WO2016086527A1 - 含有皮脂腺的皮肤组织及其形成方法和用途 - Google Patents

含有皮脂腺的皮肤组织及其形成方法和用途 Download PDF

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WO2016086527A1
WO2016086527A1 PCT/CN2015/071777 CN2015071777W WO2016086527A1 WO 2016086527 A1 WO2016086527 A1 WO 2016086527A1 CN 2015071777 W CN2015071777 W CN 2015071777W WO 2016086527 A1 WO2016086527 A1 WO 2016086527A1
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cells
stem cells
skin
dermal
final concentration
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French (fr)
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吴耀炯
王潇潇
王旭升
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清华大学深圳研究生院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin

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  • the present invention relates to the field of cell biology and regenerative medicine, and in particular to a method of forming a sebaceous gland and its use, and more particularly to a method of forming a sebaceous gland, a skin tissue containing a sebaceous gland, and a method of forming a sebaceous gland for use in skin regeneration.
  • the skin As the largest tissue of the human body, the skin is a barrier to contact with the external environment. When external skin damage or diseases cause skin defects, skin damage and infection are often caused. China has a large population, with 15 million burns and ulcers per year, of which 3.5 million are required for skin grafts and more than 400 million square centimeters. Therefore, finding an ideal skin substitute has always been a clinically urgent problem. With the rapid development of cell biology, materials science, biochemistry, bioengineering, and transplantation, people have a deeper understanding of the biological characteristics of epidermal keratinocytes, the processing and synthesis of artificial materials, and so on. It is possible to build tissue engineering skin.
  • the sebaceous gland is the most important accessory organ for maintaining the stability of skin structure. Sebaceous glands and sweat and exfoliated epithelial cells form a layer of sebum sweat latex film on the surface of the skin. Its thickness is 7-10 ⁇ m, which is a three-dimensional lipid structure on the skin surface. Promotes the integrity of the skin barrier. In the absence of this three-dimensional cortical structure, rough skin and dry hair will appear. Since some skins of the palms, ankles, fingers, and toes do not have sebaceous glands, skin dryness often occurs.
  • tissue engineering skin has a dermis and epidermis similar to normal skin, it lacks sebaceous glands and cannot provide nutrient and moisturizing, antibacterial and anti-oxidant protection and moisture retention. After skin transplantation, skin shrinkage and deformation are serious and appearance is easy. Problems such as chapped and susceptible to infection, while the performance of tissue-engineered skin is significantly lower than that of natural normal skin.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a method of forming a sebaceous gland.
  • epidermal stem cells derived from the epidermis of the skin and dermal cells derived from the dermis of the skin have a large potential for expansion, and after isolation and culture in vitro, epidermal stem cells are characterized by high expression of integrin CD29, CD49f and K15 ( Keratin 15), and the characteristic marker molecule expression of two stem cells in subsequent passages stable.
  • the inventors amplified the above two cells in different culture systems, and then transplanted the two cells to the skin of nude mice in proportion, and histological analysis revealed a newly formed sebaceous gland structure.
  • the invention provides a method of forming a sebaceous gland.
  • the method comprises co-culturing dermal cells and epidermal stem cells in a gel.
  • the inventors have surprisingly found that the method can form skin tissue with sebaceous glands.
  • the present application forms a method of skin tissue having sebaceous glands, which is simple, convenient, quick, easy to control, and easy to achieve large-scale production.
  • the method of sebaceous gland according to the above embodiment of the present invention may further have the following additional technical features:
  • the co-cultivation is carried out subcutaneously in the animal.
  • the regeneration of the skin tissue of the sebaceous glands is promoted, and the structure of the regenerated skin tissue is similar to that of normal skin.
  • the dermal cells comprise at least one of dermal stem cells and fibroblasts.
  • the dermal cells are fibroblasts
  • the gel contains insulin, dexamethasone, glitazone and XAV939 (3,5,7,8-tetrahydro-2-[ 4-(Trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one).
  • the chemical agent induces differentiation of epidermal stem cells into free sebaceous glands, thereby forming a sebaceous gland structure similar to normal skin.
