of ushi SF-1 by adenovirus is low, the bone marrow cells are sufficient for long-term and diverse steroid production, or SF-1 is necessary for the initiation of steroid production. However, it is not important to maintain it.

[0039] The results of flow cytometry suggested that the steroidogenic cells originated from pluripotent and immature hematopoietic Z mesenchymal stem cells.

[0040] Furthermore, it was found that the novel steroidogenic cells can control steroid secretion in response to corticotropin (ACTH). This ACTH response is dose-dependent. Even in this respect, the steroidogenic cells obtained in the present invention are considered to be beneficial. For example, when the steroid-producing bone marrow cells obtained in the present invention are transplanted to a patient with adrenal insufficiency, ACTH is secreted if the steroid in the adrenal cortex in the living body is insufficient, and the corticosteroid is secreted from the cells. On the contrary, if it is excessive, A CTH is suppressed, and as a result, corticosteroid secretion can also be suppressed.

[0041] The present inventors further examined how the novel steroidogenic cells are affected by the culture conditions. As a result of experiments, it was clarified that the novel steroid-producing cells can produce gonadal steroid hormones by culturing them in a medium containing retinoic acid or a medium containing retinoic acid and hCG (human chorionic stimulating hormone). . As a conventional technique, it has not been known before the present invention that retinoic acid induces the production of a potent gland hormone that is known to induce the production of adrenal hormone (corticosterone).

[0042] Further, when the steroid-producing cells obtained in the present invention are transplanted, it is presumed that steroids can be secreted at a high local concentration in transplantation in vivo. In other words, steroids are secreted at the necessary site as much as necessary, and the systemic steroids do not become excessive, so no serious side effects are expected.

[0043] Based on the above findings, the steroidogenic bone marrow cells obtained in the present invention were transferred to autologous cells. By using such means as transplantation, it can be applied to steroid cell transplantation for steroid hormone secretion abnormalities such as steroid hormone deficiency, allergy / autoimmune disease treatment, rejection of transplanted organ rejection, etc.! U, thought to provide a cure. That is, according to the present invention, steroid hormone secretion abnormalities such as steroid hormone deficiency, autoimmune disease, prevention of rejection of transplanted organs, etc. by transplanting the novel steroid-producing cells to a target site in the living body. A method of treatment is provided.

[0044] Further, based on the above findings, there is provided a pharmaceutical composition comprising the steroid hormone secreted from the novel steroidogenic cell obtained above and a pharmaceutically acceptable carrier. Is done. The pharmaceutical composition may be used as a “steroid” for the treatment of bronchial asthma, rheumatism and the like, as well as external use for oral atopic dermatitis. It can also be used for the treatment of steroid deficiency, allergies, autoimmune diseases and the like.

[0045] Furthermore, the pluripotency of bone marrow cells obtained in the present invention into adrenal or gonadal steroid-producing cells is an important model for elucidating the tissue, site, and cell-specific mechanism of adrenal gonadal cell differentiation. It is speculated that it can be.

[0046] Further, as described above, the steroidogenic cells obtained in one embodiment of the present invention are differentiated from pluripotent stem cells such as bone marrow stem cells, mesenchymal stem cells, hematopoietic stem cells in bone marrow cells. Presumed to be. As is known, mesenchymal stem cells are also present in fat and muscle. Therefore, it is considered that mesenchymal stem cells and hematopoietic stem cells can be separated from the steroid-producing cells of the present invention even if they are not derived from bone marrow cells. If it can be collected from sources other than bone marrow, it has the advantages of wide options and high convenience and safety.

[0047] Next, an example of generation of a novel steroidogenic cell according to the present invention will be described in the following examples.

Example 1

[0048] In order to verify whether steroidogenic cells are generated by introduction of SF-1 into bone marrow cells, the present inventors first have an adenovirus having ushi SF-1 (Adx-bSF-1). And long-term cultured bone marrow cells were infected with Adx-bSF-1. Thus, the bone marrow cell force revealed that a significant amount of various steroids were produced and steroidogenic cells were produced. Hereinafter, this verification process will be described in detail. [0049] Preparation of adenoviral vector containing SF-1

Using the Adenovirus Expression Vector Kit (Takara, Osaka, Japan), a recombinant (recombinant) vector derived from a human type 5-adenovirus vector was prepared.

