JP2006131600A - Human cell composition for hair growth acceleration - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent or a therapeutic method which has a hemokinesis improvement action in alopecia, especially in a typical androgenic alopecia, and improves the thinning of a caput (a baldness part). <P>SOLUTION: A human cell composition is provided which contains the culture fibroblasts and the vascular growth factor secretory cells of a subject and improves the skin hemokinesis and/or accelerates the hair growth of the subject. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to cell transplantation medicine, and more particularly to a cell transplantation method for promoting hair growth on the head (head) and a human cell composition therefor.

  Alopecia is a generic name for a disease or condition associated with hair loss. There are several different types of hair loss, but the most common are androgenetic alopecia (see Non-Patent Document 1, for example), alopecia areata, drug-induced alopecia, etc. It is.

  Male pattern baldness is a typical progressive loss of excessive amounts of hair from the scalp. Clearly identifiable male pattern baldness occurs in 50% of men by age 50 and 50% of women by age 60 years. Male pattern baldness is believed to result from both a genetic predisposition and the presence of significant amounts of circulating androgens. When a male hormone (androgen) binds to an androgen receptor in hair follicle cells, a substance that causes an immune response to the hair follicle is generated, and for this reason, the hair follicle in the growing phase is switched to the resting phase.

  This male hormone is mainly dihydrotestosterone (DHT), which is a kind of active male hormone produced by the action of 5α reductase on testosterone. When there is a lot of 5α reductase at the top of the head or the sensitivity to DHT is high mainly due to genetic predisposition, DHT easily binds to the receptor and the hair follicle becomes smaller and softens due to repetition of this immune reaction. It is considered.

  Current treatments for androgenetic alopecia include vasodilator minoxidil (2,4-diamino-6-piperidino-pyrimidine-3-oxide or 6- (1-piperidinyl) -2,4 -pyrimidinediamine-3-oxide (see, for example, Patent Documents 1 and 2). Minoxidil was originally developed as an antihypertensive agent, and the mechanism of hair growth promoting action is not clear, but the affected blood vessels that have fallen into ischemia leading to hair loss are expanded by pharmacological action, resulting in alleviation of ischemia. It is thought to promote hair growth.

  However, the conventional therapeutic agents and treatment methods are not effective enough, and there are problems such as temporary effects, and further development of therapeutic agents or treatment methods is desired.

U.S. Pat.No. 4,139,619 U.S. Pat.No. 4,596,812 Sawaya, M.E.Seminars in Cutaneous Medicine and Surgery 17 (4): 276-283, 1998

  The present inventor has promoted a decrease in blood flow by alopecia, especially in typical male pattern alopecia, where the skin of the head is significantly thinned and the skin thickness (volume) is lost. The problem was found to solve these two causes at the same time. That is, an object of the present invention is to provide a therapeutic agent or a therapeutic method that has an effect of improving blood flow and improves thinning of the head (head).

  In order to solve the above problems, the present invention provides a method for promoting hair growth or hair growth by using a combination of self-cultured fibroblasts and vascular growth factor-secreting cells, and a human cell composition therefor. That is, in the first aspect, the human cell composition of the present invention improves blood flow in the subject's skin and / or promotes hair growth, and comprises the subject's cultured fibroblasts and vascular growth factor And secretory cells. The vascular growth factor-secreting cell is preferably platelet or platelet-rich plasma of the subject. Alternatively, the vascular growth factor-secreting cell may be a transformed cell that secretes human vascular growth factor. Furthermore, instead of the vascular growth factor-secreting cells, an expression vector for human vascular growth factor or a sustained-release drug for human vascular growth factor can be used.

  In one embodiment of the present invention, the cultured fibroblasts are obtained by subculturing skin fibroblasts obtained by biopsy of a subject's skin in a medium containing 0.5 to 20% by volume of autoserum. A fibroblast suspension obtained by exposing the cultured fibroblasts to proteolytic enzymes.

  In another aspect, the method for promoting hair growth according to the present invention includes a cultured fibroblast suspension of a subject, platelets of the subject, transformed cells secreting human vascular growth factor, and expression of human vascular growth factor. At least one selected from the group consisting of a vector and a sustained release agent of human vascular growth factor is administered to the subject at the same time, sequentially or separately over time.

