人毛囊神经嵴干细胞的分离、培养与初步鉴定

发布时间：2018-02-13 00:07

本文关键词： 毛囊神经嵴干细胞 SOX10 Nestin 白癜风　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景白癜风(Vitiligo)是色素障碍性疾病中最为常见的一种皮肤病,其发病机制不明,可能由于各种原因导致的基底层功能性黑素细胞破坏,使得皮肤、粘膜甚至毛发的色素减退和脱失。目前,全球约有0.5%~1%的群体罹患此病。虽然白癜风的皮损无自觉症状,未给患者带来生理上的痛苦,但其心理健康和生活质量都备受影响。因此,探明白癜风的发病机制,寻求白癜风的治疗方法,具有重大的科学及社会意义。白癜风的发病机制主要包括脱色机制和复色机制两大方面,目前对白癜风发病机制的研究主要集中在脱色机制,对于同等重要的复色机制,却鲜见研究。因此,进一步探究白癜风的发病机制(包括脱色机制和复色机制),寻找白癜风治疗的新方法将意义深远。通过临床疗效观察,发现白癜风在复色时,以毛囊周围复色的模式最为常见,因此,探究由毛囊周围引起复色及其机制更有意义。众所周知,毛囊(hair follicle,HF)是哺乳动物的重要皮肤附属器,其结构虽然微小,但作用巨大。毛囊不仅能够调节毛发的生长、脱落,而且是维持机体生物功能平衡的重要的动态微型器官。有研究发现,毛囊中存在不同发育来源的干细胞,包括毛囊干细胞(hair follicle-stem cell,HF-SC)、间充质干细胞(mesenchymal stem cells,MSCs)、色素性干细胞、神经源性干细胞等等,且已有研究证实,毛囊干细胞在体外可高度增殖,并具有分化的潜能。毛囊中除了包含上述干细胞外,新近研究发现在毛囊隆突区还存在神经嵴干细胞(neuralcrest stem cells,NCSCs),NCSCs是一种具有多能性分化潜能的祖细胞,其可以分化成为不同的细胞系。本课题将从干细胞生物学的角度,以人毛囊为研究对象,证实人毛囊中存在神经嵴干细胞,并通过原代培养,将神经嵴干细胞从毛囊中分离出来,为后续将其诱导分化成黑素细胞提供依据。目的1、本实验拟证实人毛囊中存在神经嵴干细胞。2、将人毛囊内的神经嵴干细胞分离出来,体外原代培养,并进行初步鉴定。材料及方法1、标本来源于郑州大学第一附属医院整形外科、皮肤科等手术中所获取的含毛囊的头皮组织,所取皮肤组织局部无器质性病变、无色素性疾病。2、标本采用PBS及Hank’s液进行冲洗,切成小组织块,浸入0.48 U/ml的中性蛋白酶在4℃中消化过夜,次日借助解剖显微,采用显微分离术获取单个毛囊。3、采用RT-PCR检测游离毛囊组织中SOX10、Nestin的m RNA的表达。4、将游离毛囊制成冰冻切片,连续横向切片,采用免疫荧光法检测毛囊组织中SOX10、Nestin的表达。5、在无菌环境下,消化分离含毛囊的皮肤组织,修剪多余的毛干部分,置于神经嵴干细胞培养基进行原代培养,观察细胞从毛囊隆突区游离的时间,随后取出毛囊组织,将游离出的细胞消化后采用免疫荧光法鉴定细胞中SOX10、Nestin的表达。结果1、RT-PCR结果显示,游离毛囊组织中SOX10、Nestin的m RNA呈阳性表达。2、免疫荧光染色显示,毛囊组织中段SOX10、Nestin呈阳性表达,SOX10在细胞核中表达,呈绿色荧光。Nestin在细胞浆中表达,呈红色荧光。3、毛囊在原代培养时,毛囊隆突区第2~3天有细胞成团迁出,第4天细胞增殖加快。迁出的细胞呈梭形、椭圆形,胞核饱满,类似于成纤维细胞,迁出的细胞免疫荧光染色SOX10和Nestin呈阳性表达。结论1、在m RNA和蛋白水平,人毛囊中存在SOX10和Nestin的阳性表达的神经嵴干细胞。2、体外原代培养时,人毛囊可迁出细胞,经初步鉴定迁出的细胞为神经嵴干细胞。
[Abstract]:The research background of vitiligo (Vitiligo) is the most common type of skin disease thepigment disease, its pathogenesis is unknown, possibly due to various causes of basal functional destruction of melanocytes, the skin, mucosa and hair depigmentation and depigmentation. At present, about 0.5%~1% of the population suffering from the disease. Although vitiligo lesions without symptoms, not to patients with physical pain, but the psychological health and quality of life are greatly affected. Therefore, the exploration of the pathogenesis of vitiligo, vitiligo treatment method for the scientific and social significance. The pathogenesis of vitiligo mainly includes the decolorization mechanism and the mechanism of color two at present, the research on the pathogenesis of vitiligo is mainly concentrated in the decolorization mechanism for the equally important repigmented mechanism was rarely studied. Therefore, further study of vitiligo The mechanism of disease (including the decolorization mechanism and complex color mechanism) to find new ways of treatment of vitiligo, will be of far-reaching significance. Through clinical observation, found that vitiligo in complex color, with complex color patterns around the hair follicle is most common, therefore, by exploring around the hair follicle caused by complex color and its mechanism is more important. As everyone knows, the hair follicle (hair follicle, HF) is an important skin appendage in mammals, the structure of the small, but significant role. The hair follicle can not only regulate hair growth and shedding, and maintain the balance of the important biological function of dynamic micro organs. Studies have found that different