[0050] Usci SF—lZAd4BP cDNA (distributed by Prof. Kenichiro Morohashi, National Institute for Basic Biology, Okazaki National Research Institute) was digested with BamHI and EcoRI, and the recombinant cosmid vector pAxCAwt (Takara) containing the CAG promoter Inserted into Swal site. The recombinant bovine SF-1 adenoviral vector (Adx-bSF-1) is reported by Miyake et al. (See Proc. Natl. Acad. Sci. USA 93: 132, 1996) I made it.

[0051] As a negative control, an adenovirus vector into which only the j8-galactosidase gene was transferred was constructed. This vector was called Adx-LacZ.

[0052] Searching for mouse bone marrow cells 朐 ¾ Long-term roasting

A 3-month-old male B6-GFP (green fluorescence protein) mouse (C57BLZ6Tgl 4 (act-EGFP) osbY01) was donated by Yamada (Kyoto University). In another experiment, 4-month-old male 129SVJ mice were also used. The bone marrow cells were cultured using conventional techniques with some adjustments. Briefly, fresh complete bone marrow cells were recovered by pouring the bone marrow cells from the mice into the culture medium. Culture medium A consists of 2 mM ML-daltamine, lOOUZml penicillin, 100 gZml sprepmycin, 0.0125 / z gZml amphotericin (Sigma—aldrich, Irvine ゝ UK) ゝ 10-7 M hydrocortisone (Nikkenk ayaku, Japan) and Contains a-MEM containing 20% donor horse serum (Lot6603F and 7307F, ICN Biochemicals, Aurora, Ohio). The collected cells were seeded in a 75 cm 2 tissue culture flask (Nalge Nunc, Rochester, NY) and incubated with the culture medium A at 37 ° C. and 5% C02. Adherent cells were cultured for several weeks, treated with trypsin, and stored at −80 ° C. with a cell banker until use. The stored BMCs were cultured in culture medium A for 120-180 days (passage 12-18) with the goal of growing a relatively purified cell population as needed in the experiment. From this cell population, 5 × 10 5 BMCs were again seeded in a 60 mm Petri dish (Nunc) with the culture medium A. When the BMCs grow to subconfluent The cells were infected with adenovirus with about 10 plaque forming unit Z cells. As a control for all experiments, the BMCs expressed | 8-galactosidase (Adx- LacZ)

[0053] Measurement of steroid content in culture fluid secreted from BMCs

The following hormones secreted into the culture medium: Progesterone (P4), Deoxycorticosterone (DOC), Corticosterone (Β), 17 α Hydroxyprogesterone (17 α—OH P4), 11 Deoxycortisol (S), dehydroepandrosterone (DHEA), Δ 4 -androstenedione (Δ 4-A), and testosterone (T) content using commercially available RIA kit (Diagnostic Products Corp., LA) Measured in collaboration with SRL Co. Ltd. (Tokyo, Japan) (each specific RIA system was developed by SRL). The amount of P4 and DOC secreted into the culture medium was also confirmed in the presence of synthetic 124ACTH (Shionogi Co., Osaka, Japan) in the absence of Z. The detection limits for P4, DOC, Β, 17 α—OH Ρ4, S, DHEA, Δ 4— A, and T are 0.1 ng / ml, 0.02 ηg / ml, 20. Ong / ml, 0 lng / ml, 0.04 ng / ml, 0.2 ng / ml, 0.1 ng / ml, and 0.05 ngZml or less.

[0054] immunoocvtochemistrv

Perform immunocytochemistry studies of BMCs from 129VJ mice with anti-rabbit cytochrome P450scc antibodies (RDI, NJ) using Zenon Rabbit IgG labeling kit (Molecular probes, Inc., OR) or with preimmune serum as a control. went. The cells were seeded on a collagen type I membrane (Asahi technoglass, Tokyo ^ Japan) in a 35 mm petri dish and fixed with 4% paraformaldehyde at 4 ° C. for 1 hour. After that, the manufacturer's instructions were followed. Fluorescence was observed using a fluorescence microscope (BX-51; 01ympus, Tokyo, Japan).

