本文選題：組織工程皮膚 + 毛乳頭細胞　； 參考：《北京協(xié)和醫(yī)學院》2017年博士論文

【摘要】：研究背景皮膚是人體面積最大的器官,覆蓋全身,發(fā)揮著保護、感覺、調(diào)節(jié)體溫、分泌與排泄、吸收、代謝、免疫等作用。作為人體的第一道屏障,皮膚直接與外界接觸,各種物理、化學、生物等因素都能引起皮膚的損傷,不及時有效治療會給患者帶來極大痛苦,甚至威脅生命。對于皮膚缺損,傳統(tǒng)的治療方法是進行皮膚移植或皮瓣移植術(shù),但存在供體不足和免疫排斥等問題。組織工程皮膚(Tissue Engineering Skin,TES)是將種子細胞與細胞外基質(zhì)替代物混合制成的人工皮膚,可用于創(chuàng)面修復,重建皮膚功能,具有良好的發(fā)展前景�，F(xiàn)在已能制備比較成熟的表皮-真皮組織工程皮膚,基本能夠快速覆蓋創(chuàng)面,但是缺乏毛囊等皮膚附屬器。毛囊含有豐富的毛囊干細胞,在皮膚創(chuàng)傷的修復、重建中有重要作用。毛囊所生長出的毛發(fā)除了調(diào)節(jié)體溫、分泌與排泄等基本功能之外,對于人類還有影響美觀,進而進一步影響心理及社交的作用。構(gòu)建有毛發(fā)生長能力的組織工程皮膚不但能促進皮膚損傷修復,還可以重建皮膚功能,而且對于脫發(fā)等疾病有一定治療作用。研究目的1.觀察毛囊胚胎發(fā)育及毛囊干細胞動態(tài)衍變過程,探討毛囊的發(fā)生、發(fā)展過程,為尋找有誘導毛發(fā)形成能力的種子細胞提供依據(jù)。2.掌握體外構(gòu)建表皮-真皮組織工程皮膚的技術(shù)及明確移植后的體內(nèi)轉(zhuǎn)歸,探討表皮-真皮組織工程皮膚的皮膚缺損修復以及毛發(fā)生長能力。3.尋找具有誘導毛發(fā)形成能力的種子細胞,明確其能維持誘導能力的培養(yǎng)方式及構(gòu)建方式,為構(gòu)建有毛發(fā)生長能力的組織工程皮膚奠定基礎。4.構(gòu)建有毛發(fā)生長能力的組織工程皮膚并修復皮膚缺損。研究方法1.以C57BL/6不同胎齡胎鼠的皮膚為模型,通過HE染色觀察毛囊胚胎發(fā)育過程,免疫熒光染色標記相關(guān)毛囊干細胞并觀察毛囊干細胞動態(tài)衍變過程。2.分離、培養(yǎng)兒童包皮表皮細胞及真皮成纖維細胞,以自制SD大鼠鼠尾膠原為基質(zhì),應用氣-液面培養(yǎng),構(gòu)建表皮-真皮組織工程皮膚。制備大鼠背部急性全層皮膚損傷模型,將構(gòu)建的表皮-真皮組織工程皮膚覆蓋傷口,觀察其體內(nèi)轉(zhuǎn)歸。將原代培養(yǎng)的成人毛乳頭細胞與成人毛囊干細胞以上述方式制備組織工程皮膚,并進行體內(nèi)移植觀察其轉(zhuǎn)歸。3.將新鮮分離的C57BL/6新生鼠(24小時以內(nèi))真皮細胞、貼壁培養(yǎng)的原代真皮細胞、懸浮培養(yǎng)的原代真皮細胞以及懸浮培養(yǎng)后吹打成單細胞懸液,和表皮細胞與鼠尾膠原蛋白混合,植入裸鼠背部制備的皮膚全層缺損處,三周后觀察毛發(fā)生長情況并組織學染色觀察毛囊結(jié)構(gòu)。將C57BL/6成鼠胡須毛乳頭細胞P1代與新鮮分離的新生鼠表皮細胞制備成類似毛囊樣3D結(jié)構(gòu),經(jīng)3D培養(yǎng)后,植入C57BL/6小鼠腎外膜下,三周后進行觀察。4.