TW201504435A - Quality control method for hair-follicle forming composition - Google Patents

Quality control method for hair-follicle forming composition Download PDF

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TW201504435A
TW201504435A TW103120417A TW103120417A TW201504435A TW 201504435 A TW201504435 A TW 201504435A TW 103120417 A TW103120417 A TW 103120417A TW 103120417 A TW103120417 A TW 103120417A TW 201504435 A TW201504435 A TW 201504435A
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cells
hair
cell
composition
hair follicle
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Yuzo Yoshida
Tsutomu Soma
Haruyo Yamanishi
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Shiseido Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Abstract

Provided are a composition exhibiting hair-follicle induction ability, and a quality control method for said composition. A composition exhibiting high hair-follicle induction ability can be acquired by sorting CD36-expressing DS (DS: dermal sheath) cells from amongst DS cells derived from dermal sheath. In addition, the quality of the hair-follicle induction ability of the composition can be controlled by measuring the ratio of CD36-expressing DS cells within the hair-follicle forming composition.

Description

毛囊形成用組合物之品質管理方法 Quality management method for hair follicle forming composition

本發明係關於一種對於包含源自真皮毛根鞘(「DS」：dermal seath)之CD36陽性細胞之毛囊形成用組合物之毛囊誘導能力管理品質之方法。 The present invention relates to a method for managing hair follicle inducing ability of a composition for hair follicle formation comprising CD36 positive cells derived from dermal root sheath ("DS": dermal seath).

毛髮被認為在審美外觀上極為重要。因此，因先天或後天之因素所引起之脫髮對於多數人而言為深刻之苦惱。尤其於被稱為高齡化社會、壓力社會之現代社會，頭部毛髮因各種後天之原因而面臨脫髮危機之機會日益增多。為了應對此種情況而提供發揮包含促進毛髮生長或毛髮之粗髮化之生髮效果之更優異之生髮劑，作出了各種嘗試。 Hair is considered to be extremely important in aesthetic appearance. Therefore, hair loss caused by congenital or acquired factors is a deep distress for most people. Especially in modern societies known as aging society and stress society, the opportunities for head hair to face a hair loss crisis due to various acquired factors are increasing. In order to cope with such a situation, various attempts have been made to provide a hair growth agent which exhibits a hair growth effect which promotes hair growth or hair growth.

毛囊係以成熟之活體於幾乎一生中反覆自我再生之例外之器官。期待闡明該自我再生之結構與需求較高之臨床應用緊密相聯，例如藉由組織或細胞移植之脫髮治療、含有毛囊或皮脂腺之近似自然且功能性亦優異之皮膚片之構築等。近年來，對幹細胞研究之關心高漲，並且毛囊上皮幹細胞(上皮細胞)之研究迅速進展，又，對於作為毛囊特殊之間葉系細胞之毛乳頭(「DP」：dermal papilla)細胞，亦已逐漸判明其性質。判明毛乳頭細胞係發揮向毛囊上皮幹細胞傳送活化訊號以使毛囊自我再生之所謂指揮塔之作用，且於毛囊再構成評價系統中與毛囊上皮幹細胞共同不可欠缺之細胞(Kishimoto et al.,Proc.Natl.Acad.Sci.USA(1999),Vol.96,pp.7336-7341：非專利文獻1)。 Hair follicles are mature organs that replicate the exceptions of self-regeneration in almost a lifetime. It is expected that the structure of self-regeneration will be closely related to the clinical application with high demand, such as hair loss treatment by tissue or cell transplantation, construction of skin pieces containing hair follicles or sebaceous glands which are similar in nature and excellent in function. In recent years, concerns about stem cell research have increased, and the study of hair follicle epithelial stem cells (epithelial cells) has progressed rapidly. In addition, the dermal papilla ("DP": dermal papilla) cells, which are special between the hair cells of the hair follicles, have gradually evolved. Determine its nature. It was determined that the dermal papilla cell line functions as a so-called control tower for transmitting activation signals to hair follicle epithelial stem cells to self-regenerate hair follicles, and is indispensable for hair follicle epithelial stem cells in the hair follicle reconstitution evaluation system (Kishimoto et al., Proc. USA (1999), Vol. 96, pp. 7336-7341: Non-Patent Document 1).

存在於毛球內之DP、與包圍毛根之周圍之DS均與構成毛囊之大 部分之上皮系細胞不同，由源自間葉系之細胞群構成。對於DS，近年來多數報告有提示對於毛囊形成之重要性之見解。亦報告有於移植自大鼠鬍鬚之毛球部將毛乳頭切斷之毛囊之實驗中，藉由自DS使DP再生，並將切斷有下二分之一之毛囊之DS移植至小鼠，而誘導毛囊再生(Jahoda CA,et al.,Development.1992 Apr：114(4)：887-97；非專利文獻2)。又，亦報告有藉由將DS移植至人類，可誘導毛囊之再構築(Horne KA and Jahoda CA.Development.1992 Nov：116(3)：563-71；非專利文獻3)。進而，Tobin,Paus等人之小組報告如下：於小鼠毛髮週期內，發生DS與DP間之細胞之移動，於毛髮生長期，開始增殖之DP細胞率先開始結締組織鞘(DS)細胞之增殖(Tobin DJ et al.,J.Invest.Dermatol.,120：895-904,2003；非專利文獻4)。 The DP present in the hair bulb and the DS surrounding the hair root are both large and constitute the hair follicle Some of the epithelial cells are different and consist of a cell population derived from the mesenchymal lineage. For DS, most reports in recent years have hinted at the importance of hair follicle formation. It was also reported that in the experiment of transplanting hair follicles from the hair bulbs of the rat beard to the hair follicles, the DP was regenerated from DS, and the DS that cut the lower one of the hair follicles was transplanted to the mice. Inducing hair follicle regeneration (Jahoda CA, et al., Development. 1992 Apr: 114(4): 887-97; Non-Patent Document 2). Further, it has been reported that reconstitution of hair follicles can be induced by transplanting DS to humans (Horne KA and Jahoda CA. Development. 1992 Nov: 116 (3): 563-71; Non-Patent Document 3). Furthermore, the group report by Tobin, Paus et al. is as follows: During the mouse hair cycle, the movement of cells between DS and DP occurs, and in the long-term development of hair, the DP cells that begin to proliferate are the first to initiate the proliferation of connective tissue sheath (DS) cells. (Tobin DJ et al., J. Invest. Dermatol., 120: 895-904, 2003; Non-Patent Document 4).

如此，DS對於毛囊形成發揮重要之作用之可能性較高，但對於其作用機理，不明確之方面為大多數。其中，發現於DS中之一部分細胞中，血管相關因子之表現量較高。而且，已知CD36表現性之DS細胞顯示出經由HGF(Hepatocyte Growth Factor，肝細胞生長因子)之表現而促進血管內皮細胞之增殖，藉此有助於分化為毛乳頭或毛乳頭之活化之可能性，係用以使包含CD36表現性之結締組織鞘細胞之毛囊再生之組合物(專利文獻1)。 Thus, DS is highly likely to play an important role in hair follicle formation, but the most unclear aspect of its mechanism of action is. Among them, it was found that in a part of cells in DS, the expression amount of vascular-related factors was high. Moreover, it is known that CD36-expressing DS cells exhibit the proliferation of vascular endothelial cells via the expression of Hepatocyte Growth Factor (HGF), thereby facilitating the differentiation into the activation of dermal papilla or dermal papilla. The composition is a composition for regenerating hair follicles containing CD36-expressing connective tissue sheath cells (Patent Document 1).

[先前技術文獻] [Previous Technical Literature] [專利文獻] [Patent Literature]

[專利文獻1]日本專利特開2010-275256號公報 [Patent Document 1] Japanese Patent Laid-Open Publication No. 2010-275256

[非專利文獻] [Non-patent literature]

[非專利文獻1]Kishimoto et al., Proc. Natl. Acai. USA (1999), Vol. 96, pp. 7336-7341 [Non-Patent Document 1] Kishimoto et al., Proc. Natl. Acai. USA (1999), Vol. 96, pp. 7336-7341

[非專利文獻2]Jahoda CA et al., Development. 1992 Apr; 114(4): 887-97. [Non-Patent Document 2] Jahoda CA et al., Development. 1992 Apr; 114(4): 887-97.

[非專利文獻3]Horne KA and Jahoda CA. Development. 1992 Nov; 116(3): 563-71. [Non-Patent Document 3] Horne KA and Jahoda CA. Development. 1992 Nov; 116(3): 563-71.

[非專利文獻4]Tobin DJ et al., J. Invest. Dermatol., 120: 895-904, 2003 [Non-Patent Document 4] Tobin DJ et al., J. Invest. Dermatol., 120: 895-904, 2003

[非專利文獻5]J. Linder et al., Federation of American Societies for Experimental Biology, 14(2), 319(2000) [Non-Patent Document 5] J. Linder et al., Federation of American Societies for Experimental Biology, 14(2), 319 (2000)

[非專利文獻6]Machiko Iida, et al., Dev. Growth Differ., 49, 185-195 (2007) [Non-Patent Document 6] Machiko Iida, et al., Dev. Growth Differ., 49, 185-195 (2007)

[非專利文獻7]Palmqvist L, Glover CH, Hsu L, Lu M, Bossen B et al. (2005) Stem cells (Dayton, Ohio) 23(5): 663-680 [Non-Patent Document 7] Palmqvist L, Glover CH, Hsu L, Lu M, Bossen B et al. (2005) Stem cells (Dayton, Ohio) 23(5): 663-680

[非專利文獻8]Handjiski BK et al., The British journal of dermatology 131(3): 303-310 [Non-Patent Document 8] Handjiski BK et al., The British journal of dermatology 131(3): 303-310

[非專利文獻9]Cho H, Kozasa T, Bondjers C, Betsholtz C, Kehrl JH (2003) Faseb J 17(3): 440-442 [Non-Patent Document 9] Cho H, Kozasa T, Bondjers C, Betsholtz C, Kehrl JH (2003) Faseb J 17(3): 440-442

[非專利文獻10]Paquet-Fifield S, et al. (2009) The Journal of clinical investigation 119(9): 2795-2806 [Non-Patent Document 10] Paquet-Fifield S, et al. (2009) The Journal of clinical investigation 119(9): 2795-2806

[非專利文獻11]Winkler EA, Bell RD, Zlokovic BV Molecular neurodegeneration 5: 32 [Non-Patent Document 11] Winkler EA, Bell RD, Zlokovic BV Molecular neurodegeneration 5: 32

[非專利文獻12]Jones EA, Kinsey SE, English A, Jones RA, Straszynski L et al. (2002), 46(12): 3349-3360 [Non-Patent Document 12] Jones EA, Kinsey SE, English A, Jones RA, Straszynski L et al. (2002), 46(12): 3349-3360

[非專利文獻13]Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C et al. (2002), 30(7): 783-791 [Non-Patent Document 13] Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C et al. (2002), 30(7): 783-791

[非專利文獻14]Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD et al. Nature 466(7308): 829-834 [Non-Patent Document 14] Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD et al. Nature 466 (7308): 829-834

[非專利文獻15]Zheng Y, Du X, Wang W, Boucher M, Parimoo S, et al. J Invest Dermatol (2005) 124: 867-876. [Non-Patent Document 15] Zheng Y, Du X, Wang W, Boucher M, Parimoo S, et al. J Invest Dermatol (2005) 124: 867-876.

本發明之課題之目的在於提高包含源自真皮毛根鞘(DS)之CD36表現性之細胞之組合物之品質。 An object of the present invention is to improve the quality of a composition comprising cells derived from CD36 expressing dermal root sheath (DS).

真皮毛根鞘之細胞中之CD36表現性之細胞顯示出經由HGF之表現而促進血管內皮細胞之增殖，藉此有助於分化為毛乳頭或毛乳頭之活化之可能性。然而，至於CD36表現性DS細胞、與毛囊誘導能力之關係尚未被明確。 The CD36-expressing cells in the cells of the dermal root sheath show the promotion of proliferation of vascular endothelial cells via the expression of HGF, thereby contributing to the possibility of differentiation into the activation of the dermal papilla or dermal papilla. However, the relationship between CD36-expressing DS cells and hair follicle inducing ability has not been clarified.