  • the final concentration of the insulin is 50-150 ⁇ g/mL
  • the final concentration of the dexamethasone is 10 -4 -10 -6 M
  • the final concentration of the glitazone is 150-250 ⁇ M
  • the end of the XAV939 The concentration is 5-15 ⁇ M.
  • the chemical agent induces the differentiation of epidermal stem cells and fibroblasts into free sebaceous glands.
  • the final concentration of the insulin is 100 ⁇ g / mL
  • the final concentration of the dexamethasone is 10 -5 M
  • the final concentration of the glitazone is 200 ⁇ M
  • the final concentration of the XAV939 is 10 ⁇ M.
  • the chemical agent induces the differentiation of epidermal stem cells and fibroblasts into free sebaceous glands.
  • final concentration refers to the concentration of the chemical agent in the gel after adding various chemical agents to the gel containing the mixed cells.
  • the ratio of the dermal cells to the epidermal stem cells is 10:1 to 1:10.
  • the number of mixed cells is not less than 10 4 per 3 mm 2 of skin.
  • the number of mixed cells satisfies the need for regeneration of sebaceous gland skin tissue.
  • the gel is at least one selected from the group consisting of Matrigel, collagen or hydrogel.
  • the dermal cells and the epidermal stem cells are all derived from autologous, allogeneic or xenogeneic cells. It should be noted that the xenogenic-derived cells are used for the regeneration of sebaceous glands in immunodeficient animals. Thereby, the regenerated sebaceous gland skin tissue has a small rejection effect.
  • the dermal cells and the epidermal stem cells are independently cultured in a liquid medium for 0 to 30 days, wherein the dermal stem cells are suspension culture, and the cell culture medium is DMEM/F12. (3:1), 2% B27, 40 ng/ ⁇ L bFGF and 20 ng/ ⁇ L EGF; epidermal stem cells are adherent culture, and the cell culture medium is CnT-07 medium; wherein the dermal stem cells further contain liquid medium a cell active factor and an extracellular matrix molecule, wherein the cell active factor is at least one selected from the group consisting of bFGF and EGF, and the extracellular matrix molecule is at least one selected from the group consisting of hyaluronic acid and fibronectin.
  • the dermal stem cells and epidermal stem cells have high activity and small cell variation.
  • the method further comprises: identifying the oil secretion of the surface of the skin tissue containing the regenerated sebaceous glands using in situ mass spectrometry. Thereby, the secretion of oil on the surface of the skin tissue containing the regenerated sebaceous glands can be more accurately identified.
  • the present invention also provides a skin tissue containing a sebaceous gland.
  • the sebaceous glands are formed by the method described in the foregoing embodiments of the invention. The inventors have surprisingly found that the skin tissue has a structure of sebaceous glands similar to normal skin tissue.
  • the invention also provides for the use of the aforementioned method in skin regeneration.
  • the method is for regenerating a sebaceous gland or a skin tissue containing a sebaceous gland. The inventors have surprisingly found that by applying this method in skin regeneration, sebaceous glands similar to normal skin can be obtained.
  • FIG. 1 shows a photograph of the expression result of the cell surface marker molecule CD49f of epidermal stem cells according to an embodiment of the present invention
  • FIG. 2 shows a photograph of the expression result of the cell surface marker Nestin of dermal stem cells according to an embodiment of the present invention
  • Figure 3 shows a photograph of confocal microscopy of normal skin tissue sections in accordance with one embodiment of the present invention
  • FIG. 4 is a photograph showing confocal microscopic observation of a skin tissue section formed by co-transplantation of epidermal stem cells and dermal stem cells according to an embodiment of the present invention
  • Figure 5 is a photograph showing confocal microscopic observation of skin tissue sections formed by co-transplantation of epidermal stem cells and fibroblasts after induction of co-transplantation with chemical agents according to an embodiment of the present invention
  • Figure 6 is a graph showing the results of sebaceous gland secretion detected by MALDI mass spectrometry in skin tissue formed after transplantation according to one embodiment of the present invention.
