构建有毛发生长能力的组织工程皮肤的研究

发布时间：2018-05-25 07:58

本文选题：组织工程皮肤 + 毛乳头细胞　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景皮肤是人体面积最大的器官,覆盖全身,发挥着保护、感觉、调节体温、分泌与排泄、吸收、代谢、免疫等作用。作为人体的第一道屏障,皮肤直接与外界接触,各种物理、化学、生物等因素都能引起皮肤的损伤,不及时有效治疗会给患者带来极大痛苦,甚至威胁生命。对于皮肤缺损,传统的治疗方法是进行皮肤移植或皮瓣移植术,但存在供体不足和免疫排斥等问题。组织工程皮肤(Tissue Engineering Skin,TES)是将种子细胞与细胞外基质替代物混合制成的人工皮肤,可用于创面修复,重建皮肤功能,具有良好的发展前景。现在已能制备比较成熟的表皮-真皮组织工程皮肤,基本能够快速覆盖创面,但是缺乏毛囊等皮肤附属器。毛囊含有丰富的毛囊干细胞,在皮肤创伤的修复、重建中有重要作用。毛囊所生长出的毛发除了调节体温、分泌与排泄等基本功能之外,对于人类还有影响美观,进而进一步影响心理及社交的作用。构建有毛发生长能力的组织工程皮肤不但能促进皮肤损伤修复,还可以重建皮肤功能,而且对于脱发等疾病有一定治疗作用。研究目的1.观察毛囊胚胎发育及毛囊干细胞动态衍变过程,探讨毛囊的发生、发展过程,为寻找有诱导毛发形成能力的种子细胞提供依据。2.掌握体外构建表皮-真皮组织工程皮肤的技术及明确移植后的体内转归,探讨表皮-真皮组织工程皮肤的皮肤缺损修复以及毛发生长能力。3.寻找具有诱导毛发形成能力的种子细胞,明确其能维持诱导能力的培养方式及构建方式,为构建有毛发生长能力的组织工程皮肤奠定基础。4.构建有毛发生长能力的组织工程皮肤并修复皮肤缺损。研究方法1.以C57BL/6不同胎龄胎鼠的皮肤为模型,通过HE染色观察毛囊胚胎发育过程,免疫荧光染色标记相关毛囊干细胞并观察毛囊干细胞动态衍变过程。2.分离、培养儿童包皮表皮细胞及真皮成纤维细胞,以自制SD大鼠鼠尾胶原为基质,应用气-液面培养,构建表皮-真皮组织工程皮肤。制备大鼠背部急性全层皮肤损伤模型,将构建的表皮-真皮组织工程皮肤覆盖伤口,观察其体内转归。将原代培养的成人毛乳头细胞与成人毛囊干细胞以上述方式制备组织工程皮肤,并进行体内移植观察其转归。3.将新鲜分离的C57BL/6新生鼠(24小时以内)真皮细胞、贴壁培养的原代真皮细胞、悬浮培养的原代真皮细胞以及悬浮培养后吹打成单细胞悬液,和表皮细胞与鼠尾胶原蛋白混合,植入裸鼠背部制备的皮肤全层缺损处,三周后观察毛发生长情况并组织学染色观察毛囊结构。将C57BL/6成鼠胡须毛乳头细胞P1代与新鲜分离的新生鼠表皮细胞制备成类似毛囊样3D结构,经3D培养后,植入C57BL/6小鼠肾外膜下,三周后进行观察。4.将成人头皮来源毛乳头细胞、儿童包皮成纤维细胞、人神经嵴细胞诱导的毛乳头细胞分别复合儿童包皮表皮细胞为种子细胞,进行2D或3D的构建及培养方式,将其移植裸鼠体内,验证不同种子细胞毛发形成能力的异同,寻找能稳定诱导毛发形成的种子细胞。5.以儿童包皮来源的表皮细胞和成纤维细胞为种子细胞,使用3D打印的方法在表皮-真皮组织工程皮肤的真皮层中打印毛囊样3D结构,构建有毛发生长能力的组织工程皮肤;此外,还以人神经嵴细胞诱导的毛乳头细胞混合儿童包皮来源的表皮细胞为种子细胞直接构建有毛发生长能力的组织工程皮肤;将构建的组织工程皮肤移植裸鼠体内修复皮肤缺损并观察有无毛发生长能力。研究结果1.毛囊胚胎发育及相关毛囊干细胞动态衍变过程(1)HE染色显示:C57BL/6小鼠从胚胎发育E14.5天开始出现表皮局部增厚,形成基板,真皮细胞在下方聚集;E15.5天增厚的表皮细胞逐渐形成毛芽,基板下方聚集的真皮细胞开始被形成的毛芽包裹;E16.5-E17.5天毛芽进一步发育,真皮细胞逐渐被毛芽包裹,形成毛囊的毛乳头结构;E18.5天至出生后,继续完成毛囊的胚胎发育,出生时毛囊的结构基本形成。(2)毛囊干细胞免疫荧光标记显示:随着毛囊发育和延长,标记的毛囊干细胞呈时空动态表达。2.体外构建的表皮-真皮组织工程皮肤(1)体外构建的表皮-真皮组织工程皮肤气-液面培养5天后,HE染色可观察到表皮和真皮两层结构,且表皮呈现复层化。表皮细胞及成纤维细胞状态良好,分布均匀。胶原蛋白交织成网将成纤维细胞固定在基质中。移植3周后,皮肤创口处完全愈合,与周围组织紧密连接,未见毛发;组织学显示缺损处皮肤形成表皮、真皮双层结构,且表皮复层化,未见毛囊结构。(2)将原代培养的成人毛乳头细胞与成人毛囊干细胞以上述方式制备的组织工程皮肤植入裸鼠体内三周后也未见毛发生长,组织学染色观察未见毛囊结构。3.寻找具有诱导毛囊形成能力的种子细胞(1)新鲜分离的C57BL/6新生鼠真皮细胞和表皮细胞与鼠尾胶原蛋白混合,植入裸鼠背部制备的皮肤全层缺损处三周后可见黑色毛发生长。(2)贴壁培养的原代真皮细胞组,三周后未见毛发生长,组织学染色未见毛囊结构;而在悬浮培养的原代真皮细胞组,三周后可见黑色毛发生长;悬浮培养的原代真皮细胞吹打成单细胞悬液组,三周后无毛发生长。提示3D悬浮培养可保留新生鼠原代真皮细胞的诱导毛发形成能力。(3)C57BL/6成鼠胡须毛乳头细胞P1代与新鲜分离的新生鼠表皮细胞制备成类似毛囊样3D结构,经3D培养后,植入C57BL/6小鼠肾外膜下三周后可见黑色毛发生长,组织学染色观察见毛囊结构。(4)成人毛乳头细胞P1与新鲜分离的儿童包皮表皮细胞制备成类似毛囊样3D结构,经3D培养后,移植至裸小鼠背部皮肤皮下观察3周,见白色毛纤维生长。(5)儿童包皮成纤维细胞P1与新鲜分离的儿童包皮表皮细胞制备成类似毛囊样3D结构,经3D培养后,移植至裸小鼠背部皮下观察3周,见白色毛纤维生长。植入皮内,导丝引导,三周后有白色毛纤维长出,经线粒体DNA检测毛发含有人类源性的DNA。(6)人神经嵴细胞诱导的毛乳头细胞、新鲜分离的新生鼠表皮细胞与鼠尾胶原蛋白混合,植入裸鼠背部皮下能长出黑色毛发。人神经嵴细胞诱导的毛乳头细胞、新鲜分离的儿童包皮来源表皮细胞与鼠尾胶原蛋白混合,植入裸鼠背部皮下能长出黑色毛发。4.构建带有毛囊的表皮-真皮组织工程皮肤(1)使用3D打印方法在表皮-真皮组织工程皮肤的真皮层中打印含儿童包皮来源表皮细胞和成纤维细胞的毛囊样3D结构,将构建的组织工程皮肤移植到裸鼠背部全层皮肤缺损处,三周后可见白色毛发。(2)人神经嵴细胞诱导的毛乳头细胞与新鲜分离的儿童包皮表皮细胞混合制备组织工程皮肤,皮下移植及移植到裸鼠背部全层皮肤缺损处,三周后可见黑色毛发。研究结论1.毛囊胚胎发育及相关毛囊干细胞动态衍变过程毛囊胚胎发育进一步确认了毛囊的胚胎发育是表皮细胞与真皮细胞相互作用的结果,E14.5-E17.5是C57BL/6毛囊形态学发育的关键时间。2.构建表皮-真皮组织工程皮肤(1)体外构建的表皮-真皮组织工程皮肤经气-液面培养5天后呈现表皮、真皮双层结构,表皮呈现复层化。体外移植至裸鼠全层皮肤创伤模型能使皮肤创口的愈合良好。(2)培养的人毛乳头细胞以单细胞的方式接种于真皮层中表皮-真皮组织工程皮肤体内移植后不具备形成毛发的能力。3.寻找能够诱导毛发形成的种子细胞(1)能够诱导毛发形成的细胞制备成类似毛囊样3D结构,并经3D培养后,可较好的维持其诱导毛囊形成的能力。(2)儿童包皮的成纤维细胞与表皮细胞制备成类似毛囊样的3D结构具有诱导毛发形成的能力,但是所生长出的毛发不含黑色素。(3)人神经嵴细胞诱导的毛乳头细胞具有良好的诱导毛发形成的能力,并且长出的毛发是含黑色素的。4.构建带有毛囊的表皮-真皮组织工程皮肤(1)使用3D打印机将能诱导毛发形成的种子细胞在表皮-真皮组织工程皮肤的真皮层上接种成含种子细胞的3D结构,体内移植3周后可构建带有白色毛发的表皮-真皮组织工程皮肤。(2)人神经嵴细胞诱导的毛乳头细胞与新鲜分离的儿童包皮细胞制备成表皮-真皮组织工程皮肤皮下移植及移植到裸鼠体内,可构建带有黑色毛发的表皮-真皮组织工程皮肤。