本發明者等人發現：DS細胞為異質之細胞群體，故而藉由進行繼代培養，而不具有毛囊誘導能力之細胞亦增殖，進而隨著進行繼代培養可產生喪失毛囊誘導能力之細胞。因此，著眼於如下方面：有藉由確認用以形成包含CD36表現性之DS細胞之毛囊之組合物具有適當之毛囊誘導能力，而進行組合物之品質管理的必需性。作為品質管理之方法，亦有可能於三維培養皮膚模型中直接確認毛囊誘導能力，但就時間、工夫、及/或成本之方面而言欠佳。因此，本發明者等人對於用以形成毛囊之組合物與毛囊誘導能力之關係進行努力研究，結果發現：於該組合物中所含之CD36陽性細胞中，被認為與毛囊誘導能力有關之鹼性磷酸酶(ALP)活性較高(圖3、圖4及圖5)。已知ALP活性可用作生長期之毛乳頭之標記，但另一方面，於真皮毛根鞘中，亦僅於毛髮週期之生長期之初期可見活性(非專利文獻6)。可綜合該等見解而成立如下假說：DS細胞中，尤其是CD36陽性細胞可分化為毛乳頭或使毛乳頭活化。本發明者等人進而對於CD36陽性DS細胞之基因表現進行研究，結果驚奇地發現：CD36陽性DS細胞與CD36陰性DS細胞相比，高表現以RGS5為代表之複數種間葉系幹細胞相關因子(圖6及7)。藉此，可知CD36陽性細胞亦具有作為間葉系幹細胞之能力即 自我增殖性與多能性，可理解具有該等能力之細胞發揮毛囊之再生能力即毛囊誘導能力。進而，本發明者等人將包含CD36陽性DS細胞之移植物、與包含CD36陰性DS細胞之移植物分別移植至免疫缺陷小鼠，並對毛囊誘導能力進行調查，結果於CD36陽性DS細胞群中，顯示明顯高之毛囊誘導能力(圖8及9)。藉此，完成對於藉由將CD36陽性細胞之比率設為指標之包含源自真皮毛根鞘之CD36陽性細胞之毛囊形成用組合物之毛囊誘導能力管理品質的方法。 The present inventors have found that DS cells are heterogeneous cell populations, and thus cells which are not capable of hair follicle inducing ability are also proliferated by subculture, and cells which have lost hair follicle inducing ability can be produced by subculture. Therefore, attention has been paid to the necessity of performing quality management of the composition by confirming that the composition for forming a hair follicle containing DS36 expressing DS cells has an appropriate hair follicle inducing ability. As a method of quality management, it is also possible to directly confirm hair follicle inducing ability in a three-dimensional culture skin model, but it is not preferable in terms of time, labor, and/or cost. Therefore, the present inventors conducted an effort to study the relationship between the composition for forming a hair follicle and the hair follicle inducing ability, and as a result, found that among the CD36-positive cells contained in the composition, it is considered to be related to the hair follicle inducing ability. The activity of phosphatase (ALP) is high (Fig. 3, Fig. 4 and Fig. 5). It is known that ALP activity can be used as a marker for the dermal papilla in the growth phase, but on the other hand, in the dermal root sheath, activity is also observed only in the early stage of the hair cycle (Non-Patent Document 6). The findings can be combined to establish the hypothesis that DS cells, especially CD36-positive cells, can differentiate into dermal papillae or activate the dermal papilla. The present inventors further studied the gene expression of CD36-positive DS cells, and surprisingly found that CD36-positive DS cells have higher expression of multiple mesenchymal stem cell-associated factors represented by RGS5 than CD36-negative DS cells. Figures 6 and 7). Therefore, it can be seen that CD36-positive cells also have the ability to function as mesenchymal stem cells. Self-proliferation and pluripotency, it is understood that cells having such ability exert the ability to regenerate hair follicles, that is, hair follicle-inducing ability. Further, the inventors of the present invention transplanted a graft containing CD36-positive DS cells and a graft containing CD36-negative DS cells to immunodeficient mice, and investigated hair follicle inducing ability, and the results were in a CD36-positive DS cell population. , showing a significantly higher hair follicle inducing ability (Figures 8 and 9). Thereby, a method for managing the hair follicle inducing ability of the hair follicle-forming composition containing CD36-positive cells derived from the dermal root sheath by setting the ratio of the CD36-positive cells to the index is completed.

因此，本發明案包含以下發明： Accordingly, the present invention encompasses the following inventions:

[1]一種品質管理方法，其係管理包含源自真皮毛根鞘之CD36陽性細胞之毛囊形成用組合物之毛囊誘導能力之品質的方法，且包括：取得組合物中之一部分或全部之細胞群；及於以DS細胞中之CD36之表現作為指標且CD36表現高於特定值之情形時，決定該組合物之毛囊誘導能力。 [1] A method for quality management, which is a method for managing the quality of hair follicle inducing ability of a composition for hair follicle formation derived from CD36-positive cells derived from a dermal root sheath, and comprising: obtaining a part or all of a cell population in the composition. And when the expression of CD36 in DS cells is used as an index and the CD36 performance is higher than a specific value, the hair follicle inducing ability of the composition is determined.

[2]如項目[1]中記載之品質管理方法，其中上述指標為DS細胞中之CD36陽性DS細胞之比率，且於該比率為特定比率以上之情形時，決定具有毛囊誘導能力。 [2] The quality management method according to item [1], wherein the indicator is a ratio of CD36-positive DS cells in DS cells, and when the ratio is a specific ratio or more, it is determined to have hair follicle inducing ability.

[3]如項目[2]中記載之品質管理方法，其包括：於DS細胞中之CD36陽性DS細胞之比率為特定比率以下之情形時，對上述組合物實施對CD36陽性DS細胞進行篩選之細胞分選，而取得特定比率以上之CD36陽性DS細胞。 [3] The quality management method according to item [2], which comprises: screening the CD36-positive DS cells for the above composition when the ratio of CD36-positive DS cells in the DS cells is below a specific ratio; The cells were sorted to obtain CD36-positive DS cells above a certain ratio.

[4]如項目1至3中任一項記載之品質管理方法，其包括：進而取得組合物中之一部分之細胞群；針對該細胞群測定鹼性磷酸酶活性；及於以鹼性磷酸酶活性作為指標且鹼性磷酸酶活性高於特定值之情形時，決定該組合物具有毛囊誘導能力。 [4] The quality management method according to any one of items 1 to 3, comprising: further obtaining a cell population of a part of the composition; measuring alkaline phosphatase activity against the cell population; and using alkaline phosphatase When the activity is used as an indicator and the alkaline phosphatase activity is higher than a specific value, it is decided that the composition has hair follicle inducing ability.

[5]如項目[1]至[4]中任一項記載之品質管理方法，其包括：於進而以RGS5表現性DS細胞之比率作為指標且該比率高於特定值之情形時，決定該組合物具有較高之毛囊誘導能力。 [5] The quality management method according to any one of the items [1] to [4], wherein, when the ratio of the RGS5 expressive DS cells is further used as an index and the ratio is higher than a specific value, the determination is made The composition has a higher hair follicle inducing ability.

[6]如項目[1]至[5]中任一項記載之品質管理方法，其中上述源自真皮毛根鞘之細胞為源自真皮毛根鞘杯之細胞。 [6] The quality management method according to any one of [1] to [5] wherein the cells derived from the dermal root sheath are cells derived from a dermal root sheath cup.

[7]一種組合物，其係包含源自真皮毛根鞘(DS)之CD36陽性細胞之毛囊形成用組合物，且DS細胞中之CD36陽性DS細胞之比率為特定值以上。 [7] A composition comprising a composition for forming a hair follicle derived from CD36-positive cells of a dermal root sheath (DS), wherein a ratio of CD36-positive DS cells in DS cells is a specific value or more.

[8]如項目[6]中記載之組合物，其中上述源自真皮毛根鞘之細胞為源自真皮毛根鞘杯(DSC)之細胞。 [8] The composition according to [6], wherein the cell derived from the dermal root sheath is a cell derived from a dermal root sheath cup (DSC).

藉由使用管理本發明之包含源自真皮毛根鞘之CD36陽性細胞之毛囊形成用組合物之毛囊誘導能力之品質的方法，而使移植前之組合物之品質之管理變得容易。 By managing the quality of the hair follicle inducing ability of the composition for hair follicle formation comprising the CD36-positive cells derived from the dermal root sheath of the present invention, the quality of the composition before transplantation can be easily managed.

圖1係表示毛囊組織之結構的模式圖。 Fig. 1 is a schematic view showing the structure of hair follicle tissue.

圖2係使用抗CD36抗體之毛囊的螢光染色照片。 Figure 2 is a fluorescent staining photograph of hair follicles using anti-CD36 antibodies.

圖3係表示CD36陽性DS細胞與CD36陰性DS細胞中之鹼性磷酸酶活性之差異的照片。 Figure 3 is a photograph showing the difference in alkaline phosphatase activity between CD36-positive DS cells and CD36-negative DS cells.

圖4係將CD36陽性DS細胞與CD36陰性DS細胞中之鹼性磷酸酶活性進行數值化而表示的表。 Fig. 4 is a table showing the alkaline phosphatase activity in CD36-positive DS cells and CD36-negative DS cells.

圖5係表示源自CD36陽性之DSC之細胞、與源自CD36陰性之DSC之細胞中之鹼性磷酸酶活性之差異的照片。 Fig. 5 is a photograph showing the difference in alkaline phosphatase activity between cells derived from CD36-positive DSC and cells derived from CD36-negative DSC.

圖6係於CD36陽性DS細胞與CD36陰性DS細胞中，將CD36與RGS5免疫染色而表示的照片。 Figure 6 is a photograph showing the immunostaining of CD36 and RGS5 in CD36-positive DS cells and CD36-negative DS cells.

圖7係表示CD36陽性DS細胞、與CD36陰性DS細胞中之間葉系幹 細胞表現性基因之差的表。 Figure 7 shows the relationship between CD36-positive DS cells and CD36-negative DS cells. A table showing the difference in cell expression genes.

圖8係表示將使人類DS細胞(CD36陰性或陽性)、小鼠真皮細胞、及小鼠表皮細胞混合而成之移植物移植至免疫缺陷小鼠之皮膚，移植後之毛囊再生能力的照片。 Fig. 8 is a photograph showing the ability of a graft obtained by mixing human DS cells (CD36 negative or positive), mouse dermal cells, and mouse epidermal cells to the skin of an immunodeficient mouse, and hair follicle regeneration ability after transplantation.

圖9係表示於使人類DS細胞(CD36陰性或陽性)、小鼠真皮細胞、及小鼠表皮細胞混合而成之移植物中，使小鼠真皮細胞之細胞數發生變化，並移植至免疫缺陷小鼠之皮膚，移植後之毛囊再生能力的照片。 Figure 9 is a diagram showing changes in the number of cells of mouse dermal cells in a graft obtained by mixing human DS cells (CD36 negative or positive), mouse dermal cells, and mouse epidermal cells, and transplanted to immunodeficiency Photograph of mouse skin, hair follicle regeneration ability after transplantation.

本發明提供一種對於以DS細胞中之CD36表現為指標且包含源自真皮毛根鞘之CD36陽性細胞之毛囊形成用組合物管理毛囊誘導能力之品質的方法。該方法係以下之：可藉由取得組合物中之一部分或全部之細胞群，於以DS細胞中之CD36之表現作為指標且CD36表現高於特定值之情形時，決定該組合物之毛囊誘導能力，而對於毛囊誘導能力管理品質。 The present invention provides a method for managing the quality of hair follicle inducing ability of a composition for hair follicle formation comprising CD36-positive cells derived from dermal root sheath as an indicator of CD36 expression in DS cells. The method is as follows: the hair follicle induction of the composition can be determined by obtaining a part or all of the cell population in the composition, when the expression of CD36 in the DS cell is used as an index and the CD36 expression is higher than a specific value. Ability to manage quality for hair follicle-inducing ability.

所謂本發明中所使用之源自真皮毛根鞘之CD36陽性細胞，係指源自真皮毛根鞘之細胞中之於細胞表面表現CD36之細胞，係指可藉由抗CD36抗體進行識別之細胞。另一方面，所謂CD36表現性細胞，係指表現CD36基因之細胞，只要經轉印之CD36之mRNA(messenger Ribonucleic Acid，信息核糖核酸)可藉由PCR(Polymerase Chain Reaction，聚合酶鏈反應)等而檢測出即可，CD36陽性細胞與CD36表現性細胞雖於嚴密之意義上不同，但亦有以相同之意義而使用之情形。 The CD36-positive cells derived from the dermal root sheath used in the present invention refer to cells which express CD36 on the cell surface in cells derived from the dermal root sheath, and which are cells which can be recognized by an anti-CD36 antibody. On the other hand, the CD36 expression cell refers to a cell which expresses the CD36 gene, and the transferable CD36 mRNA (messager ribonucleic acid) can be PCR (Polymerase Chain Reaction). However, it can be detected that CD36-positive cells and CD36-expressing cells are different in the sense of strictness, but they are also used in the same sense.

