人多潜能毛囊干细胞的分离培养及其向神经元样细胞诱导分化的研究

发布时间：2018-01-15 18:14

  本文关键词：人多潜能毛囊干细胞的分离培养及其向神经元样细胞诱导分化的研究　出处：《吉林大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 毛囊 干细胞 多潜能性

【摘要】： 疾病对人类的健康和生存构成重大威胁、是世界各国普遍面临的最重要的社会问题。干细胞移植技术的问世和进展为传统治疗手段认为是“不治之症”的患者带来了新的生机和希望。但干细胞的来源、自我更新潜能的维持、向功能细胞分化的潜能以及移植治疗的安全性、有效性一直是干细胞基础理论和实际应用研究中急待解决的关键科学问题。 毛囊是皮肤的附属器之一,起源于表皮与间充质间的相互作用,终生自我更新,是存在于成体内的胚胎样器官。新近研究发现,毛囊中除了含有表皮干细胞和黑色素干细胞外,还含有具有多向分化潜能的间充质干细胞。这些多潜能干细胞具有自我更新和定向分化成其它细胞的能力。与其他类型间充质干细胞相比,毛囊干细胞具有来源丰富,获取方便,不受年龄性别限制,不涉及伦理问题等优点。 首先,本研究探索了一种简便可行的获取毛囊多潜能干细胞的方法,通过应用组织块培养法,从拔取的人毛发中分离培养出多潜能毛囊干细胞,并应用免疫细胞化学法结合流式细胞仪技术,对毛囊多潜能干细胞的表型进行了分析鉴定,结果显示多潜能毛囊干细胞表达间充质干细胞相对特异的表面标记物CD29、CD44、CD90、CD105(SH-2)、CD73(SH-4)表面抗原,不表达CD31、CD34、CD45、HLA-DR、K15等表面抗原。其次,本实验初步建立了毛囊多潜能干细胞传代、扩增、冻存与复苏的方法。最后,本实验成功诱导多潜能毛囊干细胞向成骨细胞、脂肪细胞分化。探索了多潜能毛囊干细胞向神经样细胞诱导分化的条件,通过免疫细胞化学法检测诱导前后神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-L)、胶质纤维酸性蛋白(GFAP)、神经巢蛋白(nestin)、神经微管蛋白Ⅲ(β-tubulinⅢ)、S100钙结合蛋白B(S100B)及环核苷磷酸二酯酶(CNPase)的表达水平。认为双丁酰环磷腺苷(Dibutyryl Cyclic AMP)联合3-异丁基-1-甲基黄嘌呤(IBMX)是目前本实验条件下诱导人毛囊干细胞向神经元细胞分化最为有效的方法。为后期利用多潜能毛囊干细胞移植修复损伤神经、帕金森及其它神经系统病变奠定了必要的前期工作基础。
[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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