developmental sources exist in the hair follicle stem cells, including hair follicle stem cells (hair follicle-stem cell, HF-SC), mesenchymal stem cells (mesenchymal stem cells, MSCs), pigment stem cells, neural stem cells and so on, and it has been proved that the high hair follicle stem cells in vitro The degree of proliferation, differentiation and has potential. In addition to containing the hair follicle stem cells, recent research found that there are neural crest stem cells in the bulge area (neuralcrest stem, cells, NCSCs, NCSCs) is a kind of pluripotent differentiation potential of progenitor cells, which can differentiate into different cell lines. Subject from the stem cell biology, with human hair follicle as the research object, confirmed that neural crest stem cells exist in human hair follicles, and the primary cultured neural crest stem cells will be isolated from the hair follicle, for the follow-up will be induced to differentiate into melanocytes. Objective to provide the basis for the 1, this study confirmed that neural crest stem cells.2 human hair follicle, human hair follicle neural crest stem cells isolated and cultured in vitro, and preliminary identification. Materials and methods 1 samples were collected from the First Affiliated Hospital of Zhengzhou University, Department of Dermatology surgery In the scalp with hair follicle tissue, the skin tissue local no organic disease, no pigment disease.2, PBS and Hank were detected by s liquid to wash, cut into small tissue pieces were immersed in 0.48 U/ml neutral protease digestion overnight at 4 DEG C, the next day with the help of microscopic anatomy, using microscope the separation of.3 by SOX10 to get a single hair follicle, hair follicle tissue RT-PCR detection, the expression of.4 m RNA Nestin, the hair follicle and made into frozen sections, continuous transverse sections, SOX10 hair follicle tissue by immunofluorescence detection, the expression of.5 Nestin, in a sterile environment, digested with hair follicle skin tissue, pruning excess hair dry, cell culture medium and cultured in neural crest stem cells, observe the free from the hair follicle bulge time, then remove the hair follicle tissue, will free cells after digestion by immunofluorescence cell identification In SOX10, the expression of Nestin. Results 1, RT-PCR results showed that SOX10 hair follicle tissue, the positive expression of Nestin m RNA.2, immunofluorescence staining showed that hair follicle tissue of SOX10, Nestin positive expression, the expression of SOX10 in the nucleus, showed green fluorescence in cytoplasm.Nestin expression of red fluorescence.3, hair follicle in primary culture, the bulge area of the first 2~3 days cells group to move out, fourth days faster. Cell proliferation of migrating cells were spindle shaped, oval, full nucleus, similar to fibroblast cells, immunofluorescence staining of SOX10 and Nestin from the positive conclusion. In 1, m the RNA and protein levels, the positive expression of SOX10 and Nestin in human hair follicles in the neural crest stem cells.2, primary cultured in vitro, human hair follicle cells can move out, after preliminary identification of migrating cells for neural crest stem cells.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.41

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