CD36抗原亦稱為血小板反應蛋白(thrombospondin)受體。CD36係於脊椎動物之多數細胞型之表面可見之膜內在性蛋白質，亦作為 FAT(脂肪)、SCARB3、GP88、糖蛋白質IV(gpIV)或糖蛋白質IIIb(gpIIIb)所眾所周知。CD36係細胞表面蛋白質之B類清道夫(scavenger)受體家族之成員。CD36係除血小板反應蛋白以外，與膠原蛋白(collagen)、熱帶瘧原蟲所寄生之紅血球、氧化低密度脂蛋白質、天然脂蛋白質、氧化磷脂質、長鏈脂肪酸等多數之配位體結合。於使用基因改造嚙齒動物之最近之研究中，於脂肪酸或糖代謝、心臟病、味覺、及腸內之食物性脂肪輸送中，可確認CD36之明確之作用。CD36可參與葡萄糖失耐、粥狀動脈硬化、動脈高血壓病、糖尿病、心肌症、及阿爾茨海默氏病。 The CD36 antigen is also known as the thrombospondin receptor. CD36 is a membrane-intrinsic protein that is visible on the surface of most cell types of vertebrates. FAT (fat), SCARB3, GP88, glycoprotein IV (gpIV) or glycoprotein IIIb (gpIIIb) are well known. A member of the class B scavenger receptor family of CD36 line cell surface proteins. In addition to thrombospondin, CD36 binds to a large number of ligands such as collagen, red blood cells parasitized by Plasmodium falciparum, oxidized low density lipoprotein, natural lipoprotein, oxidized phospholipid, and long-chain fatty acid. In a recent study using genetically engineered rodents, the clear role of CD36 was confirmed in fatty acid or sugar metabolism, heart disease, taste, and intestinal fat delivery. CD36 can be involved in glucose intolerance, atherosclerosis, arterial hypertension, diabetes, cardiomyopathy, and Alzheimer's disease.

已知CD36表現性之DS細胞顯示出經由HGF之表現而促進血管內皮細胞之增殖，藉此有助於分化為毛乳頭或毛乳頭之活化之可能性，係用以使包含CD36表現性之結締組織鞘細胞之毛囊再生之組合物(專利文獻1)。進而根據本發明者等人，認為CD36陽性DS細胞顯示出較高之鹼性磷酸酶活性(圖3及圖4)，且顯示出以RGS5為代表之複數種間葉系幹細胞相關因子(NGFR、Nestin、ALCAM、PDGFRβ)之表現量較高，故而表現CD36之DS細胞亦具有作為間葉系幹細胞之能力即自我增殖性與多能性。藉由CD36陽性DS細胞之該等能力，可發揮毛囊之再生能力即毛囊誘導能力。然而，即便為以CD36為標記所選擇之包含CD36陽性細胞之毛囊形成用組合物，亦為其毛囊誘導能力受培養條件等影響而有不同者，有未達成所需之毛囊誘導能力之情況。因此，可藉由本發明之品質管理方法，管理毛囊形成用組合物之毛囊誘導能力。 It is known that CD36-expressing DS cells exhibit the promotion of proliferation of vascular endothelial cells via the expression of HGF, thereby facilitating the differentiation into the activation of dermal papilla or dermal papilla, and is used to make CD36 expressive connectives. A composition for regenerating hair follicles of tissue sheath cells (Patent Document 1). Further, according to the present inventors, it is considered that CD36-positive DS cells exhibit higher alkaline phosphatase activity (Fig. 3 and Fig. 4), and a plurality of mesenchymal stem cell-associated factors (NGFR, represented by RGS5) are shown. Nestin, ALCAM, and PDGFRβ have higher expression levels, so DS cells expressing CD36 also have the ability to function as mesenchymal stem cells, ie, self-proliferation and pluripotency. By the ability of CD36-positive DS cells, the ability to regenerate hair follicles, that is, hair follicle-inducing ability, can be exerted. However, even a hair follicle-forming composition containing CD36-positive cells selected by using CD36 as a marker has different hair follicle-inducing ability depending on culture conditions and the like, and may have a hair follicle-inducing ability which is not required. Therefore, the hair follicle inducing ability of the composition for hair follicle formation can be managed by the quality management method of the present invention.

CD36之表現可藉由細胞生物學之領域中所知之使用抗體之任意方法進行檢測，亦可使用定量PCR進行檢測。作為使用抗體之方法，可列舉：西方墨點(western blot)或螢光免疫染色。CD36陽性細胞之比率可藉由進行螢光免疫染色後利用目視或使用市售之程式進行計數而 決定，亦可藉由流式細胞儀(flow cytometry)而算出比率。 The expression of CD36 can be detected by any method known in the field of cell biology using antibodies, or by quantitative PCR. As a method of using an antibody, Western blot or fluorescent immunostaining can be mentioned. The ratio of CD36-positive cells can be counted by visual inspection or using a commercially available program after performing fluorescent immunostaining. It is decided that the ratio can also be calculated by flow cytometry.

成為指標之CD36之表現係記載為「特定值」，於CD36之表現超過該特定值之情形時，可決定具有毛囊誘導能力。關於特定值，可根據被視為必需之毛囊誘導能力而適當選擇，並不規定於特定值以下不產生毛囊誘導能力。CD36表現之特定值可根據使用之CD36表現之計測方法而變化。若為業者，則可藉由進行對三維培養皮膚模型或實驗動物之投予，而根據各計測系統而適當決定成為指標之CD36之表現之值。另一方面，就為使毛髮再生而進行向人類之移植之觀點而言，成為指標之值應經由對人類之體外或體內試驗而決定。指標較佳為DS細胞中之CD36陽性DS細胞之比率，成為指標之比率可就達成所需之毛髮生長之觀點、與達成所需之毛密度之觀點而適當選擇。 The expression of CD36 which is an indicator is described as "specific value", and when the performance of CD36 exceeds the specific value, it is determined that hair follicle inducing ability is determined. Regarding the specific value, it can be appropriately selected depending on the hair follicle inducing ability deemed to be necessary, and it is not prescribed that the hair follicle inducing ability is not generated below a specific value. The specific value of CD36 performance may vary depending on the measurement method used for CD36 performance. In the case of the manufacturer, the three-dimensional culture skin model or the experimental animal can be administered, and the value of the performance of the CD 36 serving as the index can be appropriately determined according to each measurement system. On the other hand, from the viewpoint of transplanting hair to humans, the value of the index should be determined by an in vitro or in vivo test on humans. The index is preferably a ratio of CD36-positive DS cells in DS cells, and the ratio of the index can be appropriately selected from the viewpoint of achieving desired hair growth and achieving the desired hair density.

於本發明中進行品質管理之毛囊形成用組合物包含CD36陽性細胞，可為由DS培養細胞而成之培養物，亦可藉由使用利用抗CD36抗體之細胞分選技術，藉此自作為異質之細胞群之DS細胞群對CD36表現性之細胞進行篩選而取得。作為細胞分選之方法，只要可基於表現之蛋白質將細胞分離則可為任意之方法，可為使用磁珠之方法(MACS)，亦可為使用流式細胞儀之方法(FACS)。本發明中接受品質管理之組合物可對源自CD36表現性之DS之細胞進行細胞分選並將所篩選之細胞供於培養而使其增殖而製成組合物，亦可為將所篩選之細胞群直接製成組合物者。 The hair follicle-forming composition for quality management in the present invention comprises CD36-positive cells, which may be cultures in which cells are cultured by DS, or may be heterologous by using a cell sorting technique using an anti-CD36 antibody. The DS cell population of the cell population was obtained by screening CD36 expressing cells. As a method of cell sorting, any method can be used as long as the cells can be separated based on the expressed protein, and it can be a method using a magnetic bead (MACS) or a method using a flow cytometer (FACS). The composition for quality management in the present invention can perform cell sorting on cells derived from CD36 expressing DS and culture the cells to be cultured to proliferate to prepare a composition, or can be selected as a composition. The cell population is directly made into a composition.

根據本發明之品質管理方法，於CD36表現低於特定值之情形時，亦可將該組合物廢棄。進而，於另一態樣中，於CD36表現低於特定值之情形時，亦可進而包括如下步驟：藉由進行使用CD36抗體之細胞分選，而取得CD36表現較高之組合物。於該情形時，必須再次藉由本發明之品質管理方法而確認毛囊誘導能力。 According to the quality management method of the present invention, when the CD36 exhibits a value lower than a specific value, the composition can be discarded. Further, in another aspect, when the CD36 exhibits a value lower than a specific value, the method further includes the step of obtaining a composition having a high CD36 performance by performing cell sorting using the CD36 antibody. In this case, the hair follicle inducing ability must be confirmed again by the quality management method of the present invention.

本發明之品質管理方法亦可以間葉系幹細胞相關因子之表現作 為指標，決定組合物是否具有較高之毛囊誘導能力。以間葉系幹細胞相關因子之表現作為指標之品質管理方法可加入至本發明之以CD36表現作為指標之品質管理方法中而進行，亦可獨立地進行。 The quality management method of the present invention can also be used for the expression of mesenchymal stem cell related factors. As an indicator, it is determined whether the composition has a high hair follicle inducing ability. The quality management method using the expression of the mesenchymal stem cell-related factor as an indicator can be added to the quality management method of the present invention using the CD36 expression as an index, or can be carried out independently.

於本發明中，所謂「間葉系幹細胞相關因子」，係指RGS5、PDGFβ、NGFR、NESTIN、ALCAM等，認為該等因子係於間葉系幹細胞中進行表現，有助於作為間葉系幹細胞之能力即自我增殖性與多能性。因此，可藉由將該等因子之表現設為指標，而確認包含CD36陽性細胞之毛囊形成用組合物之毛囊誘導能力。 In the present invention, the "mesenchyelia stem cell-associated factor" refers to RGS5, PDGFβ, NGFR, NESTIN, ALCAM, etc., and these factors are considered to be expressed in mesenchymal stem cells, and contribute to mesenchymal stem cells. The ability is self-proliferation and pluripotency. Therefore, the hair follicle inducing ability of the hair follicle-forming composition containing CD36-positive cells can be confirmed by using the expression of these factors as an index.

已知作為間葉系幹細胞相關因子之一種之RGS5為G蛋白質訊號調節蛋白質，存在於細胞質內且主要於血管周圍之外被細胞中進行表現(非專利文獻9)。又，近年報告有皮膚中之血管外被細胞發揮作為一種間葉系幹細胞之作用，於皮膚再生時參與再構成(非專利文獻10)。因此，可理解表現RGS5之細胞具有作為外被細胞之性質，且作為間葉系幹細胞發揮作用。進而，本發明者等人發現：RGS5於CD36陽性之DS細胞中進行高表現(圖6及7)，故而亦可將RGS5之表現用作毛囊誘導能力之指標，決定該組合物進而具有較高之毛囊誘導能力。 RGS5, which is a kind of mesenchymal stem cell-associated factor, is a G protein signal-regulating protein, and is present in cells in the cytoplasm and mainly expressed in cells (Non-Patent Document 9). In addition, it has been reported in recent years that extravascular extracellular cells in the skin function as a mesenchymal stem cell, and participate in reconstitution during skin regeneration (Non-Patent Document 10). Therefore, it is understood that cells expressing RGS5 have properties as exogenous cells and function as mesenchymal stem cells. Further, the present inventors have found that RGS5 is highly expressed in CD36-positive DS cells (Figs. 6 and 7), so that the expression of RGS5 can also be used as an indicator of hair follicle inducing ability, and it is determined that the composition is further high. Hair follicle inducing ability.

作為間葉系幹細胞相關因子之一種之PDGFR(platelet-derived growth factor receptor，血小板源性生長因子)beta為血小板源性生長因子受體，報告有血管外被細胞特殊之表現，可理解與RGS5同樣地作為間葉系幹細胞發揮作用(Winkler EA,Bell RD,Zlokovic BV Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling.Molecular neurodegeneration 5：32：非專利文獻11)。 PDGFR (platelet-derived growth factor receptor), a kind of mesenchymal stem cell-associated factor, is a platelet-derived growth factor receptor, and it has been reported to have extracellular appearance. It is understood to be the same as RGS5. Ground functions as mesenchymal stem cells (Winkler EA, Bell RD, Zlokovic BV Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling. Molecular neurodegeneration 5:32: Non-Patent Document 11).