  According to the hair growth promoting method of the present invention, it is possible to increase the skin thickness (volume) by transplanting self fibroblasts, and at the same time, effectively achieve angiogenesis by the continuous action of vascular growth factor. Can do. It is considered that the synergistic action of these two effects can promote hair growth at the hair loss site, particularly the head (head).

  The hair growth promoting method of the present invention uses the following two means in combination. The first is to use vascular growth factor-secreting cells to regenerate blood vessels in the skin to improve skin blood flow and supply nutrients to the hair follicles. It is known that platelets and the like have an important role in situations related to vascular regeneration such as wound treatment. Platelets in peripheral blood are generated by fragmenting megakaryocytes present in the bone marrow. Platelets do not have a nucleus for replication, and their lifetime is about 11 days. Platelets are involved in the hemostatic mechanism and secrete several blood clotting factors. Platelets also secrete several cytokines that are involved in wound treatment. These cytokines are a group of growth factors (PDGF, TGF-β, VEGF, FGF, PF-4 / β-TG, etc.) released from α granules among the secretory granules of platelets, and these cytokines having vascular proliferation action Induces blood vessels in the wound and accelerates treatment. In the present invention, “vascular growth factor” refers to the various growth factors secreted from platelets and various cells. Here, PDGF is a platelet derived growth factor, and has an angiogenesis, that is, an action of forming a new blood vessel from a microvessel that already exists. PDGF consists of two polypeptides (PDGF-A chain and PDGF-B chain) having very high homology. The A chain and the B chain are encoded by different genes, and the product of the B chain gene is 93% homologous to p28sis, which is a sarcoma virus oncogene (v-sis) product. The A chain and B chain genes have many structural similarities, such as a size and structure of about 24 kb, a point consisting of seven exons, and how to use the sixth exon. PDGF in which the sixth exon is translated exists in a form that interacts with heparan sulfate proteoglycans existing in the vicinity of the matrix and cells, while PDGF lacking the sixth exon is secreted extracellularly. For the A chain, two types of mRNA are produced depending on whether the sixth exon is used or the seventh exon is used. As a result, two types of proteins are generated. However, at present, there is no difference between these biological activities.

  TGF-β is transforming growth factor β, VEGF is vascular endothelial growth factor, and FGF is fibroblast growth factor. These all have angiogenic effects. Therefore, in the present invention, these cytokines are collectively referred to as “vascular growth factor”. Vascular growth factors such as VEGF and FGF act directly on receptors present in endothelial cells to cause the formation and reconstruction of blood vessels. For example, VEGF has a strong specificity for endothelial cells because only vascular endothelial cells express VEGF receptors. In contrast, the PDGF receptor is widely expressed in many cells, so it exerts an angiogenic effect by attracting other cells that secrete factors that directly act on endothelial cells, such as monocytes and fibroblasts. It may be. The nucleotide sequence and amino acid sequence of these human-derived cytokines are all known and can be easily produced using recombinant DNA technology.

For example, platelet rich plasma (PRP) can be used as a cell that secretes such a vascular growth factor. PRP is conditioned plasma rich in platelets that is about 10 times more concentrated in platelets. There have been many reports, mainly in Europe, as therapeutic agents for skin ulcers, and wound ulcer treatment effects have been obtained against the background of good blood flow improvement effects. In the present invention, “platelet-rich plasma (PRP)” is a broad term used in its general sense, and is a platelet in which a higher concentration of platelets than peripheral blood is resuspended in plasma. Typically, it contains 500,000 to 1.2 million platelets / mm ³ or more.

  Moreover, you may use the transformed cell which secretes the said vascular growth factor. In the present specification, a “transformed cell” is obtained by introducing a gene encoding one or more of the above-mentioned vascular growth factors so that it can be expressed in a host cell. Here, the host cell is preferably a human-derived cultured cell. Examples of a method for introducing a gene into cultured cells include an electroporation method, a calcium phosphate method, and a lipofection method. Alternatively, the gene expression vector can be directly introduced into a living body. The expression vector can be introduced regardless of whether the transgene is a plasmid DNA (naked plasmid DNA) or a recombinant viral vector. The administration method when using plasmid DNA is not particularly limited. A method in which DNA is dissolved in physiological saline and injected as it is, a method using a carrier such as a liposome, various polymers, and a gene gun that sprays DNA coated on fine gold particles at a high pressure to reach the skin, Various introduction methods such as a powder jet and similar high-pressure injection gene introduction device, electroporation, and sonoporation can be applied.