  • ex vivo skin stem cells are isolated and cultured from the skin of human C57 mice or human scalp tissues in the following examples;
  • the dermal stem cell culture medium is composed of DMEM/F12 (3: 1) (purchased from Invitrogen) containing 2% B27 (purchased from Invitrogen), 40 ng/ ⁇ L bFGF (Peprotech) and 20 ng/ ⁇ L EGF (Peprotech);
  • fibroblast culture medium is DMEM containing 10% fetal bovine serum Accutase was purchased from sigma; 3M membrane was Tegaderm TM Film; suspension cell culture dish was purchased from Guangzhou Jiete Biofiltration Products Co., Ltd., and the product number was MCD-000-090.
  • Adherent cell culture dish purchased from Corning. Matrigel was purchased from BD. The experimental animals were purchased from the Guangdong Medical Laboratory Animal Center.
  • Identification of epidermal stem cells The identification of cell surface markers is as follows: Take the first, third, and fifth representative skin cells for cell climbing, and perform immunofluorescence staining of integrin CD29, CD49f and K15. The coloration of the cells. It was observed that the integrin CD29, CD49f and K15 of epidermal stem cells were highly expressed, and the specific results are shown in Fig. 1.
  • the primary cells of the dermal stem cells were passed through a 200 mesh sieve, and then centrifuged at 300 ⁇ g for 10 min, and the supernatant was discarded, and the primary cells were suspended in the dermal stem cell culture medium.
  • the dermal stem cell culture medium suspension primary cells were seeded in an non-adherent culture dish.
  • dermal stem cells to induce epidermal stem cells to form sebaceous gland structures, the specific process is as follows:
  • Example 1 The epidermal stem cells obtained in Example 1 were cultured, and the third representative skin stem cells were digested with Accutase, and a suspension of epidermal stem cells having a total cell number of 1 million was prepared in PBS.
  • Example 2 The dermal stem cells obtained in Example 2 were cultured, and a total of 1 million cell suspensions of the third generation dermal stem cells were prepared in PBS.
  • BALB/c nude mice were anesthetized with 1% sodium pentobarbital, and a wound having an area of about 7 mm 2 was made on the skin of the back with a puncher.
  • Example 1 The epidermal stem cells obtained in Example 1 were cultured, and the third representative skin stem cells were digested with Accutase, and a cell suspension of 2 million was prepared in PBS.
  • mice were anesthetized with 1% sodium pentobarbital, and a wound having an area of about 7 mm 2 was made on the skin of the back with a puncher.
  • Example 3 and Example 4 The skin tissue regions of the stem cells transplanted in the experimental group and the control group in Example 3 and Example 4 were respectively cut out, fixed in 4% paraformaldehyde overnight, immersed in PBS for 3 hours, and dehydrated in 30% sucrose for 8 hours.
  • Tissue sections were permeabilized for 10 minutes with 0.2-0.5% triton X-100 (in PBS).
  • Tissue sections were blocked with 2% BSA for 30 minutes and then washed twice with PBS.
  • the tissue dyeing sample with good sealing is placed in a 4 degree refrigerator and can be stored for more than 2 weeks.
  • the method for identifying the oil secretion ability of the sebaceous glands of the skin tissue after transplantation in Example 4 by in situ mass spectrometry is specifically as follows:
  • mice transplanted with the stem cell skin of Example 4 were anesthetized with 1% sodium pentobarbital, and the back skin was scrubbed with 100% ethanol to wash off all the oily substances on the skin surface.
  • tissue sections were pretreated by a 50% ACN aqueous solution containing 0.1% TFA to prepare a 15 g/L CHCA matrix solution.
  • the pump was used to spray 100 ⁇ l of the substrate solution evenly on the surface of the tissue at 5 ⁇ l/s.
  • the substrate solution was sprayed again and circulated twice.
  • tissue section of the sprayed substrate was placed at room temperature, and the solvent was evaporated to precipitate crystals, which were then dried in a desiccator for 2 hours.
  • the dried tissue sections were attached to a MALDI stainless steel target plate with a conductive tape and sent to a mass spectrometer for analysis.
  • Mass spectrometry conditions Randomly take 3 points of each tissue section, add 1000 scan data to each point to obtain the mass spectrum, and add 3 points to obtain the total mass spectrum. Detection by positive ion reflection mode, mass scanning range m/z 1 00-1 000; positive ion linear mode detection, mass scanning range m/z 100-2000.
  • the results are shown in Fig. 6.