[Abstract]:Background skin is the largest body area of the body, covering the body, playing the protection, feeling, regulating body temperature, secretion and excretion, absorption, metabolism, immunity and so on. As the first barrier of the human body, skin is directly exposed to the outside world, various physical, chemical, biological and other factors can cause skin damage, and not timely and effective treatment will be given to patients. The traditional treatment for skin defects is skin graft or skin flap transplantation, but there is a shortage of donor and immune rejection. The tissue engineering skin (Tissue Engineering Skin, TES) is a artificial skin that mixed seed cells with extracellular matrix substitutes and can be used in the wound. To repair and reconstruct the skin function, it has a good prospect. It is now possible to prepare the mature epidermis dermal tissue engineering skin, which can cover the wound quickly, but lack the hair follicle and other skin appendages. The hair follicle contains rich hair follicle stem cells. It has an important role in the repair of skin trauma. Hair follicles have hair growth. Besides regulating the basic functions such as body temperature, secretion and excretion, it also affects the beauty of human beings and further affects the psychological and social role. The construction of tissue engineering skin with hair growth ability can not only promote the repair of skin damage but also reconstruct the skin function, and have certain therapeutic effect on hair loss and other diseases. Objective 1. to observe the development of hair follicle embryo and the dynamic evolution process of hair follicle stem cells, and to explore the occurrence and development of hair follicle. In order to find the seed cells that have the ability to induce hair formation, it provides a basis for.2. to master the skin of epidermis dermal tissue engineering skin in vitro, and to clarify the outcome of the body after transplantation, and to explore the epidermis dermal tissue engineering skin. Skin defect repair and hair growth ability.3. search for the seed cells that have the ability to induce hair formation, identify the ways and ways to maintain the ability to induce the induction, establish the foundation for the construction of tissue engineering skin with hair growth ability and build the tissue engineering skin with hair growth ability and repair the skin defect by.4.. Method 1. the development process of hair follicle embryo was observed by HE staining in the skin of C57BL/6 fetal rats of different gestational age. The hair follicle stem cells were labeled by immunofluorescence staining and the dynamic evolution process of hair follicle stem cells was observed by.2. separation. The skin cells and dermal fibroblast cells were cultured in children, and the homemade SD rat tail