[0055] Real-time PCR quantification

LightCycler (Poche Diagnostics) for quantitative analysis of various steroidogenic enzymes, including StAR, ACTH receptor (ACTH—R), and P450scc, P450cl7, P450C11, P450501, P450ald, 3 j8— HSD, and 17 j8—HSD type 3 GmbH, Mannheim, Germany). The cultured BMCs and Y-1 Total RNA was isolated from cells using RNasy mini kit (Qiagen) and from mouse testis and adrenal gland using Isogen (Wako Pure Chemical Industries, Osaka Japan). First-strand complementary DNA was synthesized using 5 μg of total RNA as a template, and PCR was performed in the LightCycler according to the manufacturer's instructions. The sense Z antisense primer used was reported by Mukai et al. (Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible Expression of glucocorticoid—synthesizing g enes; Eur. J. Biochem. 269, 69-81, 2002 ). PCR conditions are available upon request. The threshold was measured when the fluorescence intensity determined in LightCycler Software Ver. 3.5 was in the geometric phase of amplification. The product was subjected to a 2% agarose gel. The nucleotide sequence of each PCR product was confirmed by direct sequencing using appropriate primers. The relative expression levels of the mRNA were calibrated against their β-actin and its ratio to mouse adrenal cortex Υ-1 cells, or mouse testis or adrenal gland, which were the controls.

[0056] Flow cytometry

The basic protocol of flow cytometry followed a known method. Briefly, 3 Χ105BMCs を (phycoerythrin) -binding anti-mouse c—kit ゝ CDl lb, CD34, CD44, CD45, and Sea-1 monoclonal antibodies (BD Biosciences, Japan), or isotype-matched PE-binding rat IgG (BD Biosciences) and incubation at 4 ° C for 30 minutes. The cells were analyzed with a FACScan flow cytometer (Becton Dickinson).

[0057] Statistics

1 factor ANOVA (ANOVA) was used for statistical evaluation. P <0. 0

5 was considered statistically significant.

[0058] Adx—bSF— 1 infection of long-term cultured bone marrow cells

(1) Measurement of steroid content in culture fluid secreted from BMCs

In order to verify whether steroidogenic cells are generated by introducing SF-1 into bone marrow cells, the present inventors used the above-described method to adenovirus with ushi SF-1 (Adx — BSF— Created 1). As a result of experiments, the present inventors have clarified that various steroids are produced when long-term cultured bone marrow cells are infected with Adx-bSF-1.

[0059] Long-term (123 days) cultured bone marrow cells of male GFP mice were infected with Adx-bSF-1 or Adx-LacZ as a control, and cultured for 7 days. Thereafter, the amount of steroid in the culture medium cultured for 4 days was measured. Figure 1 shows the results. Figures l (a) to (h) show progesterone (P4), deoxycorticosterone (DOC), corticosterone (Β), and 17α-hydroxyprogesterone, respectively, in long-term cultures of GFP mice. (17 α—OHP4), 11 Deoxycortisol (S), Dehydroepiandrosterone (DHEA), Δ 4 Androstenedione (Δ 4— A), and Testosterone (T) showing the basal secretion amount, The The values in the figure are indicated by the average value person SD (n = 3). S and L indicate BMCs transfected with Adx-bSF-l and BMCs transfected with Adx-LacZ, respectively. As shown in Figure 1, Adx—bSF 1 infected bone marrow cells contain significant amounts of progesterone (P4), deoxycorticosterone (DOC), corticosterone (Β), 17 α hydroxyprogesterone (17 α -OH). Ρ4), 11 Doxycortisol (S), Dehydroepiandrosterone (D ΗΕΑ), Δ 4—Androstenedione (Δ 4— Α), and Testosterone (Τ), but Adx—LacZ infected cells Did not produce. In Adx-LacZ-infected control cell cultures, all steroids except S were not detected. The trace amount of S detected in the control medium is probably a cross-reaction (9.5%) of S antibody to hydrocortisone, and no steroid precursors are likely to be produced.

