【作者】 徐红；

【导师】 李玉林；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 创伤引起的皮肤组织缺损,传统的治疗方法是利用自体断层皮片移植,但存在供皮量有限和造成额外损伤的障碍。利用体外培养的人工皮肤即组织工程皮肤作为移植的来源可以从根本上解决这个问题,但是如何获得种子细胞却一直困扰着人们。随着近年来对干细胞研究的不断深入,以干细胞作为种子细胞构建组织工程皮肤成为当前研究的热点之一。皮肤干细胞作为种子细胞构建组织工程皮肤是较好的选择。皮肤干细胞主要有表皮干细胞和毛囊干细胞。目前,表皮干细胞作为种子细胞构建组织工程皮肤已经应用于临床烧伤患者,但仍存在排异反应和供皮量有限等问题。随着医学生物学的发展,皮肤组织工程的深入,毛囊干细胞作为一种新的种子细胞逐渐受到人们的关注。越来越多的研究表明毛囊中也存在具有多向分化潜能的干细胞,如果能建立完善的培养扩增体系,只需要一定数目的头发,即可培养出表皮的膜片,供给丰富,损伤小,不造成创面,无免疫排异反应,并可实现个体化治疗,可为临床皮肤缺损的修复开辟新的领域和提供更多的选择方法。有关毛囊干细胞分离培养的研究,目前在多数实验中以鼠的触须毛囊、外科获取的人头皮样本为毛囊干细胞的组织来源。样本经中性蛋白酶(dispaseⅡ)处理,分离去除表皮,从真皮中拔取并收集生长期的毛囊组织,进行毛囊干细胞组织块培养,或者用胰酶/EDTA处理收集的毛囊组织,获得单细胞悬液培养毛囊干细胞。本研究旨在从拔出的毛发中培养出毛囊干细胞,以求建立一种新的毛囊干细胞的获取方法。实验中,我们以直接拔取的人毛发作为组织来源,进行人毛囊干细胞原代培养方法的探索,并对其进行鉴定。通过比较几种不同的毛囊干细胞原代培养方法,最终采用0.25%胰酶+0.05%EDTA消化毛囊组织,以丝裂霉素处理的人成纤维细胞为饲养层与毛囊组织及其来源的细胞共培养,可以获得毛囊组织来源的铺路石样细胞。经鉴定,此方法获得的毛囊组织来源的铺路石样细胞中含有一定数量的毛囊干细胞。更多还原

【Abstract】 Stem cells (SCs) have the unique capability to self-renew and the potentiality of high proliferate. They can differentiate into cell lineages that constitute their tissue of origin.The hair follicle bulge area is an abundant, easily accessible source of actively growing pluripotent adult stem cells. It is part of the hair follicle outer sheath that is in continuity with the arrector muscle and under with the sebaceous gland. Hair follicle stem cells which located in the hair follicle bulge are adult stem cells. We call it hair follicle stem cells. Hair follicle stem cells possess stem cell characteristics, including multipotency, high proliferation potential and ability to enter quiescence.Lineage analysis has demonstrated that all epithelial layers within the adult follicle and hair originate from bulge cells. Therefore, the hair follicle stem cells appear to be responsible for regenerating the hair follicle in each hair cycle. The hair follicle bulge stem cells are potentially bipotent as they can give rise to not only cells of the hair follicle, but also epidermal cells. Other experiments have also provided new evidence that the upper outer root sheath of whisker follicles of adult mice contain multipotent stem cells, which can differentiate into hair follicle matrix cells, sebaceous gland basal cells and epidermis.The lack of unique markers to identify hair follicle stem cells in the bulge area has hindered the study of hair follicle stem cells. So far, studies on the marks of hair follicle stem cells are focus on integrin, keratin、CD34、CD200、p63、CD71、Cx43 and so on. We use these marks isolate and identify hair follicle stem cells.According to the literature, in most of experiences they choose the mouse’s vibrissae follicles and human scalp sample as the source of hair follicle stem cells. First, using dispaseⅡto deal with the tissue sample, and then, separating and removing the epidermis. Last, plucking and collecting the follicles from the dermis. In this way, they can separate and culture the hair follicle stem cells.Our experiment is studying on the hair follicle stem cells’segregation, culture and identification. In experience, we pluck the hair follicles from the volunteer’s head. In order to search for the means of hair follicle stem cells’primary culture,we had carried out many different methods. Luckily, we first use 0.25%trypsin and 0.05%EDTA to digest the human hair follicles, and then co-culture the human hair follicle cells and human fibroblasts which were treated by mitomycin. In this way, we successfully obtain the pavement-like hair follicle hair follicle stem cells.More and more people are paying close attention to hair follicle stem cells. If we can set up a consummate system of hair follicle stem cells’cultivation and amplification, maybe we can construct the epidermis leather-diaphragm using a few hair follicles to repair the clinical cutaneous deficiency.更多还原