提示有作為間葉系幹細胞相關因子之一種之NGFR(nerve growth factor receptor，神經生長因子受體，p75NTR)屬於LNGFR(Low Affinity Nerve Growth Factor Receptor，低親和力神經生長因子受體) 及TNFR(Tumor Necrosis Factor Receptors，腫瘤壞死因子受體)超家族，並作為於中樞神經系統及末梢神經系統之細胞中進行表現之分子被發現，與神經細胞之產生、生存、分化有關。近年來，於神經系統以外，作為於間葉系幹細胞、骨髄基質細胞中進行表現之標記因子被識別(Jones EA,Kinsey SE,English A,Jones RA,Straszynski L et al.(2002)Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells.Arthritis and rheumatism 46(12)：3349-3360；非專利文獻12及Quirici N,Soligo D,Bossolasco P,Servida F,Lumini C et al.(2002)Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.Experimental hematology 30(7)：783-791；非專利文獻13)。 It is suggested that NGFR (nerve growth factor receptor, p75NTR), which is a kind of mesenchymal stem cell related factor, belongs to LNGFR (Low Affinity Nerve Growth Factor Receptor). And TNFR (Tumor Necrosis Factor Receptors) superfamily, and found as molecules in the central nervous system and peripheral nervous system cells, and is related to the production, survival and differentiation of nerve cells. In recent years, markers other than the nervous system, which are expressed in mesenchymal stem cells and osteophyte stromal cells, have been identified (Jones EA, Kinsey SE, English A, Jones RA, Straszynski L et al. (2002) Isolation and characterization Of bone marrow multipotential mesenchymal progenitor cells. Arthritis and rheumatism 46(12): 3349-3360; Non-Patent Document 12 and Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C et al. (2002) Isolation of bone marrow mesenchymal Stem cells by anti-nerve growth factor receptor antibodies. Experimental hematology 30 (7): 783-791; Non-patent document 13).

作為細胞內中間徑長絲之一種之Nestin亦作為於神經系統中進行表現之神經幹細胞之標記而被識別，但近年來，作為亦於各種間葉系幹細胞中進行表現之因子而報告(Mendez-Ferrer S,Michurina TV,Ferraro F,Mazloom AR,Macarthur BD et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature 466(7308)：829-834；非專利文獻14)。ALCAM(Activaed leukocyte cell adhesion molecule)亦同樣地，可知於間葉系幹細胞中之表現。 Nestin, which is one of the intracellular medial filaments, is also recognized as a marker of neural stem cells expressed in the nervous system, but in recent years, it has been reported as a factor that is also expressed in various mesenchymal stem cells (Mendez- Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466 (7308): 829-834; Non-Patent Document 14). Similarly, ALCAM (Activaed leukocyte cell adhesion molecule) is known to be expressed in mesenchymal stem cells.

間葉系幹細胞相關因子之表現可藉由於細胞生物學之領域中所知之使用抗體之任意方法進行檢測，亦可使用定量PCR進行檢測。作為使用抗體之方法，可列舉：西方墨點或螢光免疫染色。RGS5陽性細胞之比率可藉由於進行螢光免疫染色後利用目視或使用市售之程式進行計數而決定，亦可藉由流式細胞儀而算出比率。 The expression of mesenchymal stem cell-associated factors can be detected by any method known in the field of cell biology using antibodies, and can also be detected using quantitative PCR. As a method of using an antibody, Western blot or fluorescent immunostaining can be mentioned. The ratio of RGS5-positive cells can be determined by visual inspection or by using a commercially available program after performing fluorescent immunostaining, and the ratio can also be calculated by flow cytometry.

成為指標之間葉系幹細胞相關因子之表現係記載為「特定值」，於間葉系幹細胞相關因子之表現超過該特定值之情形時，可決定具有毛囊誘導能力。可用於本發明之間葉系幹細胞相關因子可分別獨立地 使用，或將其等組合而使用。關於特定值，可根據被視為必需之毛囊誘導能力而適當選擇，並不規定於特定值以下不產生毛囊誘導能力。間葉系幹細胞相關因子表現之特定值可根據使用之間葉系幹細胞相關因子表現之計測方法而變化。若為業者，則可藉由進行對三維培養皮膚模型或實驗動物之投予，而根據各計測系統而適當決定成為指標之間葉系幹細胞相關因子之表現之值。另一方面，就為使毛髮再生而進行向人類之移植之觀點而言，成為指標之值應經由對人類之體外或體內試驗而決定。指標較佳為DS細胞中之間葉系幹細胞相關因子表現性DS細胞之比率，成為指標之比率可就達成所需之毛髮生長之觀點、與達成所需之毛密度之觀點而進行選擇。 The expression of the phylogenetic stem cell-related factor between the indicators is described as "specific value", and when the expression of the mesenchymal stem cell-related factor exceeds the specific value, it is determined that the hair follicle-inducing ability is determined. The leaf line stem cell related factors that can be used in the present invention can be independently Use, or combine them for use. Regarding the specific value, it can be appropriately selected depending on the hair follicle inducing ability deemed to be necessary, and it is not prescribed that the hair follicle inducing ability is not generated below a specific value. The specific value of the expression of the mesenchymal stem cell-associated factor may vary depending on the measurement method using the expression of the leaf line stem cell-related factor. In the case of the manufacturer, the three-dimensional culture skin model or the experimental animal can be administered, and the value of the expression of the leaf cell stem cell-related factor between the indicators can be appropriately determined according to each measurement system. On the other hand, from the viewpoint of transplanting hair to humans, the value of the index should be determined by an in vitro or in vivo test on humans. The index is preferably a ratio of the leaf cell stem cell-related factor-expressing DS cells in the DS cells, and the ratio of the index can be selected from the viewpoint of achieving the desired hair growth and achieving the desired hair density.

根據本發明之品質管理方法，於間葉系幹細胞相關因子表現低於特定值之情形時，亦可將該組合物廢棄。進而，於另一態樣中，於間葉系幹細胞相關因子表現低於特定值之情形時，亦可進而包括如下步驟：藉由進行使用CD36抗體之細胞分選，而取得間葉系幹細胞相關因子之表現較高之組合物。於該情形時，必須再次藉由本發明之品質管理方法，而確認毛囊誘導能力。 According to the quality management method of the present invention, when the mesenchymal stem cell-related factor exhibits a value lower than a specific value, the composition can also be discarded. Further, in another aspect, when the mesenchymal stem cell-related factor exhibits a value lower than a specific value, the method further includes the step of obtaining mesenchymal stem cell correlation by performing cell sorting using the CD36 antibody. A composition with a higher performance factor. In this case, the hair follicle inducing ability must be confirmed again by the quality management method of the present invention.

根據本發明之品質管理方法，可均勻地生產具有毛囊誘導能力之組合物，故而本發明之品質管理方法亦可稱為製造方法。 According to the quality management method of the present invention, a composition having hair follicle inducing ability can be uniformly produced, and thus the quality management method of the present invention can also be referred to as a manufacturing method.

所謂毛囊誘導能力，係指於皮膚或三維培養皮膚模型中，促進毛囊形成之能力，進而亦係指使處於毛髮週期之退化期或休止期之毛囊活化並向生長期誘導之能力。具有毛囊誘導能力之組合物可於移植後經由形成毛囊而促進毛髮生長，進而可增大毛髮密度或毛髮直徑。 The so-called hair follicle inducing ability refers to the ability to promote the formation of hair follicles in a skin or three-dimensional culture skin model, and further refers to the ability to activate hair follicles in the degenerative or resting phase of the hair cycle and induce them into the growth phase. The composition having hair follicle inducing ability can promote hair growth by forming hair follicles after transplantation, thereby increasing hair density or hair diameter.

鹼性磷酸酶係指於鹼性條件下使磷酸酯化合物水解之酵素。已知胚胎幹細胞(ES細胞)、胚胎生殖細胞(EG細胞)、誘導多能性幹細胞(iPS細胞)等具有多能性之未分化細胞具有鹼性磷酸酶活性，可用作多能性標記(非專利文獻7)。另一方面，已知於生長期之毛乳頭細胞 中，特異地可見鹼性磷酸酶活性(非專利文獻8)，進而，已知於DS區域之一部分，於生長期之初期鹼性磷酸酶活性較高(非專利文獻6)。根據該等見解，可知ALP活性提示與毛髮生長之關聯，毛乳頭或DS等之毛囊中之間葉系細胞中之ALP活性顯示與毛髮生長誘導能相關，我們確認實際上於毛囊誘導能力降低之毛乳頭細胞中，ALP活性下降(未公佈實驗結果)。綜合該等見解，認為於DS區域中具有鹼性磷酸酶活性之細胞可分化為毛乳頭或使毛乳頭活化，毛囊誘導能力較高。於本實施例中，可知藉由CD36抗體所細胞分選之CD36陽性細胞與CD36陰性細胞相比，顯示出較高之ALP活性(圖3、圖4、及圖5)，顯示出CD36陽性細胞與毛囊誘導能力之關係。 Alkaline phosphatase refers to an enzyme that hydrolyzes a phosphate compound under alkaline conditions. It is known that embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells) and other pluripotent undifferentiated cells have alkaline phosphatase activity and can be used as pluripotency markers ( Non-patent document 7). On the other hand, dermal papilla cells known to be in the growing season In the first part of the DS region, it is known that the alkaline phosphatase activity is high (Non-Patent Document 6). Based on these findings, it is known that ALP activity is associated with hair growth, and ALP activity in phylum cells between hair follicles such as dermal papilla or DS shows a correlation with hair growth-inducing ability, and we confirmed that the hair follicle-inducing ability is actually reduced. ALP activity was decreased in dermal papilla cells (experimental results were not published). Based on these findings, it is considered that cells having alkaline phosphatase activity in the DS region can differentiate into dermal papillae or activate the dermal papilla, and the hair follicle inducing ability is high. In the present example, it was found that CD36-positive cells sorted by CD36 antibody showed higher ALP activity than CD36-negative cells (Fig. 3, Fig. 4, and Fig. 5), showing CD36 positive cells. Relationship with hair follicle inducing ability.

因此，於本發明之更進一步之態樣中，本發明之品質管理方法亦可以ALP活性作為指標，決定包含CD36陽性細胞之毛囊形成用組合物是否具有較高之毛囊誘導能力。以ALP活性作為指標之品質管理方法可加入至以本發明之CD36表現作為指標之品質管理方法中而進行，亦可獨立地進行。 Therefore, in a still further aspect of the present invention, the quality management method of the present invention can also determine whether the hair follicle-forming composition containing CD36-positive cells has a high hair follicle inducing ability by using ALP activity as an index. The quality management method using ALP activity as an index can be added to the quality management method using the CD36 performance of the present invention as an index, or can be carried out independently.

ALP活性之測定只要為可用於細胞生物學之領域之ALP活性測定方法，則可使用任意之方法。作為ALP活性測定方法之一例，可藉由向細胞中添加作為基質之萘酚ASMX磷酸酯，藉由鹼性磷酸酶之作用而產生萘酚衍生物，並將該萘酚進行重氮鎓染色，藉此產生紫色之顯色，而進行測定。作為測定細胞中之ALP活性之方法，有使用染色法或細胞溶解液之方法，但ALP存在於細胞膜、細胞內，故而無法使用活細胞進行評價，無法於活細胞中對顯示高ALP活性即高毛囊誘導活性者進行篩選、濃縮。因此，於以ALP活性作為指標之毛囊形成用組合物之管理方法中，包括自組合物中取得一部分之細胞群之步驟。於該步驟中，可進而包括如下步驟：對細胞數進行計測，於培養皿或標本玻片等配置特定數量之細胞。亦可於取得一部分之細胞群後，對所 配置之特定數之細胞進行ALP染色，藉由吸光度之測定而決定ALP活性。此時，可使細胞溶解而測定ALP活性，亦可使細胞固定而測定ALP活性。另一方面，亦可不藉由吸光度，而藉由算出經染色之細胞數之比率而決定ALP活性。經染色之細胞數之比率可利用目視進行觀察而算出，亦可使用市售之程式而算出。 As long as it is a method of measuring ALP activity which can be used in the field of cell biology, any method can be used. As an example of the ALP activity measuring method, a naphthol derivative can be produced by the action of an alkaline phosphatase by adding a naphthol ASMX phosphate as a matrix to the cells, and the naphthol can be subjected to diazonium dyeing. Thereby, the color development of purple was produced, and the measurement was performed. As a method for measuring the ALP activity in a cell, there is a method of using a staining method or a cell lysate. However, ALP is present in a cell membrane or a cell, so that it cannot be evaluated using a living cell, and it is not possible to display a high ALP activity in a living cell. The hair follicle-inducing active is screened and concentrated. Therefore, in the method for managing a composition for forming a hair follicle using ALP activity as an index, a step of obtaining a part of the cell population from the composition is included. In this step, the method further includes the steps of: measuring the number of cells, and arranging a specific number of cells on the culture dish or the specimen slide. Can also obtain a part of the cell population, ALP staining is performed on a specific number of cells, and ALP activity is determined by measurement of absorbance. At this time, the ALP activity can be measured by dissolving the cells, and the cells can be fixed to measure the ALP activity. On the other hand, the ALP activity can be determined by calculating the ratio of the number of stained cells without using absorbance. The ratio of the number of stained cells can be calculated by visual observation, and can also be calculated using a commercially available program.