[0060] Effect of Adx-bSF-1 expression on steroid hormone synthase in bone marrow

(1) RT-PCR measurement (effect on mRNA expression)

Next, a real-time reverse transcriptase-polym erase chain reaction (RT-PCR) was performed on the cocoon filaments used in the experiment. The result is shown in Fig.2. Figures 2 (a) to (f) are graphs showing the results of real-time PCR for StAR, P450scc, 3 j8- HSD, P450cl1, P450cl7, 17 j8- HSD type 3 and ACTH-R, respectively. In addition, the actual specific PCR band in the agarose gel amplified above 40 centimeters and stained with ethidium bromide is shown below the figure. The relative mRNA expression level is j8— Calibrated with actin. S and L indicate BM Cs transfected with Adx-bSF-1 and BMCs transfected with Adx-LacZ, respectively. Adx— LacZ-infected target Itoda force, etc. Significant PCR products of StAR, P450scc, 3 j8—HSD, P450cl1, 17 j8—HSD type 3 were not obtained. The value was expressed as the average scrutiny SD (n = 3). * Indicates P <0. 05. From these graphs, the steroid hormone synthetase ster oidogenic acute regulatory protein (StAR), P450scc, 3 j8—hydroxysteroid dehydrogenase (3 j8—USD;), P450cl l, P450cl7, and 17 j8—USD type 3 MRNA expression was observed in bone marrow cells at 11 days after Adx-bSF-1 infection. On the other hand, it was not observed in Adx-LacZ-infected cells. P450ald mRNA could not be verified. Adrenocorticotropic hormone receptor (ACTH-R) was also expressed in Adx-LacZ-infected cells but the expression level was very low (2Z1000 of adrenal expression) and was further decreased in Adx bSF-1 infected cells .

[0061] That is, the relative ratio to the expression of control Y-1 cells is expressed in the case of StAR, P450scc, and 3 β HSD; for the expression of the mouse adrenal gland, it is expressed in the case of P450cl l and ACTH-R. It is expressed in the case of P450cl7 and 17 | 8—HSD type 3 for mouse testis expression. From the target cells infected with Adx—LacZ, significant PCR products of StAR, P450s cc, 3 j8—HSD, P450cl1, 17 j8—HSD type 3 were obtained. From the above results, it was found that expression of bSF-1 in the bone marrow cells enhances steroid hormone synthase mRNA expression.

[0062] Similar results were observed in 129VJ mice, suggesting little difference by pedigree (data not shown).

[0063] (2) Observation by immunocytochemistry (effect on protein expression)

The results of immunostaining of 129VJ mouse bone marrow cells using the antibodies described above are shown in FIG. Fig. 3 reveals the expression of P450scc, a steroid hormone synthase. Green fluorescent cells are positive for P450scc. As a negative control, preimmune serum and cells did not react (data not shown). That is, it was found that the expression level of steroid hormone synthase protein is enhanced by expressing bSF-1 in the bone marrow cells. [0064] In the present invention, the steroidogenic cells simultaneously produce a mixture of adrenal and gonadal steroids, that is, DOC, B, DHEA, Δ4-A, and T. Since P450cl7, which is a steroid hormone synthase, is expressed in human adrenal gland but not in mouse adrenal gland, the prominent expression of P450cl 7 in the bone marrow cells in the present invention is due to the mixed production of steroids. It suggests that it will occur beyond.

[0065] From these results, the bone marrow cells are pluripotent in steroid-producing cells, and there is a common source of steroid-producing tissue; that is, the bone marrow cell-derived steroid obtained in the present invention. It was suggested that the producer cells may be stem cells. Then, this assumption was examined using flow cytometry.

[0066] # fine q force Differentiated steroids, fine is (; notification rfn. / 曰 H)

(1) Analysis of the bone marrow cell surface marker by flow cytometry

The results of analyzing the bone marrow cell surface markers using flow cytometry are shown in FIG. The flow cytometry experiment was performed prior to adenovirus infection using the same BMC s used in the experiment of FIG. Figures 4 (a) to (f) show cell surface markers c-kit, CDl lb, CD34, CD44, CD45, and Sea-1. From the experimental results, CD45 specific for hematopoietic cells and CD34 specific for hematopoietic progenitor cells were negative. CD1 lb, a marker for monocytes and macrophages, was also negative. Although the mouse mesenchymal stem cell marker is not completely clear and controversial, the potential marker CD44 was negative in the bone marrow cells. On the other hand, hematopoietic and mesenchymal stem / progenitor cell markers c kit and Sea-1 were positive.

[0067] Although the bone marrow cells obtained in the present invention are heterogeneous, steroidogenic cells were probably pluripotent and originated from immature stem cells. Long-term cultured bone marrow cells were treated with 0.05 mM ascorbic acia, 10 mM β-glycerophosphate ^ and 0.1 μΜ dexameth asone, so that they were separated like osteoblasts stained with alkaline phosphatase (data shown). ) This proves that long-term cultured bone marrow cells have mesenchymal properties!