  The second means is treatment for thinning of the skin at the hair loss site (decrease of collagen fibers in the scalp). For this purpose, biopsy material collected from a subject is cultured in a culture medium so as to be substantially free of immunogenic protein, and the proliferated autologous fibroblast suspension is injected subcutaneously or into subcutaneous tissue. How to do is known. The self-fibroblast used here may be any cultured cell as long as it can improve thinning of the skin. Specifically, intraoral fibroblasts, mucosal fibroblasts, and cultured chondrocytes may be used.

In a preferred embodiment, suspensions obtained by culturing and growing autologous dermal fibroblasts can be used. Skin fibroblast culture starts with, for example, a biopsy material of about 3 mm × 5 mm of skin collected from a subject. Before starting the culture, the biopsy is repeatedly washed with antibiotics and antifungal agents. The epidermis and subcutaneous adipocyte-containing tissue are then removed, the resulting culture is substantially free of non-fibroblasts, and the dermis sample is minced with a scalpel or scissors. Place specimen pieces one at a time on the dry surface of the tissue culture flask with forceps and allow them to adhere for 5-10 minutes, then slowly add a small amount of medium with care not to peel off the attached tissue fragments. After 24 hours of incubation, add additional media to the flask. When culturing is initiated using a 75 cm ² tissue culture flask, the initial volume of medium is 3-4 ml. Establishment of a cell line from a biopsy specimen usually takes 2 to 3 weeks, and in order to proliferate, the cells can be detached from the initial culture container at an appropriate time.

  It is desirable that the tissue piece adheres to the bottom of the culture vessel at the initial stage of culture. Non-adherent tissue pieces should be replanted into a new container. Stimulating and growing fibroblasts by briefly exposing tissue cultures to serine proteases produced by genetic engineering techniques and having trypsin-like activity that does not contain animal-derived components, using techniques known to those skilled in the art Can do. Since the exposure to serine protease is very short, fibroblasts attached to the walls of the culture vessel are not released. Immediately after the culture is established and approaching confluence, a sample of fibroblasts can be removed for cryopreservation. Since the number of passages in a normal human fibroblast cell culture is limited, it is preferable to cryopreserve fibroblasts with fewer passages than fibroblasts with more passages.

  Fibroblasts can be frozen in any freezing medium suitable for storing them. Good effects are obtained when a medium consisting of 70% by volume growth medium, 20% by volume human serum (from the same individual as the cells to be stored) and 10% by volume dimethyl sulfoxide (DMSO) is used. When secondary culture is initiated using thawed cells, a suspension for use in the same subject can be obtained without the inconvenience of obtaining a second specimen.

Any tissue culture method suitable for seeding skin fibroblasts from a biopsy specimen can be used for cell growth to practice the present invention. The medium is any medium suitable for growing primary fibroblast cultures. In many instances, 0.5% to 20% (v / v) serum is added to the medium to promote fibroblast proliferation. High concentrations of serum promote faster proliferation of fibroblasts. In a preferred embodiment, the serum is human serum (from the same individual as the cells to be stored), which is added to a final concentration of 10% of the medium. The medium is, for example, high glucose DMEM (“complete medium” supplemented with 2 mM glutamine, 110 mg / L sodium pyruvate, 10% (v / v) human autoserum (from the same individual as the cells to be stored) and antibiotics. ]). Cells are passaged into new flasks by serine protease treatment. Add 1/3 aliquots to each flask for growth. A three-bottom T-150 flask (total culture area 450 cm < ² >) is suitable for the practice of the present invention. Three-bottom T-150 (total culture area 450 cm ² ) has the ability to inoculate about 6 × 10 ⁶ cells and produce about 1.8 × 10 ⁷ cells.