成為指標之ALP活性之值係記載為「特定值」，於ALP活性超過該特定值之情形時，可決定具有毛囊誘導能力。關於特定值，可根據被視為必需之毛囊誘導能力而適當選擇，並不規定於特定值以下不產生毛囊誘導能力。ALP活性之特定值可根據使用之鹼性磷酸酶活性測定方法而變化，若為業者，則可藉由進行對三維培養皮膚模型或實驗動物之投予，而根據活性測定方法而適當決定成為指標之ALP活性。另一方面，就為使毛髮再生而進行向人類之移植之觀點而言，成為指標之值應經由對人類之體外或體內試驗而進行決定。 The value of the ALP activity that is the index is described as "a specific value", and when the ALP activity exceeds the specific value, the hair follicle inducing ability can be determined. Regarding the specific value, it can be appropriately selected depending on the hair follicle inducing ability deemed to be necessary, and it is not prescribed that the hair follicle inducing ability is not generated below a specific value. The specific value of the ALP activity can be changed according to the method for measuring the alkaline phosphatase activity to be used, and if it is a manufacturer, it can be appropriately determined as an index according to the activity measurement method by administering a three-dimensional culture skin model or an experimental animal. ALP activity. On the other hand, from the viewpoint of transplanting hair to humans, the value of the index should be determined by an in vitro or in vivo test on humans.

根據本發明之品質管理方法，於ALP活性低於特定值之情形時，亦可將該組合物廢棄。進而，於另一態樣中，於ALP活性低於特定值之情形時，亦可進而包括如下步驟：藉由進行使用CD36抗體之細胞分選，而取得ALP活性較高之組合物。於該情形時，必須再次藉由本發明之品質管理方法，而確認毛囊誘導能力。 According to the quality management method of the present invention, when the ALP activity is lower than a specific value, the composition can be discarded. Further, in another aspect, when the ALP activity is lower than a specific value, the method further includes the step of obtaining a composition having a high ALP activity by performing cell sorting using the CD36 antibody. In this case, the hair follicle inducing ability must be confirmed again by the quality management method of the present invention.

市售有各種用於測定ALP活性之試劑盒，例如可使用BM purple(Roche)或LabAssay ALP(Wako)等進行染色。然而，於通常ALP活性之計測時，必須將細胞進行固定或溶解，故而無法對於全部組合物測定ALP活性，而對一部分細胞群進行取樣，供於ALP活性測定。 Various kits for measuring ALP activity are commercially available, and for example, staining can be carried out using BM purple (Roche) or LabAssay ALP (Wako). However, in the usual measurement of ALP activity, it is necessary to immobilize or dissolve the cells, so that it is not possible to measure ALP activity for all the compositions, and a part of the cell population is sampled for ALP activity measurement.

毛囊係包圍毛髮之組織，與毛髮一同構成毛髮器官。毛囊包含：由上皮性成分構成之內毛根鞘及外毛根鞘、及由結締組織性成分構成之真皮毛根鞘(DS)，其邊界係由基底膜隔開(圖1)。於毛髮器官之最深部存在膨脹為球狀之毛球，於毛球中以包圍毛乳頭之方式存在 毛母細胞，藉由毛母細胞進行增殖、分化、角化而形成毛髮。認為尤其是包圍毛球部之真皮毛根鞘(DS)中之接近毛乳頭之基底部位被稱為真皮毛根鞘杯(DSC：Dermal Sheath Cup)，存在於DSC之細胞有助於分化為毛乳頭及毛乳頭之活化。 The hair follicle surrounds the tissue of the hair and forms a hair organ together with the hair. The hair follicle comprises: an inner root sheath and an outer root sheath composed of an epithelial component, and a dermal root sheath (DS) composed of a connective tissue component, the boundary of which is separated by a basement membrane (Fig. 1). In the deepest part of the hair organ, there is a bulb that expands into a globular shape, which exists in the hair bulb to surround the nipple. Hair cells, which are proliferated, differentiated, and keratinized by hair cells to form hair. It is considered that the base portion of the dermal root sheath (DS) surrounding the hair bulb, which is close to the dermal papilla, is called the Dermal Sheath Cup (DSC), and the cells present in the DSC contribute to differentiation into the dermal papilla and Activation of the dermal papilla.

所謂本發明中之「毛囊形成」，係指毛髮器官中形成毛囊之情況，係指於三維培養皮膚模型中形成毛囊、或於哺乳動物尤其是人類中形成毛囊之情況。於向哺乳動物移植藉由本發明所品質管理之組合物之情形時，可重新形成毛囊，亦可對處於休止期之毛囊起作用，促進毛囊之再活化。有時亦對毛囊之新形成與毛囊之再活化進行區別、或不區別，而使用毛囊之再生之用語。藉由本發明所品質管理之組合物可經由毛囊之形成而促進新毛髮之擴展及/或毛髮生長。將組合物可促進毛囊之形成之能力稱為毛囊誘導能力。 The "hair follicle formation" in the present invention refers to the case where hair follicles are formed in a hair organ, and refers to a case where hair follicles are formed in a three-dimensional culture skin model or hair follicles are formed in a mammal, especially a human. In the case of transplanting a composition managed by the quality of the present invention to a mammal, the hair follicle can be reformed, and the hair follicle in the resting period can be acted upon to promote reactivation of the hair follicle. Sometimes the new formation of hair follicles is distinguished from or not differentiated from the reactivation of hair follicles, and the term "regeneration" of hair follicles is used. The composition managed by the quality of the present invention can promote the expansion of new hair and/or hair growth via the formation of hair follicles. The ability of a composition to promote the formation of hair follicles is referred to as hair follicle inducing ability.

真皮毛根鞘(DS)細胞係存在於真皮毛根鞘之細胞，係間葉系細胞之一種。真皮毛根鞘(DS：Dermal seath)有時亦稱為結締組織性毛鞘或結締組織鞘，係包圍上皮性之外毛根鞘之真皮性組織。藉由本發明所品質管理之組合物中所含之DS細胞較佳為存在於處於真皮毛根鞘(DS)中尤其是鄰接於毛乳頭之基底部位至毛乳頭之高度附近之區域即真皮毛根鞘杯(DSC)區域之細胞。DS細胞可為藉由初代培養自組織所取得之細胞，亦可為藉由繼代培養所取得之細胞，又，亦可為自成體幹細胞、iPS細胞、及ES細胞進行分化誘導所獲得之細胞。就進行移植之觀點而言，較佳為藉由繼代培養使自移植對象之DS所取得之細胞增殖之細胞。 The dermal root sheath (DS) cell line is present in cells of the dermal root sheath, one of the mesenchymal cells. The dermal root sheath (DS: Dermal seath), sometimes referred to as the connective tissue sheath or connective tissue sheath, surrounds the dermal tissue of the epithelial outer root sheath. Preferably, the DS cells contained in the composition managed by the quality of the present invention are present in a region of the dermal root sheath (DS), particularly adjacent to the base of the dermal papilla, to the vicinity of the height of the dermal papilla, ie, the dermal sheath cup. Cells in the (DSC) region. The DS cells may be cells obtained by self-organization in primary culture, cells obtained by subculture, or differentiation induced by autologous stem cells, iPS cells, and ES cells. cell. From the viewpoint of transplantation, it is preferred to subculture cells in which cells obtained from the transplanted subject are proliferated.

DS細胞係與DP細胞同樣地被分類為間葉系細胞，DP被認為是源自DS尤其是DSC，毛髮生長期之DP增殖率先使DS增殖，故而認為DS尤其是DSC供給DP細胞(Tobin DJ et al.,J.Invest.Dermatol.,120：895-904,2003；非專利文獻4)。認為DS係由異質之細胞群構成，於毛髮 週期之休止期至生長期之間伴隨***與移動而下降，其一部分化為毛乳頭(DP)，開始毛髮之擴展。 The DS cell line is classified as mesenchymal cells in the same way as DP cells, and DP is considered to be derived from DS, especially DSC. The long-term DP proliferation rate of hair growth first causes DS to proliferate, so it is considered that DS, especially DSC, supplies DP cells (Tobin DJ). Et al., J. Invest. Dermatol., 120: 895-904, 2003; Non-Patent Document 4). It is believed that the DS system consists of heterogeneous cell populations in the hair. The cycle from the rest period to the growth phase declines with division and movement, and part of it becomes the dermal papilla (DP), which begins to expand hair.

藉由本發明所品質管理之組合物中所含之DS細胞可源自所有哺乳動物例如人類、黑猩猩、其他靈長類、家畜動物，例如狗、貓、兔、馬、綿羊、山羊、牛、豬、其他實驗用動物，例如大鼠、小鼠、豚鼠，更佳為裸小鼠、免疫缺陷小鼠(SCID mouse)、裸大鼠之表皮，但就向人類之移植、或製造研究用三維培養皮膚模型之觀點而言，較佳為源自人類之細胞。又，該表皮部位可為有毛部位例如頭皮，亦可為無毛部位例如***。 The DS cells contained in the composition managed by the quality of the present invention may be derived from all mammals such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs. Other experimental animals, such as rats, mice, guinea pigs, more preferably nude mice, immunodeficient mice (SCID mouse), the epidermis of nude rats, but transplanted to humans, or three-dimensional culture for manufacturing research From the viewpoint of the skin model, it is preferably a cell derived from humans. Further, the skin portion may be a hairy portion such as a scalp or a hairless portion such as a foreskin.

DS細胞及DP細胞亦可利用任意方法進行單離，例如可藉由生檢而取得毛囊組織，藉由解剖將外毛根鞘或內毛根鞘去除，將目標之部位例如DS尤其是DSC、或DP進行分離後，經由酵素處理，於培養基中進行培養。作為酵素處理，可使用用於細胞分離之任意酵素，例如可使用膠原酶或釋放酶(liberase，Roche Applied Science)。若為業者，則可適當選擇適於培養目標之細胞之培養基，例如可於培養DS、DSC、及DP時，使用用於培養間葉系細胞之培養基。該培養基亦可為含血清培養基或無血清培養基之任一種，就進行向人類之移植之觀點而言，較佳為無血清培養基。 DS cells and DP cells can also be isolated by any method. For example, hair follicle tissue can be obtained by biopsy, and the outer root sheath or inner hair root sheath can be removed by dissection, and the target site such as DS, especially DSC, or DP can be removed. After separation, the cells are cultured in an enzyme medium. As the enzyme treatment, any enzyme for cell separation can be used, and for example, collagenase or release enzyme (liberase, Roche Applied Science) can be used. For the manufacturer, a medium suitable for culturing the target cells can be appropriately selected. For example, when culturing DS, DSC, and DP, a medium for culturing mesenchymal cells can be used. The medium may be either serum-containing medium or serum-free medium, and is preferably a serum-free medium from the viewpoint of transplantation to humans.

用以形成藉由本發明所品質管理之毛囊之組合物之目的在於：於移植後之皮膚中，或於三維培養皮膚中使毛囊形成或再生，而形成毛髮器官，其目的在於最終地促進毛髮之擴展及/或毛髮生長。認為有助於毛囊之形成之細胞係未必藉由單一之細胞，而藉由異質之細胞群共同地發揮作用而使毛囊形成或再生，故而用以形成藉由本發明所品質管理之毛囊之組合物可僅為DS細胞，亦可包含除DS細胞以外而存在於上皮及毛髮器官中之任意細胞。作為DS細胞以外之細胞，亦可包含：DS細胞以外之間葉系細胞例如毛乳頭(DP)細胞、或上皮系 細胞例如內毛根鞘或外毛根鞘之細胞。 The composition for forming a hair follicle managed by the quality of the present invention is to form or regenerate hair follicles in the skin after transplantation or in three-dimensional culture skin to form a hair organ, the purpose of which is to finally promote hair. Expansion and / or hair growth. It is considered that a cell line contributing to the formation of hair follicles does not necessarily have a single cell, but the hair follicle is formed or regenerated by a heterogeneous cell population, thereby forming a composition of hair follicles managed by the quality of the present invention. It may be only DS cells, and may also include any cells present in epithelial and hair organs other than DS cells. The cells other than the DS cells may further include: phyllodes cells other than the DS cells, such as dermal papilla (DP) cells, or epithelial cells. Cells such as cells of the inner root sheath or outer root sheath.