  • the red line represents the oil secreted by the normal sebaceous gland
  • the green line represents the oil secreted by the regenerated sebaceous gland.
  • the mass spectrometry results show that the regenerated sebaceous gland has a similar oil secretion capacity as the normal sebaceous gland.
  • the method for forming sebaceous glands of the present invention can effectively form skin tissue having sebaceous glands, and the method is simple, convenient, quick, easy to control, and easy to achieve large-scale production.

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Abstract

提供了含有皮脂腺的皮肤组织及其形成方法和用途，其中，形成皮脂腺的方法包括：在凝胶中对真皮细胞和表皮干细胞进行共培养。

Description

含有皮脂腺的皮肤组织及其形成方法和用途
优先权信息
本申请请求2014年12月02日向中国国家知识产权局提交的、专利申请号为201410723513.5的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及细胞生物学和再生医学领域，具体地，涉及形成皮脂腺的方法及其应用，更具体地，涉及形成皮脂腺的方法，含有皮脂腺的皮肤组织以及形成皮脂腺的方法在皮肤再生中的用途。
背景技术
皮肤作为人体最大的组织，是与外界环境接触的屏障，当外界损伤或疾病等因素造成皮肤缺损时，常常造成皮肤的损伤和感染。我国人口众多，每年烧伤与溃疡患者达到1500万人，其中需要进行皮肤移植的病例在350万人，皮肤需求量在4亿平方厘米以上。因此，寻找一种理想的皮肤代用品一直是临床上一个急需解决的难题。随着细胞生物学、材料学、生物化学、生物工程学、移植学的飞速发展，人们对表皮角质形成细胞的生物学特性、人工材料的加工与合成等有了更深入的认识，使在体外构建组织工程皮肤成为可能。
皮脂腺是维护皮肤结构功能稳定最重要的附属器官，因为皮脂腺与汗液以及脱落的上皮细胞等在皮肤表面形成一层皮脂汗液乳胶膜，其厚度7-10μm，其为皮肤表面三维脂质结构，可以促进皮肤屏障的完整性。如果缺少这层三维皮质结构，将会出现皮肤粗糙和毛发枯槁。由于手掌、足跖和手指、足趾的部分皮肤因为没有皮脂腺，所以经常出现皮肤干裂现象。而现在的组织工程皮肤虽然有和正常皮肤类似的真皮和表皮层，但缺乏皮脂腺，无法为皮肤提供营养滋润、抗菌抗氧化的保护以及水分保持，皮肤移植后，出现皮肤收缩变形严重、外表易干裂及易感染的等问题，同时组织工程皮肤的性能明显低于天然正常皮肤。
然而，现有的组织工程皮肤仍有待改进。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种形成皮脂腺的方法。
需要说明的是，本发明是基于发明人的下列工作而完成的：
发明人发现，来源于皮肤表皮的表皮干细胞和来源于皮肤真皮的真皮细胞，均具有大量扩增的潜力，并且在体外分离培养后，表皮干细胞的特征为高表达整合素CD29，CD49f以及K15(角质蛋白15)，而且在后续的传代中两种干细胞的特征标记性分子表达 稳定。发明人将上述两种细胞分别在不同的培养体系中进行扩增，然后将两种细胞按比例移植到裸鼠皮肤，经组织学分析，发现有新形成的皮脂腺结构。