[0068] Confirmation of ACTH in long-term cultured bone marrow cells Since the expression of ACTH-R was confirmed above, the ACTH reactivity of bone marrow cells was examined by measuring P4 and DOC. Infect long-term (100 days) cultured bone marrow cells of male GFP mice with Adx-bSF-1 or Adx-LacZ, and stimulate with 2.4 nM to 2.4 M ACTH every 3-4 days after infection. Steroids were measured. Adx— bSF— 1 young ίma Adx— It was infected with LacZ (DayO), then itoda tsukuma on days 0, 4, 7, 11, 14, 18, 21, 25, 28 2. 4ηΜ 力 et al. 2 Treated with 4 μΜ ACTH. Before adding ACTH, the culture solution was collected and the steroid concentration was measured. FIG. 5 is a graph showing the results. FIGS. 5 (a) and (b) show the effect of ACTH on the secretion of progesterone (P4) and DOC from cultured BMCs collected from GFP mice, respectively. The values in the figure are indicated by the mean valuer SD (n = 3); a, b, c, d, and e are A CTH of 0, 2.4, 24, 240 nM and 2.4 / z M, respectively. Is shown. * Indicates P <0. 05 and ** indicates P <0. 01 relative to the control (without ACTH). From the experimental results, ACTH produced these steroids in a dose-dependent manner in Adx-bSF-1-infected bone marrow cells. However, it was not generated in Adx-LacZ infected cells (data not shown).

[0069] These results indicate that the introduction of bSF-1 into long-term cultured bone marrow cells can be separated into steroidogenic cells that secrete various steroids under ACTH-stimulated as well as basal conditions. .

[0070] In addition, mRNA induction of steroidogenic enzymes P450scc, 3β-HSD, P450c21, P450cl1, and 17j8-HSD by 2.4 M ACTH was also confirmed by real-time PCR. However, mRNA induction of ACTH-R was not confirmed (Fig. 6). Cells used in the experiment were treated with 2.4 μΜ ACTH on days 0, 4, and 7 after infection with Adx-bSF-1 (DayO) and cultured for 4 days. On day 11, the total RNA of the cells was extracted and subjected to real-time PCR. Figures 6 (a) to (f) are respectively P450scc, 3 | 8— HSD, P450c21, P450cl l in the presence of 2.4 M ACTH (lower right oblique column) and absence (white column). , 17 j8-HSD type 3 and ACTH-R mRNA expression. The values in the figure are indicated by the average value person SD (n = 3). Relative mRNA expression levels were calibrated with ι8-actin. The relative ratio to the expression of control Y-1 cells is expressed in the case of P4 50scc and 3 j8- HSD; P450c21, P45 for mouse adrenal glands It is expressed in the case of Ocl l and ACTH-R; it is expressed in the case of 17 j8-HSD type 3 for mouse testis. * Indicates P <0. 05 and ** indicates P <0.01 relative to the control (in the absence of ACTH).

[0071] Long-term steroids from bone marrow sputum 'cattle.

We examined the duration of bone marrow cell steroidogenesis following Adx—bSF—1 infection. Long-term (180 days) cultured bone marrow cells were infected with Adx-bSF-1 or Adx-LacZ, and P4 and DOC in the culture were measured every 3-4 days. The results are shown in Fig. 7. Figs. 7 (a) and 7 (b) show the results of progesterone (P4) and deoxycorticosterone (DOC) in the culture medium of long-term cultured BMCs from GFP mice, respectively. Shows the time course of basal secretions (time course). The value in the figure shows the average value of duplicate (duplicate) dishes. The black column shows the steroid secreted by the cell force infected with Adx-bSF-1. Secretion of P4 and DOC was not detectable in cultures from BMCs infected with Adx-LacZ (data not shown). From Figure 7, it is clear that P4 and DOC production lasted at least 112 days. Considering the half-life of adenovirus (2-3 weeks), this long-term steroid production is unexpected.