At the end of the incubation, the cells are detached from the tissue culture flask with serine protease and the pelleted by centrifugation is resuspended in saline. Is a three-base T-0.99 flasks, grown to full capacity, about 10 ⁷ cells are produced, which is sufficient to produce a suspension of about 0.3 ml. The human cell composition of the present invention can be prepared by mixing the fibroblast suspension and vascular growth factor-secreting cells in an appropriate ratio. A preferable mixing ratio is about 1: 0.1 to 100, more preferably about 1: 1 to 10 in terms of the number of cells. That is, about 10 ⁷ to 10 ⁹ cells / ml of vascular growth factor-secreting cells can be used for a suspension of about 10 ⁷ cells / ml.

The human cell composition of the present invention can be injected into a subject's hair loss site, particularly the head, by a conventional surgical technique. Specifically, if desired, a local anesthetic-containing tape (for example, penless (trade name)) is attached to the desired injection site and left for about 15 minutes. If you are in a hurry, this process can be omitted. Next, the cell suspension prepared by the above-described method is injected subcutaneously, for example, with a syringe equipped with a 27 gauge injection needle. As a guide for the amount to be injected, 1 ml (3 × 10 ⁷ cells) is an amount that can be injected into about 6 to 10 cm ² of the skin surface area. Specifically, about 0.3 ml to 0.6 ml is used for the entire unilateral lower eyelid. The human cell composition may contain other additives as long as it does not interfere with cell transplantation medicine. For example, a local anesthetic is added to suppress aggregation of fibroblasts in the suspension. Can do. The cell aggregation inhibitory action by the local anesthetic has been discovered by the present inventor and has been filed as Japanese Patent Application No. 2004-229466, the contents of which are incorporated herein by reference. For example, by adding about 0.1 to 5% of an amide type local anesthetic such as lidocaine, mepivacaine, dibucaine, bupivacaine, and procaine, or an ester type local anesthetic such as cocaine, procaine, chlorocaine, and tetracaine. It has the effect of suppressing the aggregation of the cells in the cell suspension and facilitating the injection into the subject, and at the same time reducing the pain of the subject. The human cell composition of the present invention preferably contains only self-cultured cells and self-derived biological materials such as platelets. However, other foreign proteins, for example, for gelation depending on the purpose. Of course, additives such as collagen and fibrin having reduced immunogenicity may be used.

  The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention, and functionally equivalent methods and ingredients are included within the scope of the invention.

Human dermis-derived fibroblasts were subcultured to produce the following two types of cultured skin models. Dermal fibroblasts (3 × 10 ⁷ cells) suspended in a 10-fold concentration MEM medium for 8 volumes of acid-soluble collagen type I (Cellmatrix- (registered trademark) IA; Nitta Gelatin Co., Ltd.) derived from porcine tendon / Ml) is added and mixed well, and then 1 volume of collagen gel reconstitution buffer solution (0.05N NaOH solution containing 2.2% NaHCO ₃ and 200 mM HEPES) is added to adjust the pH to about 7.4. And heated at 37 ° C. for about 30 minutes to prepare a collagen gel. The gelled collagen concentration was adjusted to 3 mg / ml at the final concentration. This dermal fibroblast collagen gel (collagen dermis substitute) is placed in a culture vessel and contains PRP (a number of platelets similar to dermal fibroblasts) (minimum essential medium containing 20% fetal bovine serum) α (Gibco Oriental Co., Ltd.) added and cultured at 37 ° C. in the presence of 5% carbon dioxide (P group), and collagen dermis substitute cultured in a culture solution containing no PRP (Group C) Was made.

  About each of P group and C group, the culture supernatant of 48 hours after culture | cultivation was collect | recovered, and the amount of VEGF was quantified by ELISA. That is, each culture supernatant was added to a 96-well plate coated with an anti-human VEGF monoclonal antibody (Quantikine (registered trademark), R & D Systems Inc.). After washing the wells, peroxidase-labeled anti-human VEGF polyclonal antibody was added, and after further washing the wells, the substrate solution was added and the plate was incubated for 10 minutes at room temperature, and the absorbance at 620 nm was measured. As a result, the VEGF concentration in the culture supernatant was 182.2 ± 14.5 pg / ml (n = 4) in the P group and 17.8 ± 3.7 pg / ml (n = 4) in the C group. . Although a part of VEGF detected at a high concentration in the P group seems to be derived from PRP, the following experiment was conducted to confirm whether VEGF was also expressed in dermal fibroblasts.