因而，根据本发明的一个方面，本发明提供了一种形成皮脂腺的方法。根据本发明的实施例，该方法包括：在凝胶中对真皮细胞和表皮干细胞进行共培养。发明人惊奇的发现，该方法可以形成具有皮脂腺的皮肤组织。根据本发明的实施例，本申请形成具有皮脂腺的皮肤组织的方法，操作简单、方便快捷、容易控制、易于实现规模化生产。
另外，根据本发明上述实施例的成皮脂腺的方法，还可以具有如下附加的技术特征：
根据本发明的实施例，所述共培养是在动物的皮下进行的。由此，促进皮脂腺的皮肤组织的再生，并且再生的皮肤组织的结构与正常皮肤类似。
根据本发明的实施例，所述真皮细胞包括真皮干细胞和成纤维细胞的至少之一。
根据本发明的实施例，所述真皮细胞为成纤维细胞，并且在所述凝胶中含有胰岛素、***、格列酮和XAV939(3,5,7,8-四氢-2-[4-(三氟甲基)苯基]-4H-噻喃并[4,3-d]嘧啶-4-酮)。由此，化学试剂诱导表皮干细胞分化为自由皮脂腺，进而形成与正常皮肤类似的皮脂腺结构。
所述胰岛素的终浓度为50-150μg/mL，所述***的终浓度为10-4-10-6M，所述类格列酮的终浓度为150-250μM，所述XAV939的终浓度为5-15μM。由此，化学试剂诱导表皮干细胞和成纤维细胞分化为自由皮脂腺的效果好。
根据本发明实施例，所述胰岛素的终浓度为100μg/mL，所述***的终浓度为10-5M，所述类格列酮的终浓度为200μM，所述XAV939的终浓度为10μM。由此，化学试剂诱导表皮干细胞和成纤维细胞分化为自由皮脂腺的效果更佳。
其中，需要说明的是，本申请中所使用的术语“终浓度”是指将各种化学试剂加入含混合细胞的凝胶后，该化学试剂在凝胶中的浓度。
根据本发明的实施例，所述真皮细胞和所述表皮干细胞混合的数量比为10:1～1:10。
根据本发明的实施例，对于每3mm2皮肤，混合细胞的数量不少于104个。由此，混合细胞的数量满足皮脂腺皮肤组织再生的需求。
根据本发明的实施例，所述凝胶为选自Matrigel、胶原或者水凝胶的至少一种。
根据本发明的实施例，所述真皮细胞和所述表皮干细胞均为来源于自体、同种异体或者异种的细胞。其中需要说明的是，所述来源于异种的细胞用于免疫缺陷动物的皮脂腺再生。由此，再生的皮脂腺皮肤组织排异作用小。
根据本发明的实施例，所述真皮细胞和所述表皮干细胞分别独立地预先在液体培养基中被贴壁培养0～30代，其中，真皮干细胞为悬浮培养，细胞培养基组成为DMEM/F12(3:1)，2％B27，40ng/μL bFGF和20ng/μL EGF；表皮干细胞为贴壁培养，细胞培养基为CnT-07培养基；其中，所述真皮干细胞的液体培养基中进一步含有细胞活性因子和细胞外基质分子，其中所述细胞活性因子为选自bFGF、EGF的至少一种，所述细胞外基质分子为选自透明质酸、纤维粘连蛋白的至少一种。由此，真皮细胞和表皮干细胞的活性高，细胞变异小。
根据本发明的实施例，该方法进一步包括：利用原位质谱鉴定含再生皮脂腺的皮肤组织表面的油脂分泌。由此，能够更加精确的鉴定所述含再生皮脂腺的皮肤组织表面的油脂分泌情况。
根据本发明的另一方面，本发明还提供了一种含有皮脂腺的皮肤组织。根据本发明的实施例，所述皮脂腺是通过前述的本发明实施例的所述的方法形成的。发明人惊奇地发现，该皮肤组织具有与正常皮肤组织类似的皮脂腺的结构。
根据本发明的再一方面，本发明还提够了前述的方法在皮肤再生中的用途。根据本发明实施例，所述方法用于再生皮脂腺，或含有皮脂腺的皮肤组织。发明人惊奇地发现，在皮肤再生中应用该方法，可以获得具有与正常皮肤类似的皮脂腺。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的表皮干细胞的细胞表面标志分子CD49f的表达结果的图片；
图2显示了根据本发明一个实施例的真皮干细胞的细胞表面标志Nestin的表达结果的图片；
图3显示了根据本发明一个实施例的正常皮肤组织切片的共聚焦显微镜观察的图片；