於在藉由本發明所品質管理之組合物中包含DP細胞之情形時，組合物中所含之DS細胞相對於DP細胞之使用之比率並無特別限定，較佳為於組合物中以1：10~10：1而含有，更佳為以1：3~3：1而含有。進而，相對於DS細胞與DP細胞之總量，亦可以1：10~10：1而含有上皮系細胞，進而較佳為以1：1~10：1而含有，進而更佳為以1：1~3：1而含有，最佳為以1：1而含有。 In the case where DP cells are contained in the composition managed by the quality of the present invention, the ratio of the DS cells contained in the composition to the DP cells is not particularly limited, and is preferably 1: in the composition. It is contained in 10 to 10:1, and more preferably in the range of 1:3 to 3:1. Further, the epithelial cells may be contained in the range of 1:10 to 10:1, and more preferably in the range of 1:1 to 10:1, and more preferably 1: in the total amount of the DS cells and the DP cells. It is contained in 1 to 3:1, and is preferably contained in 1:1.

所謂毛乳頭(DP)細胞，係指作為間葉系細胞而位於毛囊基底部，發揮向毛囊上皮幹細胞傳送活化訊號以使毛囊自我再生之所謂之指揮塔之作用的細胞。僅含有活化毛乳頭細胞之毛乳頭細胞調製品例如可藉由使用基因轉殖小鼠之Kishimoto et al.,Proc.Natl.Acad.Sci.USA(1999),Vol.96,pp.7336-7341中記載之方法進行調製。 The term "hair papilla (DP) cell" refers to a cell that acts as a mesenchymal cell in the base of the hair follicle as a mesenchymal cell and functions as a so-called cell tower that transmits an activation signal to the hair follicle epithelial stem cells to self-regenerate the hair follicle. A dermal papilla cell preparation containing only activated dermal papilla cells can be obtained, for example, by using a genetically-transferred mouse, Kishimoto et al., Proc. Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341. The method described in the method is modulated.

「上皮系細胞」係構成皮膚之表皮或上皮之大部分之細胞，由接於真皮之一層基底細胞產生。若以小鼠為例，則作為上皮系細胞，可較佳地使用源自新生兒(或胎兒)之上皮系細胞，可為源自成熟之皮膚例如休止期毛髮之上皮或生長期毛髮之上皮之細胞，亦可為處於角質形成細胞之形態之細胞之培養物。此種細胞可利用業者眾所周知之方法由所需之供體動物之皮膚進行調製。 "Epithelial cells" are cells that form the epidermis or epithelium of the skin and are produced by basal cells attached to one layer of the dermis. In the case of a mouse, for example, a neonatal (or fetal) epithelial cell can be preferably used as an epithelial cell, and can be derived from a mature skin such as a resting epithelium or a growing epithelium. The cells may also be cultures of cells in the form of keratinocytes. Such cells can be modulated from the skin of the desired donor animal using methods well known to those skilled in the art.

藉由本發明所品質管理之組合物中所含之DP細胞或上皮系細胞可與DS細胞同樣地源自所有哺乳動物例如人類、黑猩猩、其他靈長類、家畜動物，例如狗、貓、兔、馬、綿羊、山羊、牛、豬、其他實驗用動物，例如大鼠、小鼠、豚鼠，更佳為裸小鼠、免疫缺陷小鼠、裸大鼠之皮膚，但就向人類之移植、或製造研究用三維培養皮膚模型之觀點而言，較佳為源自人類之細胞。 The DP cells or epithelial cells contained in the composition managed by the quality of the present invention can be derived from all mammals such as humans, chimpanzees, other primates, livestock animals, such as dogs, cats, rabbits, like DS cells. Horses, sheep, goats, cows, pigs, other experimental animals, such as rats, mice, guinea pigs, more preferably the skin of nude mice, immunodeficient mice, nude rats, but transplanted to humans, or From the viewpoint of manufacturing a three-dimensional culture skin model for research, it is preferably a cell derived from humans.

將藉由本發明所品質管理之組合物移植至受體動物之方法可藉由其本身公知之移殖方法。例如，參照Weinberg et al,J.Invest Dermatol.Vol.100(1993),pp.229-236。另外，作為另一方法，藉由如下方法進行移植：將藉由本發明所品質管理之組合物利用注射針等細胞移植用裝置投予至皮膚。可選擇任意之皮膚區域，但就用於人類之觀點而言，較佳為將組合物移植至頭皮尤其是頭皮中毛髮較少之區域。移植至頭皮之上皮或表皮之細胞有向受到損傷之毛囊或休眠毛囊移動，促進毛囊之活化之情況，亦有促進新毛囊之形成之情況。於以毛髮生長為目的而移植至包含人類之動物之情形時，或可根據醫師或獸醫而適當決定適當之方法。 The method of transplanting a composition managed by the quality of the present invention to a recipient animal can be carried out by a method known per se by itself. For example, see Weinberg et al, J. Invest Dermatol. Vol. 100 (1993), pp. 229-236. Further, as another method, transplantation is carried out by administering a composition managed by the quality of the present invention to the skin using a device for cell transplantation such as an injection needle. Any skin area can be selected, but from the standpoint of human use, it is preferred to transplant the composition to the scalp, especially the area of the scalp where hair is less. Cells transplanted to the epithelium or epidermis of the scalp may move toward damaged hair follicles or dormant hair follicles, promote the activation of hair follicles, and promote the formation of new hair follicles. In the case of transplantation to a human-containing animal for the purpose of hair growth, an appropriate method may be appropriately determined according to a physician or a veterinarian.

進而，可藉由使根據本發明所品質管理之組合物包含於三維培養皮膚模型中，而提供擔載再生毛囊之三維培養皮膚模型。然而，於該情形時，需要成為毛髮生長之指揮塔之毛乳頭細胞。三維培養皮膚模型可利用業者眾所周知之方法(Exp.Cell Res.Amano S.et al.,(2001),Vol.271,pp.249-362)例如以如下所述之方式進行製作。三維培養皮膚模型以1×106~108個/cm2之量分別含有DSc及DPc，較佳為以1.0~1.5×107個/cm2之量含有，更佳為以約1.27×107個/cm2之量含有。 Further, a three-dimensional culture skin model carrying the regenerated hair follicle can be provided by including the composition managed according to the present invention in a three-dimensional culture skin model. However, in this case, it is necessary to become a dermal papilla cell of a hair growth control tower. The three-dimensional culture skin model can be produced, for example, in the manner as described below by a method well known to the manufacturer (Exp. Cell Res. Amano S. et al., (2001), Vol. 271, pp. 249-362). The three-dimensional culture skin model contains DSc and DPc, respectively, in an amount of 1 × 10 6 to 10 8 /cm 2 , preferably 1.0 to 1.5 × 10 7 /cm 2 , more preferably about 1.27 × 10 It is contained in an amount of 7 pieces/cm 2 .

三維培養皮膚模型於先前技術中所知之三維培養皮膚模型之製法中，可藉由使用藉由本發明所品質管理之組合物而進行製造。例如將人類纖維母細胞適當量分散於0.1%膠原蛋白溶液/DMEM(Dulbecco's Modified Eagle Medium，細胞培養基)/10%FBS(Fetal Bovine Serum，胎牛血清)中，分注於培養皿，並立即於37℃之CO2培養箱中靜置。凝膠化後，自培養皿壁面及底面將凝膠剝離，使其於培養皿中浮游。一面搖動膠原蛋白凝膠一面進行培養，使凝膠收縮至約五分之一而製成真皮模型。將真皮模型放置於不鏽鋼柵格上，並於其上放置玻璃環，將0.4ml分散於KGM(表皮細胞培養用培養基)之人類培養表皮細胞(1.0×106細胞數/ml)注入至玻璃環內，進行培養。此時，將藉由本發明所品質管理之組合物同時進行混 合並注入。作為人類培養表皮細胞之代替，亦可使用小鼠新生兒表皮細胞。以使真皮模型之上部暴露於空氣中之程度將DMEM-KGM-5%FBS+Ca2+之培養基添加至培養皿內，並進行培養，約一週後觀察皮膚樣本，判定毛囊原基形成之有無與再現性。 The three-dimensional culture skin model can be produced by using a composition managed by the quality of the present invention in the method of producing a three-dimensional culture skin model known in the prior art. For example, the appropriate amount of human fibroblasts is dispersed in 0.1% collagen solution / DMEM (Dulbecco's Modified Eagle Medium, cell culture medium) / 10% FBS (Fetal Bovine Serum, fetal bovine serum), dispensed in a Petri dish, and immediately It was allowed to stand in a 37 ° C CO 2 incubator. After gelation, the gel was peeled off from the wall surface and the bottom surface of the dish to float in the dish. The dermal model was made by culturing while shaking the collagen gel to shrink the gel to about one-fifth. The dermal model was placed on a stainless steel grid, and a glass ring was placed thereon, and 0.4 ml of human cultured epidermal cells (1.0×10 6 cells/ml) dispersed in KGM (epidermal cell culture medium) was injected into the glass ring. Inside, culture. At this time, the composition managed by the quality of the present invention is simultaneously mixed and injected. As a substitute for human cultured epidermal cells, mouse neonatal epidermal cells can also be used. The medium of DMEM-KGM-5%FBS+Ca 2+ was added to the culture dish to the extent that the upper part of the dermis model was exposed to the air, and cultured. After about one week, the skin sample was observed to determine whether or not the hair follicle primordium was formed. And reproducibility.

擔載有再構成毛囊之三維培養皮膚模型可用於研究、解明毛囊之再生機構或對毛髮生長、脫髮有效之藥劑、天然藥之篩選。 The three-dimensional culture skin model carrying the reconstituted hair follicle can be used to study and explain the regeneration mechanism of the hair follicle or the medicine for the hair growth and hair loss, and the screening of the natural medicine.

[實施例] [Examples] 實施例1：細胞培養 Example 1: Cell Culture

將人類頭皮組織放入至含10%牛胎兒血清之DMEM(Gibco/invitrogen)中，利用手術刀將真皮部分去除，自切斷面取出毛囊。使用精密鑷子自毛囊將包含ORS之毛幹去除並將DS區域及DSC區域取出。對於單離DS及單離DSC，於37℃下進行40分鐘膠原酶(sigma公司)處理後，於包含medium(介質)-1(含10%牛胎兒血清、EGF、bFGF、0.00075%之β巰基乙醇、青黴素100units/ml(力價)、鏈黴素0.1mg/ml(力價)、雙性黴素B 0.25μg/ml(力價)之日水纖維母細胞用低血清培養基)之膠原蛋白塗佈之35mm碟(Iwaki)上進行靜置培養。經過1週後，確認增殖後，作為DS細胞而用作實驗樣品。 The human scalp tissue was placed in DMEM (Gibco/invitrogen) containing 10% fetal bovine serum, and the dermis was partially removed using a scalpel, and the hair follicle was taken out from the cut surface. Remove the hair shaft containing the ORS from the hair follicle using precision tweezers and remove the DS area and the DSC area. For isolated DS and isolated DSC, after 40 minutes of collagenase (sigma) treatment at 37 ° C, containing medium (medium)-1 (containing 10% fetal fetal serum, EGF, bFGF, 0.00075% of β-mercapto Ethanol, penicillin 100units/ml (force price), streptomycin 0.1mg/ml (force price), amphotericin B 0.25μg/ml (force price), daily water fibroblasts with low serum medium) collagen The culture was carried out on a coated 35 mm dish (Iwaki). After one week, after confirming proliferation, it was used as an experimental sample as a DS cell.

經單離之DS細胞及DSC細胞係藉由medium-1而進行7~10天靜置培養。其後，使用胰蛋白酶實施細胞之繼代。培養條件係於37℃、5%CO2之條件下，將膠原蛋白塗佈之燒瓶T-75(Iwaki)用作培養容器。供於實驗之各細胞係使用經1次~3次繼代者，繼代中，於實驗時將MesenPro RS medium(Life technologies)用作培養基，每2~3天交換培養基。 The isolated DS cells and DSC cell lines were statically cultured for 7 to 10 days by medium-1. Thereafter, the passage of the cells was carried out using trypsin. The culture conditions were carried out using a collagen-coated flask T-75 (Iwaki) as a culture vessel under the conditions of 37 ° C and 5% CO 2 . For each cell line used for the experiment, one to three subcultures were used, and in the experiment, MesenPro RS medium (Life technologies) was used as a medium, and the medium was exchanged every 2 to 3 days.