將成人頭皮來源毛乳頭細胞、兒童包皮成纖維細胞、人神經(jīng)嵴細胞誘導的毛乳頭細胞分別復合兒童包皮表皮細胞為種子細胞,進行2D或3D的構(gòu)建及培養(yǎng)方式,將其移植裸鼠體內(nèi),驗證不同種子細胞毛發(fā)形成能力的異同,尋找能穩(wěn)定誘導毛發(fā)形成的種子細胞。5.以兒童包皮來源的表皮細胞和成纖維細胞為種子細胞,使用3D打印的方法在表皮-真皮組織工程皮膚的真皮層中打印毛囊樣3D結(jié)構(gòu),構(gòu)建有毛發(fā)生長能力的組織工程皮膚;此外,還以人神經(jīng)嵴細胞誘導的毛乳頭細胞混合兒童包皮來源的表皮細胞為種子細胞直接構(gòu)建有毛發(fā)生長能力的組織工程皮膚;將構(gòu)建的組織工程皮膚移植裸鼠體內(nèi)修復皮膚缺損并觀察有無毛發(fā)生長能力。研究結(jié)果1.毛囊胚胎發(fā)育及相關(guān)毛囊干細胞動態(tài)衍變過程(1)HE染色顯示:C57BL/6小鼠從胚胎發(fā)育E14.5天開始出現(xiàn)表皮局部增厚,形成基板,真皮細胞在下方聚集;E15.5天增厚的表皮細胞逐漸形成毛芽,基板下方聚集的真皮細胞開始被形成的毛芽包裹;E16.5-E17.5天毛芽進一步發(fā)育,真皮細胞逐漸被毛芽包裹,形成毛囊的毛乳頭結(jié)構(gòu);E18.5天至出生后,繼續(xù)完成毛囊的胚胎發(fā)育,出生時毛囊的結(jié)構(gòu)基本形成。(2)毛囊干細胞免疫熒光標記顯示:隨著毛囊發(fā)育和延長,標記的毛囊干細胞呈時空動態(tài)表達。2.體外構(gòu)建的表皮-真皮組織工程皮膚(1)體外構(gòu)建的表皮-真皮組織工程皮膚氣-液面培養(yǎng)5天后,HE染色可觀察到表皮和真皮兩層結(jié)構(gòu),且表皮呈現(xiàn)復層化。表皮細胞及成纖維細胞狀態(tài)良好,分布均勻。膠原蛋白交織成網(wǎng)將成纖維細胞固定在基質(zhì)中。移植3周后,皮膚創(chuàng)口處完全愈合,與周圍組織緊密連接,未見毛發(fā);組織學顯示缺損處皮膚形成表皮、真皮雙層結(jié)構(gòu),且表皮復層化,未見毛囊結(jié)構(gòu)。(2)將原代培養(yǎng)的成人毛乳頭細胞與成人毛囊干細胞以上述方式制備的組織工程皮膚植入裸鼠體內(nèi)三周后也未見毛發(fā)生長,組織學染色觀察未見毛囊結(jié)構(gòu)。3.尋找具有誘導毛囊形成能力的種子細胞(1)新鮮分離的C57BL/6新生鼠真皮細胞和表皮細胞與鼠尾膠原蛋白混合,植入裸鼠背部制備的皮膚全層缺損處三周后可見黑色毛發(fā)生長。(2)貼壁培養(yǎng)的原代真皮細胞組,三周后未見毛發(fā)生長,組織學染色未見毛囊結(jié)構(gòu);而在懸浮培養(yǎng)的原代真皮細胞組,三周后可見黑色毛發(fā)生長;懸浮培養(yǎng)的原代真皮細胞吹打成單細胞懸液組,三周后無毛發(fā)生長。提示3D懸浮培養(yǎng)可保留新生鼠原代真皮細胞的誘導毛發(fā)形成能力。(3)C57BL/6成鼠胡須毛乳頭細胞P1代與新鮮分離的新生鼠表皮細胞制備成類似毛囊樣3D結(jié)構(gòu),經(jīng)3D培養(yǎng)后,植入C57BL/6小鼠腎外膜下三周后可見黑色毛發(fā)生長,組織學染色觀察見毛囊結(jié)構(gòu)。(4)成人毛乳頭細胞P1與新鮮分離的兒童包皮表皮細胞制備成類似毛囊樣3D結(jié)構(gòu),經(jīng)3D培養(yǎng)后,移植至裸小鼠背部皮膚皮下觀察3周,見白色毛纖維生長。