[0072] Also, bSF-1 RT-PCR was performed using RNA extracted from cell forces obtained on days 0, 14, 21, and 49 in FIG. Electrophoresis was performed on a 1.5% agarose gel, and ethidium bromide was used for staining. Fig. 8 shows the results. Y1, V, (1), S and L are Y-1 cells (negative control), Adx-bSF1 (positive control), BMCs before infection, Adx-bSF, respectively. -BMCs transfected with 1 and BMCs transfected with Adx-LacZ. In the experiment of expression of bSF-1 derived from Adx-bSF-1, the expression of endogenous mouse SF-1 could not be confirmed, and only adenovirus-derived ushi SF-1 was detected (FIG. 8). This result suggests that long-lasting steroid production is caused by adenovirus-derived ushi SF-1, which is not derived from endogenous mouse SF-1.

[0073] Gonadal hormone-producing cattle. Induction of cell differentiation

The present inventors further examined how the steroidogenic cells of the present invention are affected by the culture conditions. Similar to the method described above, long-term cultured bone marrow cells (LTBMCs) Infection with urine SF-1-expressing virus or control virus, and retinoic acid (A TRA), retinoic acid and hCG (human chorionic stimulating hormone) during viral infection and medium exchange. day 0, 4, 7, 11, 14), dayl 4-18 [Honolemon in the cultured medium was measured. The results are shown in FIGS. 9A and 9B (when ATRA is added to the medium) and FIGS. 10A and 1 OB (when ATRA and hCG are added to the medium). The experimental results revealed that the production of corticosterone (adrenal hormone) and testosterone (gonad hormone) was promoted. In particular, since the production of testosterone was markedly promoted (from Fig. 9B), the steroid-producing cells of the present invention were induced to differentiate into gonadal hormone-producing cells when cultured in a medium containing retinoic acid. It was suggested.

[0074] Although it is known that retinoic acid stimulates adrenal cells and promotes the production of adrenal hormones, it was first demonstrated by the present invention that it induces the production of gonadal hormones.

[0075] Certain preferred embodiments and examples of the invention have been described and illustrated above. The invention is not limited to these embodiments or examples. SUMMARY OF THE INVENTION Various modifications can be made without departing from the scope.

Brief Description of Drawings

[0076] [Fig. 1] Fig. 1 (a) to (h) are long-term cultures from GFP mice, pgestesterone (P4), deoxycorticosterone (DOC), corticostero (B), 17 α hydroxyprogesterone (17 α— ΟΗΡ4), 11 deoxycortisol (S), dehydroepandrosterone (DHEA), Δ 4—androstenedine (Δ 4—A), And a graph showing the basal secretion amount of testosterone (T).

FIG. 2 (a) to (f) are graphs showing the results of real-time PCR of StAR, P450scc, 3 j8- HSD, P450cl 1, P450cl 7, 17 j8-HSD type 3 and ACTH-R, respectively.

[FIG. 3] FIG. 3 is a view showing an immunostained image obtained by using an anti-cytochrome P450scc antibody of BMCs collected from a 129VJ mouse.

[Figure 4] Figures 4 (a) to (f) show the results of flow cytometric analysis of c-kit, CD ib, CD34, CD44, CD45, and Sea-1 expression, which are surface markers in cultured BMCs, respectively. . [FIG. 5] FIGS. 5 (a) and 5 (b) are graphs showing the effect of ACTH on the secretion of progesterone (P4) and DOC from cultured BMCs in which GFP mouse power was also collected.

[Fig.6] Figures 6 (a) to (f) are respectively 2. P450scc, 3 j8— HSD, P450c21, P450cl l, in the presence of ACTH (lower right slanted column) and in the presence (white column), 17 β

— Graph showing real-time PCR for HSD type 3 and ACTH-R.

[Figure 7] Figures 7 (a) and (b) show the time courses of the basal secretions of progesterone (P4) and deoxycorticosterone (DOC) in the culture medium of long-term cultured BMCs from GFP mice, respectively. The graph which shows (time course).

FIG. 8 shows the expression of AdSF-bSF-1 derived bSF-1 on agarose gel.

[9] FIG. 9, 129VJ mouse Adx-BSF-l infection BMCs obtained from retinoic acid (concentration 0~: L0 ^_4 M) corticosterone production amount when cultured in medium containing the (a), Tesutosu The figure which shows teron production amount (b).