collagen was used as matrix. The skin tissue engineering skin of epidermis and dermis was constructed with gas liquid surface culture. The model of acute full skin injury on the back of the rat was prepared. The wound of the epidermis dermal tissue engineering skin was covered by the constructed skin. The original adult dermal papilla cells and adult hair follicle stem cells were prepared to prepare the tissue engineering skin. In vivo transplantation of.3., the fresh isolated C57BL/6 neonatal rat (less than 24 hours), primary dermis cells cultured, primary dermis in suspension culture and suspension culture were blown into single cell suspension, and epidermal cells were mixed with rat tail collagen, and the skin full layer defect was implanted in the back of nude mice. At three weeks, the hair growth was observed and the hair follicle structure was observed by histological staining. The P1 generation of the C57BL/6 rat Hu Xumao papilla cells and the fresh isolated neonatal rat epidermal cells were prepared to form a hair follicle like 3D structure. After 3D culture, the C57BL/6 mice were implanted under the renal outer membrane. After three weeks, the adult dermal papilla cells from the adult scalp were observed by.4.. The dermal papilla cells induced by the human neural crest cells and the human dermal papilla cells induced by human neural crest cells are the seed cells, respectively, for the construction and culture of 2D or 3D. They are transplanted to the nude mice to verify the similarities and differences of the hair formation ability of different seed cells, and to find the seed cells that can induce the formation of hair stable.5. for children. Skin derived epidermal cells and fibroblasts are seed cells, using 3D printing method to print hair follicle like 3D structure in the dermis of epidermis tissue engineering skin to construct hair growth ability tissue engineering skin. In addition, the dermal papilla cells induced by human neural crest cells are mixed with epidermal cells derived from children's foreskin. Construction of tissue engineering skin with hair growth ability directly for seed cells; repair skin defects in nude mice and observe without hair growth in nude mice. Results 1. hair follicle embryo development and related hair follicle stem cell dynamic evolution process (1) HE staining showed that C57BL/6 mice from embryonic development E14.5 days The epidermis was thickened locally, forming the basal plate, the dermal cells gathered at the bottom; the thickened epidermal cells of the E15.5 days gradually formed the hair buds, and the dermal cells gathered below the substrate were wrapped up in the formed hair buds; the hair buds were further developed in E16.5-E17.5 days and the dermal cells were gradually wrapped by the hair buds, forming the hair nipple structure of the hair follicles; E18.5 days came out. After birth, the embryo development of the hair follicle was completed and the structure of the follicle was formed at birth. (2) the hair follicle stem cell immunofluorescence markers showed that with the development and extension