(5)兒童包皮成纖維細胞P1與新鮮分離的兒童包皮表皮細胞制備成類似毛囊樣3D結(jié)構(gòu),經(jīng)3D培養(yǎng)后,移植至裸小鼠背部皮下觀察3周,見白色毛纖維生長。植入皮內(nèi),導絲引導,三周后有白色毛纖維長出,經(jīng)線粒體DNA檢測毛發(fā)含有人類源性的DNA。(6)人神經(jīng)嵴細胞誘導的毛乳頭細胞、新鮮分離的新生鼠表皮細胞與鼠尾膠原蛋白混合,植入裸鼠背部皮下能長出黑色毛發(fā)。人神經(jīng)嵴細胞誘導的毛乳頭細胞、新鮮分離的兒童包皮來源表皮細胞與鼠尾膠原蛋白混合,植入裸鼠背部皮下能長出黑色毛發(fā)。4.構(gòu)建帶有毛囊的表皮-真皮組織工程皮膚(1)使用3D打印方法在表皮-真皮組織工程皮膚的真皮層中打印含兒童包皮來源表皮細胞和成纖維細胞的毛囊樣3D結(jié)構(gòu),將構(gòu)建的組織工程皮膚移植到裸鼠背部全層皮膚缺損處,三周后可見白色毛發(fā)。(2)人神經(jīng)嵴細胞誘導的毛乳頭細胞與新鮮分離的兒童包皮表皮細胞混合制備組織工程皮膚,皮下移植及移植到裸鼠背部全層皮膚缺損處,三周后可見黑色毛發(fā)。研究結(jié)論1.毛囊胚胎發(fā)育及相關(guān)毛囊干細胞動態(tài)衍變過程毛囊胚胎發(fā)育進一步確認了毛囊的胚胎發(fā)育是表皮細胞與真皮細胞相互作用的結(jié)果,E14.5-E17.5是C57BL/6毛囊形態(tài)學發(fā)育的關(guān)鍵時間。2.構(gòu)建表皮-真皮組織工程皮膚(1)體外構(gòu)建的表皮-真皮組織工程皮膚經(jīng)氣-液面培養(yǎng)5天后呈現(xiàn)表皮、真皮雙層結(jié)構(gòu),表皮呈現(xiàn)復層化。體外移植至裸鼠全層皮膚創(chuàng)傷模型能使皮膚創(chuàng)口的愈合良好。(2)培養(yǎng)的人毛乳頭細胞以單細胞的方式接種于真皮層中表皮-真皮組織工程皮膚體內(nèi)移植后不具備形成毛發(fā)的能力。3.尋找能夠誘導毛發(fā)形成的種子細胞(1)能夠誘導毛發(fā)形成的細胞制備成類似毛囊樣3D結(jié)構(gòu),并經(jīng)3D培養(yǎng)后,可較好的維持其誘導毛囊形成的能力。(2)兒童包皮的成纖維細胞與表皮細胞制備成類似毛囊樣的3D結(jié)構(gòu)具有誘導毛發(fā)形成的能力,但是所生長出的毛發(fā)不含黑色素。(3)人神經(jīng)嵴細胞誘導的毛乳頭細胞具有良好的誘導毛發(fā)形成的能力,并且長出的毛發(fā)是含黑色素的。4.構(gòu)建帶有毛囊的表皮-真皮組織工程皮膚(1)使用3D打印機將能誘導毛發(fā)形成的種子細胞在表皮-真皮組織工程皮膚的真皮層上接種成含種子細胞的3D結(jié)構(gòu),體內(nèi)移植3周后可構(gòu)建帶有白色毛發(fā)的表皮-真皮組織工程皮膚。(2)人神經(jīng)嵴細胞誘導的毛乳頭細胞與新鮮分離的兒童包皮細胞制備成表皮-真皮組織工程皮膚皮下移植及移植到裸鼠體內(nèi),可構(gòu)建帶有黑色毛發(fā)的表皮-真皮組織工程皮膚。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R622

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