本文關(guān)鍵詞：人多潛能毛囊干細(xì)胞的分離培養(yǎng)及其向神經(jīng)元樣細(xì)胞誘導(dǎo)分化的研究　出處：《吉林大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

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【摘要】： 疾病對人類的健康和生存構(gòu)成重大威脅、是世界各國普遍面臨的最重要的社會問題。干細(xì)胞移植技術(shù)的問世和進(jìn)展為傳統(tǒng)治療手段認(rèn)為是“不治之癥”的患者帶來了新的生機(jī)和希望。但干細(xì)胞的來源、自我更新潛能的維持、向功能細(xì)胞分化的潛能以及移植治療的安全性、有效性一直是干細(xì)胞基礎(chǔ)理論和實(shí)際應(yīng)用研究中急待解決的關(guān)鍵科學(xué)問題。 毛囊是皮膚的附屬器之一,起源于表皮與間充質(zhì)間的相互作用,終生自我更新,是存在于成體內(nèi)的胚胎樣器官。新近研究發(fā)現(xiàn),毛囊中除了含有表皮干細(xì)胞和黑色素干細(xì)胞外,還含有具有多向分化潛能的間充質(zhì)干細(xì)胞。這些多潛能干細(xì)胞具有自我更新和定向分化成其它細(xì)胞的能力。與其他類型間充質(zhì)干細(xì)胞相比,毛囊干細(xì)胞具有來源豐富,獲取方便,不受年齡性別限制,不涉及倫理問題等優(yōu)點(diǎn)。 首先,本研究探索了一種簡便可行的獲取毛囊多潛能干細(xì)胞的方法,通過應(yīng)用組織塊培養(yǎng)法,從拔取的人毛發(fā)中分離培養(yǎng)出多潛能毛囊干細(xì)胞,并應(yīng)用免疫細(xì)胞化學(xué)法結(jié)合流式細(xì)胞儀技術(shù),對毛囊多潛能干細(xì)胞的表型進(jìn)行了分析鑒定,結(jié)果顯示多潛能毛囊干細(xì)胞表達(dá)間充質(zhì)干細(xì)胞相對特異的表面標(biāo)記物CD29、CD44、CD90、CD105(SH-2)、CD73(SH-4)表面抗原,不表達(dá)CD31、CD34、CD45、HLA-DR、K15等表面抗原。其次,本實(shí)驗(yàn)初步建立了毛囊多潛能干細(xì)胞傳代、擴(kuò)增、凍存與復(fù)蘇的方法。最后,本實(shí)驗(yàn)成功誘導(dǎo)多潛能毛囊干細(xì)胞向成骨細(xì)胞、脂肪細(xì)胞分化。探索了多潛能毛囊干細(xì)胞向神經(jīng)樣細(xì)胞誘導(dǎo)分化的條件,通過免疫細(xì)胞化學(xué)法檢測誘導(dǎo)前后神經(jīng)元特異性烯醇化酶(NSE)、神經(jīng)絲蛋白(NF-L)、膠質(zhì)纖維酸性蛋白(GFAP)、神經(jīng)巢蛋白(nestin)、神經(jīng)微管蛋白Ⅲ(β-tubulinⅢ)、S100鈣結(jié)合蛋白B(S100B)及環(huán)核苷磷酸二酯酶(CNPase)的表達(dá)水平。認(rèn)為雙丁酰環(huán)磷腺苷(Dibutyryl Cyclic AMP)聯(lián)合3-異丁基-1-甲基黃嘌呤(IBMX)是目前本實(shí)驗(yàn)條件下誘導(dǎo)人毛囊干細(xì)胞向神經(jīng)元細(xì)胞分化最為有效的方法。為后期利用多潛能毛囊干細(xì)胞移植修復(fù)損傷神經(jīng)、帕金森及其它神經(jīng)系統(tǒng)病變奠定了必要的前期工作基礎(chǔ)。
[Abstract]:Diseases pose a major threat to human health and survival. Stem cell transplantation is the most important social problem all over the world. The advent and progress of stem cell transplantation technology has brought new life and hope to patients who are regarded as "incurable disease" by traditional treatment methods. But the source of stem cells. The maintenance of self-renewal potential, the potential to differentiate into functional cells and the safety and efficacy of transplantation therapy have been the key scientific problems to be solved in the basic theory and practical application of stem cells. Hair follicle is one of the appendages of skin, originated from the interaction between epidermis and mesenchymal, self-renewal in life, it is an embryoid organ existing in adult body. In addition to the hair follicle contains epidermal stem cells and melanin stem cells. They also contain mesenchymal stem cells with multiple differentiation potential. These pluripotent stem cells have the ability to self-renew and differentiate into other cells, compared with other types of mesenchymal stem cells. Hair follicle stem cells have the advantages of rich sources, convenient access, no age and gender restrictions, and no ethical issues. First of all, this study explored a simple and feasible method to obtain hair follicle multipotential stem cells. By using tissue mass culture method, the multipotential hair follicle stem cells were isolated and cultured from the extracted human hair. The phenotypes of hair follicle multipotent stem cells were identified by immunocytochemistry and flow cytometry. The results showed that the multipotential hair follicle stem cells expressed a relatively specific surface marker CD29, CD4, CD90, CD105 and SH-2. CD73 SH-4) surface antigens were not expressed in CD31, CD34, CD45, HLA-DRK15 and other surface antigens. The method of passage, expansion, cryopreservation and resuscitation of hair follicle multipotential stem cells was established. Finally, the multipotential hair follicle stem cells were successfully induced to osteoblasts. Differentiation of adipocytes. The conditions of differentiation of multipotential hair follicle stem cells into neuron-like cells were explored. The neuron-specific enolase (NSEs) was detected by immunocytochemistry before and after induction. Neurofilament protein (NF-LN), glial fibrillary acidic protein (GFAP), nestins, 尾 -tubulin 鈪，

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