图4显示了根据本发明一个实施例的由表皮干细胞和真皮干细胞共同移植后形成的皮肤组织切片的共聚焦显微镜观察的图片；
图5显示了根据本发明一个实施例的由表皮干细胞和成纤维细胞混合后用化学试剂诱导共同移植后形成的皮肤组织切片的共聚焦显微镜观察的图片；
图6显示了根据本发明一个实施例的MALDI质谱检测移植后形成的皮肤组织的皮脂腺分泌结果的图片。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
本申请的实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
下面详细描述本发明的实施例，如无特殊说明，下述实施例中离体皮肤干细胞从C57小鼠的皮肤或人的头皮组织分离并培养；真皮干细胞培养基组成为DMEM/F12(3:1) (购买于Invitrogen公司)，含2％B27(购买于Invitrogen公司)，40ng/μL bFGF(Peprotech)和20ng/μL EGF(Peprotech)；成纤维细胞的培养基为DMEM含10％胎牛血清；Accutase购于sigma；3M膜为TegadermTM Film；悬浮细胞培养皿，购自于广州杰特生物过滤制品有限公司，货号为MCD-000-090。贴壁细胞培养皿，购自于Corning公司。Matrigel购自于BD公司。实验动物均购自于广东省医学实验动物中心。
实施例1
表皮干细胞的分离和培养及鉴定过程具体如下：
(1)取胎鼠全层皮肤，加入质量浓度0.25％dispase II酶4℃过夜。
(2)剥离胎鼠的皮肤表皮，将表皮剪碎后加入质量浓度0.25％胶原酶，37℃消化30min后终止反应，获得消化后的表皮干细胞的原代细胞。
(3)将获得的原代细胞过80目筛网，然后300×g离心10min，弃上清后以CnT-07(由CELLnTEC公司生产)培养基悬浮细胞。
(4)将CnT-07悬浮的原代细胞接种于铺有小鼠I型胶原的10CM细胞培养板上，孵育60min后，以0.01mol/L HBSS洗涤细胞2次，再加入CnT-07培养。
(5)待原代细胞长至60％～80％融合时，用Accutase酶消化，然后按1∶2比例传代。
(6)表皮干细胞的鉴定：即细胞表面标志物的鉴定，方法如下：取第1、3、5代表皮干细胞做细胞爬片，对其进行整合素CD29和CD49f以及K15的免疫荧光染色，观察细胞的着色情况。观察发现，表皮干细胞的整合素CD29，CD49f和K15均高表达，具体结果如图1所示。
实施例2
真皮干细胞的分离和培养及鉴定过程具体如下：
(1)取胎鼠全层皮肤，将皮肤剪碎后，加入质量浓度0.25％dispase酶4℃过夜。
(2)剥离胎鼠的表皮，分离真皮，并将真皮剪碎，加入质量浓度0.25％胰蛋白酶，37℃消化20～40min后终止反应，获得消化后的真皮干细胞的原代细胞。
(3)将真皮干细胞的原代细胞过200目筛网，然后，300×g离心10min，弃上清后，以真皮干细胞培养基悬浮原代细胞。
(4)将真皮干细胞培养基悬浮原代细胞接种于不贴壁培养皿中。
(5)待不贴壁培养皿中出现较多细胞球后，将真皮干细胞按1∶2的比例传代。
(6)真皮干细胞的鉴定：即细胞表面标志物的鉴定，具体方法如下：取第1、3、5代真皮干细胞做细胞甩片，对真皮干细胞的Nestin和Fibronectin进行免疫荧光染色，观察细胞的着色情况。免疫荧光分析发现，真皮干细胞的Nestin和Fibronectin均高表达，其中，Nestin阳性的结果如图2所示。
实施例3
利用真皮干细胞诱导表皮干细胞形成皮脂腺结构，具体过程如下：
(1)培养实施例1中获得的表皮干细胞，将第3代表皮干细胞用Accutase酶消化后，制备总细胞数100万的表皮干细胞悬液于PBS中。
(2)培养实施例2中获得的真皮干细胞，制备第3代真皮干细胞总细胞数100万细胞悬液于PBS中。