  The collagen dermis substitute was collected from the culture vessel, the culture solution was removed, and the collagen dermis substitute substantially free of platelets was homogenized in ISOGEN (Nippon Gene Co., Ltd.) to extract RNA. RT / PCR was performed using VEGF primers synthesized by Life Technologies, and the amount of VEGF mRNA was quantified. The base sequences of the primers used for the amplification reaction are sense strand: 5′-CCATGAACTTTCTGCTGTCTT-3 ′ (SEQ ID NO: 1) and antisense strand: 5′-ATCGCATCAGGGGCACACAG-3 ′ (SEQ ID NO: 2). The denaturation step (94 ° C., 1 minute); annealing step (55 ° C., 1 minute); extension step (72 ° C., 2 minutes) was repeated 30 times with 0.1 μg / μl template RNA in a thermocycler. After carrying out the amplification reaction, 1.5% agarose gel electrophoresis was performed using each reaction solution and stained with ethidium bromide. Each gel was photographed under UV irradiation at the same exposure and time. As a result, as shown in FIG. 2, it was found that VEGF mRNA was specifically expressed in the P group. The band density was measured using NIH image software, and the relative concentration ratio with the amount of GAPDH mRNA constitutively expressed in dermal fibroblasts was calculated. It was ± 0.030 and 0.332 ± 0.143 in group C. From these results, it was found that culturing dermal fibroblasts in the presence of platelets induces VEGF expression in dermal fibroblasts and has the ability to secrete VEGF.

  Two types of cultured skin models were prepared in the same manner as in Example 1, and the cultured skin (collagen dermis substitute) after 48 hours of culture was transplanted into immunodeficient mice (BALB / cA, nu) to induce induced angiogenesis and dermis-like A histological study was conducted on the maintenance of the structure. Frozen sections were prepared from mouse skin transplanted with each of P group and C group, and immunostaining for vascular endothelial cells and hematoxylin and eosin staining were performed as a comparison. The section is about 10 to 50 μm. The results are shown in FIG. In the group P (PRP addition group), there was no skin atrophy and many vascular cell inductions were observed. The reddish purple point indicated by the arrow in FIG. 3 is the vascular endothelium. On the other hand, in group C, skin atrophy was observed, and the induction of vascular cells was small. From the above results, it is considered that skin angiogenesis is clearly observed in the immunodeficient mice transplanted with the cell composition of the present invention and the skin blood flow is improved.

It is the figure which represented typically two types of cultured skin models (collagen dermis substitute) produced using collagen gel. It is the result of detecting the amount of VEGF mRNA expressed in dermal fibroblasts in a cultured skin model by RT / PCR. It is a microscope picture of the skin of the immunodeficient mouse | mouth which transplanted cultured skin.

Explanation of symbols

10 Culture Container 20 Collagen Gel 30 Dermal Fibroblast 40 Platelet (PRP)

Claims (6)

  A human cell composition for improving blood flow in the subject's skin and / or promoting hair growth, comprising cultured fibroblasts of the subject and vascular growth factor-secreting cells.   The human cell composition according to claim 1, wherein the vascular growth factor-secreting cell is platelet or platelet-rich plasma of the subject.   The human cell composition according to claim 1, wherein the vascular growth factor-secreting cell is a transformed cell that secretes human vascular growth factor.   2. The human cell composition according to claim 1, comprising a human vascular growth factor expression vector or a human vascular growth factor sustained-release agent in place of the vascular growth factor-secreting cells.   The cultured fibroblasts are subcultured in a medium containing 0.5-20% by volume autologous serum of skin fibroblasts obtained by biopsy of the subject's skin, and the cultured fibroblasts are protein. The human cell composition according to any one of claims 1 to 4, which is a fibroblast suspension obtained by exposure to a degrading enzyme. A subject's cultured fibroblast suspension,
At least one selected from the group consisting of the subject's platelets, transformed cells secreting human vascular growth factor, human vascular growth factor expression vector and sustained release drug of human vascular growth factor simultaneously and sequentially Or a method of promoting hair growth, characterized by being administered separately to the subject after a period of time.