實施例2：細胞分選 Example 2: Cell sorting

使用Mini MACS separator(Miltenyl Biotec)對細胞進行區分。操作條件係根據Miltenyl Biotec所提示之操作說明而進行。使用胰蛋白 酶溶液，將實施例1中所培養之細胞剝離後，使細胞懸浮液通過Pre-separation filter(預分離過濾器)(Miltenyl Biotec)，對細胞數進行計數。使500~800萬個細胞懸浮於100μl之Buffer(緩衝器)1(含0.5%之BSA(sigma)、2mM之EDTA之PBS溶液)中，並於其中以成為50倍稀釋之方式添加CD36抗體(Abcam，ab17044)，於冰箱中反應約15分鐘。使用Buffer1進行洗淨，藉由離心操作將細胞回收後，於80μl之Buffer1中進行再懸浮，添加20μl之抗小鼠IgG1 Microbeads(磁珠)(Miltenyl Biotec)，於冰箱中反應15分鐘。使用Buffer1進行洗淨，藉由離心操作將細胞回收後，添加500μl之Buffer1，使用Mini MACS separator(分離器)、MS Columns(管柱)(Miltenyl Biotec)，對CD36陽性細胞進行分選。將於利用Buffer1進行洗淨中所流出之細胞設為CD36陰性細胞，於3次洗淨後將藉由磁石而間接地吸附之細胞進行回收，將其設為CD36陽性細胞。將經回收之CD36陽性、陰性DS細胞、及CD36陽性、陰性DSC細胞於MesenPro RS medium(Life technologies)中懸浮後，將TypeIV膠原蛋白塗佈之燒瓶T-25(BD biosciences)用作培養容器，於37℃、5%CO2環境下培養4~8天，將所獲得者繼續用於實驗。 Cells were differentiated using a Mini MACS separator (Miltenyl Biotec). Operating conditions were carried out according to the instructions given by Miltenyl Biotec. After the cells cultured in Example 1 were peeled off using a trypsin solution, the cell suspension was passed through a Pre-separation filter (Miltenyl Biotec), and the number of cells was counted. Five to eight million cells were suspended in 100 μl of Buffer 1 (containing 0.5% BSA (sigma), 2 mM EDTA in PBS), and CD36 antibody was added thereto in a 50-fold dilution ( Abcam, ab17044), reacted in the refrigerator for about 15 minutes. After washing with Buffer1, the cells were recovered by centrifugation, resuspended in 80 μl of Buffer1, and 20 μl of anti-mouse IgG1 Microbeads (Miltenyl Biotec) was added and reacted in a refrigerator for 15 minutes. Washing was performed using Buffer1, and after the cells were recovered by centrifugation, 500 μl of Buffer1 was added, and CD36-positive cells were sorted using a Mini MACS separator (separator) and MS Columns (Miltenyl Biotec). The cells which were eluted by washing with Buffer 1 were set as CD36-negative cells, and after washing three times, the cells indirectly adsorbed by the magnets were collected and designated as CD36-positive cells. After recovering recovered CD36-positive, negative DS cells, and CD36-positive, negative DSC cells in MesenPro RS medium (Life technologies), Type IV collagen coated flask T-25 (BD biosciences) was used as a culture vessel. The culture was carried out for 4 to 8 days at 37 ° C in a 5% CO 2 atmosphere, and the obtained ones were used for the experiment.

實施例3：鹼性磷酸酶活性之測定 Example 3: Determination of alkaline phosphatase activity

將於實施例2中所分選之CD36陽性DS細胞及CD36陰性DS細胞、及CD36陽性DSC細胞及CD36陰性DSC細胞分別播種於6孔碟(BD biosciences)後，於37℃、5%CO2條件下，進行培養直至半融合狀。利用PBS進行洗淨後，利用4%PFA培養15分鐘並進行固定，利用PBS進行洗淨，添加BM purple(紫色)(Roche)並進行約30分鐘顯色反應，利用PBS進行洗淨。使用顯微鏡(Olympus)對顯示ALP活性之染色為藍色之細胞進行攝影(圖3及圖5)。 CD36-positive DS cells and CD36-negative DS cells, and CD36-positive DSC cells and CD36-negative DSC cells sorted in Example 2 were seeded in 6-well plates (BD biosciences) at 37 ° C, 5% CO 2 , respectively. Under the conditions, the culture was carried out until it was semi-fused. After washing with PBS, the cells were incubated with 4% PFA for 15 minutes, fixed, washed with PBS, and BM purple (purple) (Roche) was added thereto, and a color reaction was carried out for about 30 minutes, followed by washing with PBS. The cells stained with blue showing ALP activity were photographed using a microscope (Olympus) (Fig. 3 and Fig. 5).

將於實施例2中所分選之CD36陽性DS細胞及CD36陰性DS細胞分 別播種於6孔碟(BD biosciences)後，進行培養直至半融合狀，利用PBS進行洗淨後，向各孔中添加100μL含0.05%Triton X之PBS，使用刮刀，將細胞溶解液回收至輔助管中。反覆進行兩次凍結融解後，於4℃、15分鐘、15,000rpm之條件下進行離心處理，將上清液移至新輔助管中並將其作為樣品。繼而，使用LabAssay ALP(Wako)，根據Wako所提示之操作說明進行。藉由樣品中之ALP(鹼性磷酸酶)而使磷酸對硝基苯酯分解為對硝基苯酚與磷酸，所生成之對硝基苯酚係於鹼性側呈現黃色。藉由利用吸光讀板儀對該405nm之吸光度進行測定而求出檢體中之鹼性磷酸酶活性值。再者，基質反應條件係37度、15分鐘。求出已知ALP活性之標準溶液稀釋系之405nm之吸光度，繪製校準曲線，基於該校準曲線而算出樣品中之ALP活性並顯示(圖4)。 CD36-positive DS cells and CD36-negative DS cells sorted in Example 2 After culturing in a 6-well dish (BD biosciences), the cells were cultured until they were semi-fused, washed with PBS, and then 100 μL of PBS containing 0.05% Triton X was added to each well, and the cell lysate was recovered to the auxiliary using a spatula. In the tube. After repeatedly performing two freeze-thaws, centrifugation was carried out at 4 ° C, 15 minutes, and 15,000 rpm, and the supernatant was transferred to a new auxiliary tube and used as a sample. Then, using LabAssay ALP (Wako), follow the instructions given by Wako. The p-nitrophenyl phosphate is decomposed into p-nitrophenol and phosphoric acid by ALP (alkaline phosphatase) in the sample, and the resulting p-nitrophenol is yellow on the alkaline side. The absorbance at 405 nm was measured by an absorbance plate reader to obtain an alkaline phosphatase activity value in the sample. Further, the substrate reaction conditions were 37 degrees and 15 minutes. The absorbance at 405 nm of the standard solution dilution system of known ALP activity was determined, and a calibration curve was drawn, and ALP activity in the sample was calculated based on the calibration curve and displayed (Fig. 4).

實施例4：免疫細胞染色 Example 4: Immune cell staining

使用對於使用CD36抗體、RGS5抗體之細胞染色，將DSc播種於TypeI膠原蛋白塗佈之8孔Chamber Slide(BD biosciences)後，培養3-5天者。利用PBS洗淨後，利用4%PFA固定15分鐘，利用PBS進行洗淨，利用含有3%BSA之PBS進行30分鐘黏連處理，於將CD36抗體(Abcam，ab17044)、RGS5抗體(proteintech)利用含3%BSA之PBS分別稀釋50倍、稀釋200倍而成之第一抗體溶液中反應1小時。利用PBS洗淨4次後，於將Alexa 594標識抗小鼠IgG抗體(Life technologies)、Alexa 488標識抗兔IgG抗體(Life technologies)利用含3%BSA之PBS稀釋200倍而成之第二抗體溶液中反應1小時。為了進行核染色而於DAPI溶液中進行反應後，利用PBS洗淨4次，並藉由防褪色封固劑Vectorshield(Vector)與覆蓋玻璃進行封固。使用螢光顯微鏡(Olympus)進行觀察(圖6)。 For staining with cells using CD36 antibody, RGS5 antibody, DSc was seeded in TypeI collagen coated 8-well Chamber Slide (BD biosciences) and cultured for 3-5 days. After washing with PBS, the cells were fixed with 4% PFA for 15 minutes, washed with PBS, and incubated with PBS containing 3% BSA for 30 minutes to utilize CD36 antibody (Abeam, ab17044) and RGS5 antibody (proteintech). The PBS containing 3% BSA was diluted 50-fold and diluted 200-fold to form a first antibody solution for 1 hour. After washing 4 times with PBS, the Alexa 594-labeled anti-mouse IgG antibody (Life technologies), Alexa 488-labeled anti-rabbit IgG antibody (Life technologies) was diluted 200-fold with 3% BSA in PBS to obtain a second antibody. The solution was reacted for 1 hour. After the reaction was carried out in a DAPI solution for nuclear staining, it was washed 4 times with PBS, and sealed with a cover glass by a fade-proof mounting agent Vectorshield (Vector). Observation was carried out using a fluorescence microscope (Olympus) (Fig. 6).

實施例5：組織染色 Example 5: Tissue staining

利用冷凍組織包埋劑OTC化合物(Sakura Finetek)將人類頭皮組織 包埋，利用冷凍切片製成裝置低溫恆溫器(Leica)而製成冷凍切片載玻片。利用4%PFA固定15分鐘後，利用PBS進行洗淨，使用於PBS中添加有5%脫脂乳、1%驢血清、0.1%triton-X100之阻斷溶液反應1小時。繼而，使用將小鼠CD36抗體溶液(Abcam，ab17044)利用阻斷溶液稀釋50倍而成之第一抗體溶液，於常溫下反應1時間或於4℃下反應一晚。利用PBS進行3次洗淨後，使用將Alexa 594標識抗小鼠IgG抗體(Invitrogen)利用阻斷溶液分別稀釋200倍而成之第二抗體溶液於常溫下反應1小時。利用DAPI溶液進行反應後，利用PBS洗淨3次，藉由防褪色劑Prolong Gold Antifade Reagent與覆蓋玻璃進行封固。使用螢光顯微鏡(Olympus)進行觀察。將結果示於圖2。 Human scalp tissue using frozen tissue embedding agent OTC compound (Sakura Finetek) Embedding, frozen section slides were made by cryosectioning a device cryostat (Leica). After fixing for 15 minutes with 4% PFA, the cells were washed with PBS, and reacted with a blocking solution containing 5% skim milk, 1% sputum serum, and 0.1% triton-X100 in PBS for 1 hour. Then, the first antibody solution obtained by diluting the mouse CD36 antibody solution (Abeam, ab17044) with a blocking solution by 50-fold was used, and the reaction was carried out at room temperature for 1 hour or at 4 ° C for one night. After washing three times with PBS, the second antibody solution obtained by diluting the Alexa 594-labeled anti-mouse IgG antibody (Invitrogen) with a blocking solution by 200-fold was used for 1 hour at room temperature. After the reaction was carried out using a DAPI solution, it was washed three times with PBS, and sealed with a cover glass by a defoamer Prolong Gold Antifade Reagent. Observation was performed using a fluorescence microscope (Olympus). The results are shown in Figure 2.