(3)将(1)和(2)中的两种细胞悬液混合，300g离心5min。
(4)取离心后的细胞，弃上清，在细胞沉淀中加入10微升Matrigel，混匀，待用。
(5)将BALB/c裸鼠用1％的戊巴比妥钠麻醉，用打孔器在其背部皮肤上制造面积约7mm2的伤口。
(6)将步骤(4)中制备获得的混有皮肤干细胞的Matrigel移植到裸鼠皮肤伤口表面。
(7)用3M膜覆盖裸鼠伤口表面，然后用绷带将裸鼠伤口包扎。
(8)正常饲养裸鼠，3周后拆开绷带去除3M膜，切取细胞移植的皮肤组织，并固定染色后，观察皮脂腺结构，发现有大量皮脂腺结构形成。
实施例4
利用化学试剂诱导表皮干细胞形成皮脂腺结构，具体过程如下：
(1)培养实施例1中获得的表皮干细胞，将第3代表皮干细胞用Accutase酶消化后，制备200万的细胞悬液于PBS中。
(2)将成纤维细胞用Accutase酶消化后，制备200万的细胞悬液于PBS中。
(3)将(1)和(2)中的两种细胞悬液混合，并平均分成2份，一份作为实验组，另一份作为对照组，300g，离心5min。
(4)将上述2份离心后的细胞弃上清，分别在细胞沉淀中加入10微升Matrigel，混匀。
(5)在实验组的Matrixgel中加入适量的胰岛素，***，类格列酮以及XAV939，使其最终浓度为：胰岛素100μg/mL，***10-5M，类格列酮200μM，XAV939 10μM，将上述化学试剂充分吹吸混匀。对照组不添加上述化学试剂。
(6)将BALB/c裸鼠用1％的戊巴比妥钠麻醉，用打孔器在其背部皮肤上制造面积约7mm2的伤口。
(7)将步骤(5)中制备获得的2组混有皮肤干细胞的Matrigel分别移植到裸鼠皮肤伤口表面。
(8)用3M膜覆盖裸鼠伤口表面，然后用绷带将裸鼠伤口包扎。
(9)正常饲养裸鼠，3周后拆开绷带去除3M膜，切取细胞移植的皮肤组织，并固定染色后，观察皮脂腺结构。发现实验组有大量皮脂腺结构形成而对照组只有很少量的皮脂腺结构形成。
实施例5
对实施例3和实施例4中实验组和对照组的干细胞移植的皮肤组织进行组织形态学鉴定，具体如下：
1、制备表皮干细胞再生皮脂腺结构组织切片：
(1)分别切取实施例3和实施例4中实验组和对照组的干细胞移植的皮肤组织区域，4％多聚甲醛固定过夜，PBS浸泡3小时，30％蔗糖脱水8小时。
(2)OCT包埋组织，冰冻切片，取10μm厚度的组织切片。
(3)将组织切片自然晾干。
(4)将组织切片用PBS洗三遍，每次5分钟。
(5)用0.2-0.5％triton X-100(PBS配制)对组织切片做透膜处理10分钟。
(6)将透膜处理后的组织切片用PBS洗三遍，每次5分钟。
(7)将组织切片用2％BSA封闭30分钟，然后用PBS洗两遍。
(8)将封闭后的组织切片加入抗生物素Cy3标记的1抗，室温孵育3小时。
(9)将连接1抗的组织切片用PBS洗三遍，每次5分钟。
(10)加入0.5μg/ml DAPI(PBS配制)染色10分钟。
(11)将染色后组织切片用PBS洗三遍，去除多余的DAPI。
(12)将去除多余的DAPI的组织切片加入20μl封片剂封片。
(13)封片好的组织染色样品置于4度冰箱中，可存放2周以上。
2、组织切片的组织形态学鉴定：
采用共聚焦显微镜观察组织切片，观察发现，正常皮肤的皮脂腺，免疫荧光染色显示其有大量生物素(Biotin)合成，细胞呈现为空泡状结构；实施例3的再生皮肤组织中有皮脂腺结构的形成，其结构形态与正常皮肤中的皮脂腺类似；实施例4中有化学试剂诱导的组织(即实验组)有大量生物素合成的皮脂腺结构，而对照组几乎没有类似的结构形成，具体结果如图3-5所示。
实施例6
利用原位质谱鉴定实施例4中移植后的皮肤组织的皮脂腺的油脂分泌能力的方法具体如下：
(1)将实施例4中干细胞皮肤移植的BALB/c裸鼠用1％的戊巴比妥钠麻醉，用100％的乙醇擦洗其背部皮肤，使其皮肤表面的油脂类物质全部洗掉。