of the hair follicle, the labeled hair follicle stem cells expressed spatio-temporal dynamic expression of the epidermis dermal tissue engineering skin constructed in vitro (1) the epidermal dermal tissue engineering skin gas-liquid constructed in vitro (.2.). After 5 days of surface culture, the two layers of epidermis and dermis were observed by HE staining, and the epidermis showed a complex layer. The epidermis and fibroblasts were in good condition and distributed evenly. Collagen interwoven into the net and fixed the fibroblasts in the matrix. After 3 weeks of transplantation, the skin wound site was fully healed, closely connected to the surrounding tissue, and no hair was found; histology histology The epidermis of the skin, the dermis double layer structure, the epidermis of the epidermis and the hair follicle structure were not found. (2) the tissue engineering skin of adult adult hair papilla cells and adult hair follicle stem cells was implanted in the nude mice for three weeks, and no hair growth was found. Histological staining showed no hair follicle structure.3. search tool. The seed cells with the ability to induce hair follicle formation (1) fresh isolated C57BL/6 neonatal rat dermis and epidermal cells were mixed with rat tail collagen. Black hair grew after implantation of the full layer of skin defect in the back of nude mice for three weeks. (2) the primary dermis of adherent culture had no hair growth after three weeks and histological staining was not found. The hair follicle structure was seen, and black hair grew after three weeks in the primary dermal cell suspension culture, and the primary dermis in suspension culture was blown into a single cell suspension group and had no hair growth after three weeks. It was suggested that 3D suspension culture could retain the hair formation ability of the original dermal cells of the newborn rats. (3) the hair nipple of C57BL/6 mouse beard was fine. The cell P1 generation and fresh isolated neonatal rat epidermal cells were prepared to resemble hair follicle like 3D structure. After 3D culture, black hair growth was seen three weeks after the implantation of C57BL/6 mice under the renal outer membrane. (4) the adult dermal papilla cells P1 and fresh isolated infant epidermis cells were prepared to be similar to the hair follicle like 3D structure. After 3D culture, the skin was transplanted into the skin of the back of the nude mice for 3 weeks, and the white hair fibers were grown. (5) the children's prepuce fibroblasts, P1 and the fresh isolated children's epidermis cells, were prepared to be similar to the hair follicle like 3D structure. After 3D culture, the skin was transplanted to the back of the nude mice for 3 weeks and saw the growth of white hair fibers. After three weeks, the white hair fibers were long out. The hair papilla cells induced by human DNA. (6) human neural crest cells were detected by the mitochondrial DNA. The fresh isolated neonatal rat epidermal cells were mixed with the rat tail collagen, and the hair of the nude mice was implanted in the back of the nude mice. The