實施例6：間葉系幹細胞相關因子之定量PCR Example 6: Quantitative PCR of mesenchymal stem cell related factors

對於實施例2中所分選之CD36陽性DS細胞及CD36陰性DS細胞，使用TRIzol(Invitrogen)，使用所提供之操作說明自該等細胞中回收RNA。所回收之RNA係利用核酸定量裝置Nanodrop(Thermo scientific)測定濃度。調整RNA濃度後，使用Applied Biosystems公司之操作說明，藉由High-Capacity cDNA Reverse Transcription Kits由RNA合成cDNA。使用反應試劑LightCycler(商標)FastStart DNA MasterPLUS SYBR Green(Roche)、反應裝置LightCycler(Roche)將所合成之cDNA於鑄模中進行定量RT-PCR。組成條件係根據Roche之操作說明而進行。所使用之引子如下所述：G3PDH Forward(正向)：5'-GCACCGTCAAGGCTGAGAAC-3'，Reverse(反向)：5'-ATGGTGGTGAAGACGCCAGT-3'，CD36 Forward：5'-GAGGAACTATATTGTGCCTATTCTTTGGC-3'，Reverse：5'-CATAAAAGCAACAAACATCACCACACCAAC-3'， RGS5 Forward：5'-TGCAAAGGACTTGCAGCTTTGCC-3'，Reverse：5'-TCTGGGTCTTGGCTGGTTTCTC-3'，NGFR Forward：5'-AAGCAGAACACCGTGTGCGAG-3'，Reverse：5'-TGTAATCCAACGGCCAGGGATC-3'，Nestin Forward：5'-TCAAGATGTCCCTCAGCCTGG-3'，Reverse：5'-ACTGGGAGCAAAGATCCAAGACG-3'，PDGFRβ Forward：5'-AGCCAGAGCTGGAACAGTTG-3'，Reverse：5'-CCAGAAGGGGACAGCTGATA-3' ALCAM Forward：5'-GCCTTGGATGGTATACTGTAAATTCAGC-3' Reverse：5'-GGTACATCGTCGTACTGCACAC-3' For the CD36-positive DS cells and CD36-negative DS cells sorted in Example 2, RNA was recovered from the cells using TRIzol (Invitrogen) using the instructions provided. The recovered RNA was measured for concentration using a nucleic acid quantitative device, Nanodrop (Thermo scientific). After adjusting the RNA concentration, cDNA was synthesized from RNA by High-Capacity cDNA Reverse Transcription Kits using the instructions of Applied Biosystems. The synthesized cDNA was subjected to quantitative RT-PCR in a mold using a reagent, LightCycler (trademark) FastStart DNA MasterPLUS SYBR Green (Roche), and a reaction device, LightCycler (Roche). The composition conditions are based on Roche's operating instructions. The primers used are as follows: G3PDH Forward: 5'-GCACCGTCAAGGCTGAGAAC-3', Reverse: 5'-ATGGTGGTGAAGACGCCAGT-3', CD36 Forward: 5'-GAGGAACTATATTGTGCCTATTCTTTGGC-3', Reverse: 5'-CATAAAAGCAACAAACATCACCACACCAAC-3', RGS5 Forward: 5'-TGCAAAGGACTTGCAGCTTTGCC-3', Reverse: 5'-TCTGGGTCTTGGCTGGTTTCTC-3', NGFR Forward: 5'-AAGCAGAACACCGTGTGCGAG-3', Reverse: 5'-TGTAATCCAACGGCCAGGGATC-3', Nestin Forward: 5'-TCAAGATGTCCCTCAGCCTGG- 3', Reverse: 5'-ACTGGGAGCAAAGATCCAAGACG-3', PDGFRβ Forward: 5'-AGCCAGAGCTGGAACAGTTG-3', Reverse: 5'-CCAGAAGGGGACAGCTGATA-3' ALCAM Forward: 5'-GCCTTGGATGGTATACTGTAAATTCAGC-3' Reverse: 5'-GGTACATCGTCGTACTGCACAC- 3'

使用附屬之軟體，測定各基因之表現量。再者，G3PDH係用作內部標準，於對各基因分別定量時，對使用其之對照群之cDNA量進行修正。將結果示於圖7。 The amount of expression of each gene was measured using the attached software. Further, G3PDH was used as an internal standard, and when each gene was quantified, the amount of cDNA of the control group using the same was corrected. The results are shown in Fig. 7.

實施例7：人類DS細胞之移植實驗 Example 7: Transplantation experiment of human DS cells

為了確認CD36陽性DS細胞之毛囊再生能力，使用作為毛囊再構成評價系統之貼片分析法進行評價。移植評價對象之細胞之貼片分析法係報告於非專利文獻15中，具體而言，藉由下述方法進行。 In order to confirm the hair follicle regeneration ability of CD36-positive DS cells, evaluation was carried out using a patch assay method as a hair follicle reconstitution evaluation system. The patch analysis method for transplanting cells to be evaluated is reported in Non-Patent Document 15, and specifically, by the following method.

貼片分析方法 Patch analysis method 細胞之調製 Cell modulation

自B57B6新生兒將皮膚摘出，於分散酶(1mg/ml)、膠原酶(1mg/ml)溶液中以4℃靜置一晚，使表皮與真皮分離。藉由對經分離之表皮使用Accummax(Innovative Cell Technologies)，而使表皮細胞自表皮分散。使經分散之表皮細胞通過40μm之Nylon Streiner(尼龍過濾器)(BD biosciences)，採取表皮細胞。藉由在分散酶(1mg/ml)及膠原酶(1mg/ml)溶液中對經分離之真皮進行處理，而進而進行酵素分解， 使真皮細胞分散。使經分散之真皮細胞通過40μm之Nylon Streiner，採取真皮細胞。 The skin was removed from B57B6 newborns, and allowed to stand at 4 ° C for one night in a solution of dispase (1 mg/ml) and collagenase (1 mg/ml) to separate the epidermis from the dermis. Epidermal cells were dispersed from the epidermis by using Accummax (Innovative Cell Technologies) on the isolated epidermis. The epidermal cells were taken by passing the dispersed epidermal cells through a 40 μm Nylon Streiner (BD biosciences). The enzyme is decomposed by treating the separated dermis in a solution of dispase (1 mg/ml) and collagenase (1 mg/ml). Disperse dermal cells. The dermal cells were taken by dispersing the dermal cells through a 40 μm Nylon Streiner.

細胞之計數 Count of cells

將所調製之表皮細胞及真皮細胞、及實施例2中所調製之CD36陽性DS細胞及CD36陰性DS細胞於細胞懸浮液之一部分轉移至另一管中，添加等量之錐蟲藍(Trypan blue)溶液，利用血球計算出細胞數。將該等細胞以表1中所示之組成加以混合，製成移植物。 The prepared epidermal cells and dermal cells, and the CD36-positive DS cells and CD36-negative DS cells prepared in Example 2 were transferred to another tube in one part of the cell suspension, and an equal amount of trypan blue (Trypan blue) was added. ) Solution, using the blood cells to calculate the number of cells. The cells were mixed in the composition shown in Table 1 to prepare a graft.

移植實驗 Transplant experiment

於移植前預先利用電動剃刀對異氟醚麻醉下之NOD/SCID(Nonobese Diabetic-Severe Combined Immunodeficiency Disease，非肥胖性糖尿病和重症聯合免疫缺陷)小鼠之背後進行剃毛，於背後皮膚下，紮入18G之針，然後，使用微量吸管導入上述所調製之移植物。2週後，使用立體顯微鏡SMZ 745T(Nikon公司)觀察移植部位之皮膚外觀、及自皮膚下再構成毛囊之有無等，使用DMC-GM1(Panasonic公司)進行照相攝影。將移植表1中所示之組成之移植物所得之結果示於圖8。與不含人類DS細胞之情形相比，導入有包含人類CD36陽性DS細胞之移植物之情形時，再構成毛囊之數量明顯增多，看到較大凸起之像。另一方面，於包含人類CD36陰性DS細胞之情形時，再構成毛囊之數量與不含人類DS細胞者相比，未確認到較大之變化。又，於包含人類CD36陽性DS細胞之情形時，確認到血管被誘導至再構成毛囊之周圍，血液量較多，但於包含人類CD36陰性 DS細胞之情形、與不含人類細胞之移植物之情形時，確認到幾乎未見血管之誘導，血液量較少。 Before the transplantation, the electric razor was used to shave the NOD/SCID (Nonobese Diabetic-Severe Combined Immunodeficiency Disease) mice under isoflurane anesthesia, under the skin behind the back. The 18G needle was inserted, and then the above-mentioned prepared graft was introduced using a micropipette. Two weeks later, the appearance of the skin at the transplantation site and the presence or absence of hair follicles from the skin were observed using a stereoscopic microscope SMZ 745T (Nikon Co., Ltd.), and photographing was performed using DMC-GM1 (Panasonic Corporation). The results obtained by transplanting the graft of the composition shown in Table 1 are shown in Fig. 8. When a graft containing human CD36-positive DS cells was introduced as compared with the case where human DS cells were not contained, the number of reconstituted hair follicles was significantly increased, and a larger convex image was observed. On the other hand, in the case of including human CD36-negative DS cells, the number of reconstituted hair follicles was not confirmed to be large as compared with those without human DS cells. Moreover, in the case of including human CD36-positive DS cells, it was confirmed that blood vessels were induced to re-construct around the hair follicle, and the amount of blood was large, but was negative in human CD36. In the case of DS cells and in the case of grafts containing no human cells, it was confirmed that almost no blood vessel was induced, and the amount of blood was small.

繼而，藉由與上述移植實驗相同之方法，將表2中所示之組成之細胞懸浮液移植至NOD/SCID小鼠之背後皮膚下，於移植後經過2週時，觀察移植部位，進行照相攝影。 Then, the cell suspension of the composition shown in Table 2 was transplanted under the skin behind the NOD/SCID mice by the same method as the above-described transplantation experiment, and the graft site was observed and photographed after 2 weeks after the transplantation. photography.

將結果示於圖9。為了更明確地觀察人類CD36陽性DS細胞之毛囊再生效果，導入減少了小鼠真皮細胞之數之移植物，結果為，於任一結果中，包含人類CD36陽性DS細胞之情形與包含人類CD36陰性DS細胞之情形相比，再構成毛囊之數均明顯多。根據以上結果，顯示人類CD36陽性DS細胞係具有促進毛囊之再構成之能力即促進毛囊再生之能力之細胞群體。 The results are shown in Fig. 9. In order to more clearly observe the hair follicle regeneration effect of human CD36-positive DS cells, a graft having a reduced number of mouse dermal cells was introduced, and as a result, in any of the results, human CD36-positive DS cells were included and contained in human CD36-negative Compared with the case of DS cells, the number of reconstituted hair follicles was significantly higher. Based on the above results, it was revealed that the human CD36-positive DS cell line has a cell population that promotes the reconstitution of hair follicles, that is, the ability to promote hair follicle regeneration.

<110> 日商資生堂股份有限公司 <110> Japanese Business Shiseido Co., Ltd.

<120> 控制成型毛囊組成之質量的方法 <120> Method for controlling the quality of molded hair follicles

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<400> 14 <400> 14

Claims (6)

一種品質管理方法，其係包含源自真皮毛根鞘(DS)之CD36陽性細胞之毛囊形成用組合物之品質管理方法，且包括：取得組合物中之一部分或全部之細胞群；及於以DS細胞中之CD36之表現作為指標且CD36表現高於特定值之情形時，決定該組合物之毛囊誘導能力。 A quality management method comprising a quality management method for a hair follicle forming composition derived from CD36 positive cells of a dermal root sheath (DS), and comprising: obtaining a part or all of a cell population of the composition; The hair follicle inducing ability of the composition is determined when the expression of CD36 in the cells is used as an index and the CD36 expression is higher than a specific value. 如請求項1之品質管理方法，其中上述指標為DS細胞中之CD36陽性DS細胞之比率，且於該比率為特定值以上之情形時，決定具有毛囊誘導能力。 The quality management method according to claim 1, wherein the indicator is a ratio of CD36-positive DS cells in the DS cells, and when the ratio is a specific value or more, it is determined to have a hair follicle inducing ability. 如請求項2之品質管理方法，其包括：於DS細胞中之CD36陽性DS細胞之比率為特定值以下之情形時，對上述組合物實施對CD36陽性DS細胞進行篩選之細胞分選，而取得特定值以上之CD36陽性DS細胞。 The quality management method of claim 2, which comprises: performing cell sorting on CD36-positive DS cells by using the above composition in a case where the ratio of CD36-positive DS cells in the DS cells is below a specific value; CD36-positive DS cells with specific values above. 如請求項1至3中任一項之品質管理方法，其包括：於進而以鹼性磷酸酶活性作為指標且鹼性磷酸酶活性高於特定值之情形時，決定該組合物具有毛囊誘導能力。 The quality management method according to any one of claims 1 to 3, which comprises determining that the composition has hair follicle inducing ability when the alkaline phosphatase activity is further used as an index and the alkaline phosphatase activity is higher than a specific value. . 如請求項1至4中任一項之品質管理方法，其包括：於進而以DS細胞中之間葉系幹細胞相關因子表現作為指標且表現高於特定值之情形時，決定該組合物具有高毛囊誘導能力。 The quality management method according to any one of claims 1 to 4, which comprises determining that the composition has a high level in the case where the expression of the leaf cell stem cell-related factor in the DS cell is used as an index and the expression is higher than a specific value. Hair follicle induction ability. 如請求項1至5中任一項之品質管理方法，其中上述源自真皮毛根鞘之細胞為源自真皮毛根鞘杯之細胞。 The quality management method according to any one of claims 1 to 5, wherein the cell derived from the dermal root sheath is a cell derived from a dermal sheath cup.
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