(2)分别切取实验组和对照组的细胞移植后的皮肤组织，用皮肤活检器切取2mm直径的皮肤组织，其中包括正常皮肤和干细胞移植皮肤，将该组织片置于37℃的培养箱中孵育2小时，使其组织内的皮脂向皮肤表面分泌。
(3)利用原位质谱鉴定上述皮肤组织表面油脂的分泌前，需对组织切片进行预处理，其过程为用含0.1％TFA的50％ACN水溶液配制成15g/L的CHCA基质溶液，用蠕动泵以5μl/s推动100μl基质溶液均匀喷洒在组织表面上，待组织表面溶液将干时，再次喷洒基质溶液，循环2次。
(4)将喷好基质的组织切片放在室温下，待溶剂挥发析出结晶后放入干燥器中干燥2h。
(5)干燥后的组织切片用导电胶带贴在MALDI不锈钢靶板上，送入质谱仪分析。
(6)质谱条件随机取每片组织切片3个点，每个点累加1000次扫描数据得到质谱图，3个点累加后得到总质谱图。用正离子反射式模式检测，质量扫描范围m/z 1 00-1 000；正离子线性模式检测，质量扫描范围m/z 100-2000。
结果如图6所示，其中红线代表正常皮脂腺分泌的油脂，绿线代表再生皮脂腺分泌的油脂，质谱结果表明，再生皮脂腺有和正常皮脂腺类似的油脂分泌能力。
工业实用性
本发明的形成皮脂腺的方法，能够有效地形成具有皮脂腺的皮肤组织，并且该方法操作简单、方便快捷、容易控制、易于实现规模化生产。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (14)

  1. 一种形成皮脂腺的方法，其特征在于，包括：
    在凝胶中对真皮细胞和表皮干细胞进行共培养。
  2. 根据权利要求1的方法，其特征在于，所述共培养是在动物的皮下进行的。
  3. 根据权利要求1所述的方法，其特征在于，所述真皮细胞包括真皮干细胞和成纤维细胞的至少之一。
  4. 根据权利要求3所述的方法，其特征在于，所述真皮细胞为成纤维细胞，并且在所述凝胶中含有胰岛素、***、格列酮和XAV939。
  5. 根据权利要求4所述的方法，其特征在于，所述胰岛素的终浓度为50-150μg/mL，所述***的终浓度为10-4-10-6M，所述类格列酮的终浓度为150-250μM，所述XAV939的终浓度为5-15μM。
  6. 根据权利要求5所述的方法，其特征在于，所述胰岛素的终浓度为100μg/mL，所述***的终浓度为10-5M，所述类格列酮的终浓度为200μM，所述XAV939的终浓度为10μM。
  7. 根据权利要求1所述的方法，其特征在于，所述真皮细胞和所述表皮干细胞混合的数量比为10:1～1:10。
  8. 根据权利要求2所述的方法，其特征在于，对于每3mm2皮肤，混合细胞的数量不少于104个。
  9. 根据权利要求1所述的方法，其特征在于，所述凝胶为选自Matrigel、胶原或者水凝胶的至少一种。
  10. 根据权利要求1所述的方法，其特征在于，所述真皮细胞和所述表皮干细胞均为来源于自体、同种异体或者异种的细胞。
  11. 根据权利要求1所述的方法，其特征在于，所述真皮细胞和所述表皮干细胞分别独立地预先在液体培养基中培养0～30代，其中，真皮干细胞为悬浮培养，细胞培养基组成为DMEM/F12(3:1)，2％B27，40ng/μL bFGF和20ng/μL EGF；表皮干细胞为贴壁培养，细胞培养基为CnT-07培养基；其中，所述真皮干细胞的液体培养基中进一步含有细胞活性因子和细胞外基质分子，其中所述细胞活性因子为选自bFGF、EGF的至少一种，所述细胞外基质分子为选自透明质酸、纤维粘连蛋白的至少一种。
  12. 根据权利要求1所述的方法，其特征在于，进一步包括：利用原位质谱鉴定含再生皮脂腺的皮肤组织表面的油脂分泌。
  13. 一种含有皮脂腺的皮肤组织，其特征在于，所述皮脂腺是通过权利要求1-12任一项所述的方法形成的。
  14. 权利要求1-12任一项所述的方法在皮肤再生中的用途。
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