本文關(guān)鍵詞： 毛囊 神經(jīng)嵴干細(xì)胞 SOX10 Nestin 白癜風(fēng)　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景白癜風(fēng)(Vitiligo)是色素障礙性疾病中最為常見的一種皮膚病,其發(fā)病機(jī)制不明,可能由于各種原因?qū)е碌幕讓庸δ苄院谒丶?xì)胞破壞,使得皮膚、粘膜甚至毛發(fā)的色素減退和脫失。目前,全球約有0.5%~1%的群體罹患此病。雖然白癜風(fēng)的皮損無自覺癥狀,未給患者帶來生理上的痛苦,但其心理健康和生活質(zhì)量都備受影響。因此,探明白癜風(fēng)的發(fā)病機(jī)制,尋求白癜風(fēng)的治療方法,具有重大的科學(xué)及社會(huì)意義。白癜風(fēng)的發(fā)病機(jī)制主要包括脫色機(jī)制和復(fù)色機(jī)制兩大方面,目前對白癜風(fēng)發(fā)病機(jī)制的研究主要集中在脫色機(jī)制,對于同等重要的復(fù)色機(jī)制,卻鮮見研究。因此,進(jìn)一步探究白癜風(fēng)的發(fā)病機(jī)制(包括脫色機(jī)制和復(fù)色機(jī)制),尋找白癜風(fēng)治療的新方法將意義深遠(yuǎn)。通過臨床療效觀察,發(fā)現(xiàn)白癜風(fēng)在復(fù)色時(shí),以毛囊周圍復(fù)色的模式最為常見,因此,探究由毛囊周圍引起復(fù)色及其機(jī)制更有意義。眾所周知,毛囊(hair follicle,HF)是哺乳動(dòng)物的重要皮膚附屬器,其結(jié)構(gòu)雖然微小,但作用巨大。毛囊不僅能夠調(diào)節(jié)毛發(fā)的生長、脫落,而且是維持機(jī)體生物功能平衡的重要的動(dòng)態(tài)微型器官。有研究發(fā)現(xiàn),毛囊中存在不同發(fā)育來源的干細(xì)胞,包括毛囊干細(xì)胞(hair follicle-stem cell,HF-SC)、間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)、色素性干細(xì)胞、神經(jīng)源性干細(xì)胞等等,且已有研究證實(shí),毛囊干細(xì)胞在體外可高度增殖,并具有分化的潛能。毛囊中除了包含上述干細(xì)胞外,新近研究發(fā)現(xiàn)在毛囊隆突區(qū)還存在神經(jīng)嵴干細(xì)胞(neuralcrest stem cells,NCSCs),NCSCs是一種具有多能性分化潛能的祖細(xì)胞,其可以分化成為不同的細(xì)胞系。本課題將從干細(xì)胞生物學(xué)的角度,以人毛囊為研究對象,證實(shí)人毛囊中存在神經(jīng)嵴干細(xì)胞,并通過原代培養(yǎng),將神經(jīng)嵴干細(xì)胞從毛囊中分離出來,為后續(xù)將其誘導(dǎo)分化成黑素細(xì)胞提供依據(jù)。目的1、本實(shí)驗(yàn)擬證實(shí)人毛囊中存在神經(jīng)嵴干細(xì)胞。2、將人毛囊內(nèi)的神經(jīng)嵴干細(xì)胞分離出來,體外原代培養(yǎng),并進(jìn)行初步鑒定。材料及方法1、標(biāo)本來源于鄭州大學(xué)第一附屬醫(yī)院整形外科、皮膚科等手術(shù)中所獲取的含毛囊的頭皮組織,所取皮膚組織局部無器質(zhì)性病變、無色素性疾病。2、標(biāo)本采用PBS及Hank’s液進(jìn)行沖洗,切成小組織塊,浸入0.48 U/ml的中性蛋白酶在4℃中消化過夜,次日借助解剖顯微,采用顯微分離術(shù)獲取單個(gè)毛囊。3、采用RT-PCR檢測游離毛囊組織中SOX10、Nestin的m RNA的表達(dá)。4、將游離毛囊制成冰凍切片,連續(xù)橫向切片,采用免疫熒光法檢測毛囊組織中SOX10、Nestin的表達(dá)。5、在無菌環(huán)境下,消化分離含毛囊的皮膚組織,修剪多余的毛干部分,置于神經(jīng)嵴干細(xì)胞培養(yǎng)基進(jìn)行原代培養(yǎng),觀察細(xì)胞從毛囊隆突區(qū)游離的時(shí)間,隨后取出毛囊組織,將游離出的細(xì)胞消化后采用免疫熒光法鑒定細(xì)胞中SOX10、Nestin的表達(dá)。結(jié)果1、RT-PCR結(jié)果顯示,游離毛囊組織中SOX10、Nestin的m RNA呈陽性表達(dá)。2、免疫熒光染色顯示,毛囊組織中段SOX10、Nestin呈陽性表達(dá),SOX10在細(xì)胞核中表達(dá),呈綠色熒光。Nestin在細(xì)胞漿中表達(dá),呈紅色熒光。3、毛囊在原代培養(yǎng)時(shí),毛囊隆突區(qū)第2~3天有細(xì)胞成團(tuán)遷出,第4天細(xì)胞增殖加快。遷出的細(xì)胞呈梭形、橢圓形,胞核飽滿,類似于成纖維細(xì)胞,遷出的細(xì)胞免疫熒光染色SOX10和Nestin呈陽性表達(dá)。結(jié)論1、在m RNA和蛋白水平,人毛囊中存在SOX10和Nestin的陽性表達(dá)的神經(jīng)嵴干細(xì)胞。2、體外原代培養(yǎng)時(shí),人毛囊可遷出細(xì)胞,經(jīng)初步鑒定遷出的細(xì)胞為神經(jīng)嵴干細(xì)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R758.41

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