[Fig. 10] Fig. 10 shows Adx-bSF-l-infected BMCs collected from 129VJ mice when cultured under various culture conditions (containing cortisol, retinoic acid, and hCG (human chorionic gonadotropin)) The figure which shows corticosterone production amount (a) and testosterone production amount (b).

Claims

The scope of the claims

[I] A steroidogenic cell characterized by differentiation from a bone marrow cell by introducing a factor involved in steroid hormone synthesis into the bone marrow cell.

[2] A steroidogenic cell characterized in that the pluripotent stem cell force is differentiated by introducing a factor involved in steroid hormone synthesis into the pluripotent stem cell.

[3] In the steroidogenic cell according to claim 2,

A steroidogenic cell, wherein the pluripotent stem cell is derived from a bone marrow cell.

[4] In the steroidogenic cell according to claim 2,

The pluripotent stem cells are somatic pluripotent stem cells including mesenchymal stem cells and hematopoietic stem cells.

[5] In the steroidogenic cell according to claim 1 or 2,

A steroidogenic cell, wherein the factor involved in steroid hormone synthesis is a transcriptional regulator of steroid hormone synthase.

[6] In the steroidogenic cell according to claim 5,

A steroidogenic cell, wherein the transcriptional regulator of the steroid hormone synthase is Steroidogenic factor 1 (S F-l).

[7] In the steroidogenic cell according to claim 1 or 2,

The steroid-producing cell is characterized in that it secretes steroid hormones over a predetermined period.

[8] In the steroidogenic cell according to claim 7,

The steroidogenic cell, wherein the predetermined period is at least 2 weeks or more.

[9] In the steroidogenic cell according to claim 1 or 2,

The steroid-producing cell is a corticotrophic hormone (ACTH) responsiveness.

[10] The steroid-producing cell according to claim 9, wherein

A steroidogenic cell, wherein the ACTH reactivity is ACTH dose-dependent.

[II] In the steroidogenic cell according to claim 1 or 2, The steroidogenic cells include pregnenolone, pro gesterone, hydroxycorticosterone, 18-hydroxycorticosterone, 18-hydroxycorticosterone, 18-hydroxycorticosterone, aldosterone, aldosterone ), 丄 / α human mouth ³ r prepreg nonolone (17 — hydroxypr egnelone), 17 α hydroxyprogesterone (17 α-hydroxyprogesterone), 11 oxyconoretizole (11-deoxycortisol), cortisol (cortisol), Produces at least one steroid hormone consisting of DHEA (deh ydroepiandrosterone), androstenedione), estrone, estrone, androstenediol, testosterone, and estradiol Steroidogenic cells characterized by .

[12] In the steroidogenic cell according to claim 1 or 2,

The steroid-producing cells are those that produce gonadal steroid hormones by culturing in a retinoic acid-containing medium.

[13] In the steroidogenic cell according to claim 1 or 2,

A steroidogenic cell characterized in that the steroidogenic cell is one that produces a gonadal steroid hormone by culturing in a medium containing retinoic acid and hCG (human chorionic stimulating hormone).

[14] (a) preparing a bone marrow cell;

(b) introducing a factor involved in steroid hormone synthesis into the bone marrow cells and differentiating the bone marrow cells into steroidogenic cells;

A method for producing a steroid-producing cell, comprising:

[15] The method for producing a steroid-producing cell according to claim 14, wherein

A method for producing a steroid-producing cell, further comprising the step of culturing the bone marrow cell in a retinoic acid-containing medium, wherein the bone marrow cell is induced to differentiate into a gonadal steroid-producing cell.

[16] In the method for producing a steroid-producing cell according to claim 14,

A method for producing a steroid-producing cell, further comprising the step of culturing the bone marrow cell in a medium containing retinoic acid and hCG (human chorionic stimulating hormone), and further inducing differentiation into a gonadal steroid-producing cell.

[17] A pharmaceutical composition comprising the secreted steroid hormone secreted steroid hormone according to claim 1 or 2, and a pharmaceutically acceptable carrier.

[18] The pharmaceutical composition according to claim 17,

The pharmaceutical composition is for treating abnormal steroid hormone secretion.

[19] The pharmaceutical composition according to claim 17,

The pharmaceutical composition is for treating an autoimmune disease.

[20] The pharmaceutical composition according to claim 17,

The pharmaceutical composition, which is used as an immunosuppressant during organ transplantation.