isolated skin cells from the children are mixed with the rat tail collagen. The skin of the nude mice can be implanted in the skin of the nude mice to grow black hair.4. to construct the epidermis dermal tissue engineering skin with the hair follicle. (1) the 3D printing method is used to print the hair of the cuticle and fibroblast in the dermis of the epidermis tissue engineering skin. The constructed tissue engineered skin was transplanted to the full skin defect on the back of nude mice. White hair was seen three weeks later. (2) the dermal papilla cells induced by human neural crest cells were mixed with fresh isolated children's epidermis cells to prepare tissue engineered skin, subcutaneous transplantation and transplantation to the full skin defect of the back of nude mice for three weeks. The development of embryo development of hair follicle and the development of hair follicle stem cells in the process of hair follicle embryo development further confirm that the embryo development of hair follicle is the result of the interaction between epidermal cells and dermis cells, E14.5-E17.5 is the key time of C57BL/6 hair follicle morphology development.2. to construct epidermis dermal tissue engineering skin. Skin (1) the epidermis dermal tissue engineering skin was constructed in vitro for 5 days, which showed epidermis, dermis double layer structure and complex layer. In vitro transplantation to nude mice skin wound model could make the skin wound healing well. (2) the cultured human dermal papilla cells were inoculated in the epidermis dermal group in the dermis by single cell method. The ability to form hair after the skin graft in the skin of the fabric is.3. to find the seed cells that can induce hair formation (1) the cells that can induce hair formation are prepared to be similar to the hair follicle like 3D structure. After 3D culture, it can maintain the ability to induce the formation of hair follicles. (2) the preparation of fibroblasts and epidermal cells in children's prepuce The 3D structure similar to the hair follicle has the ability to induce hair formation, but the growing hair does not contain melanin. (3) the dermal papilla cells induced by human neural crest cells have a good ability to induce hair formation, and the hair of the hair is a melanin containing.4. construction of the epidermis dermal tissue engineering skin with hair follicles (1) using 3D The printers can induce the seed cells of hair to be inoculated into the 3D structure containing seed cells on the dermis of the epidermis tissue engineering skin. After 3 weeks in vivo transplantation, the epidermis dermal tissue engineering skin with white hair can be constructed. (2) human dermal papilla cells induced by human neural crest cells and fresh isolated children's circumcision cells The epidermis dermal tissue engineered skin is subcutaneously transplanted and transplanted into nude mice to construct epidermal dermal tissue engineering skin with black hair.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R622

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