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Cre/loxP位点特异性重组酶系统联合SV40LTAg诱导人黑素细胞可逆性永生化
Reversible Immortalization of Human Melanocytes Mediated by SV40 Large T Antigen and Cre/loxP Site-Specific Recombinase System
【作者】 王鹰;
【导师】 郝飞;
【作者基本信息】 第三军医大学 , 皮肤病与性病学, 2006, 博士
【摘要】 白癜风是一种常见的后天性局部脱色素性皮肤病,据国内外统计其发病率约为0.5~2%,且有逐年增高趋势。白癜风的病因和发病机制尚未明确,病理表现为皮损局部表皮和毛囊中的黑素细胞(melanocytes,MC)减少或缺失,从而使黑素合成减少或缺失,临床表现为境界清楚的色素脱失斑。近年来,国外学者研究表明,白癜风患者皮损局部和全身均可检测到针对MC表面抗原的自身抗体和细胞毒性CD8+T细胞,认为白癜风是一种自身免疫性脱色素性皮肤病。白癜风的治疗比较困难,目前有很多治疗方法,主要包括光化学治疗、内服药物治疗、外用药物治疗以及外科治疗,前三种治疗方法都存在疗效个体差异大、副作用大等不足,外科治疗主要包括自体表皮移植、自体MC移植和异体MC移植等方法,其中自体MC移植是疗效最肯定、副作用最小的方法,因而外科治疗已经成为白癜风治疗研究的热点。目前临床应用于移植治疗的MC主要来源于患者自身正常皮肤分离的MC体外培养。成人表皮中MC含量极少,只占表皮细胞的2%左右,正常情况下体外培养的MC生长缓慢、增殖能力极为有限,常需使用促MC生长因子如经典的霍乱毒素(cholera toxin,CT)、十四烷酰佛波醇乙酯(12-o-tetradecanoylphorbol-13-acetate,TPA)以及近年逐渐被重视的碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、转铁蛋白、氢化可的松、牛垂体提取物等。我们应用黑素细胞专用培养基M254及添加剂HMGS(包含TPA、bFGF、转铁蛋白、氢化可的松和牛垂体提取物等),进行成人正常表皮MC的分离与培养,经系列生物学鉴定细胞表型和生物学功能正常,同时观察到MC体外传代最多不超过10代,能得到的MC数量有限,远不能满足临床移植治疗所需,极大地限制了MC移植治疗的临床应用。因此,MC的来源已经成为白癜风及其它脱色素性皮肤病外科治疗发展的瓶颈,需要积极探索MC体外大量培养的有效方法和途径,为自体MC移植提供安全、有效且可大量反复使用的移植供体。组织工程学的原理是利用人工的功能性组织替代功能障碍的器官或组织,其中,细
【Abstract】 Vitiligo is a common skin disease characterized by the development of white macules and patches associated with local melanocyte loss and/or destruction. The incidence of vitiligo is approximately 0.5 to 2 percent and increasing year by year. The etiology of vitiligo is not completely known, but the observation of circulating antimelanocytic antibodies and CD8 (+) T lymphocytic infiltrations at the margins of lesions in the majority of patients has lent support to the hypothesis that it is an autoimmune disease, whch results in therapeutic difficulties. Patients affected with this ailment have received the benefit of topical and oral medications to fight depigmentation, oral 8-methoxy psoralens and sunlight exposure was initially the only really effective treatment– described over half a century ago– but now there is a whole change of therapies for treating depigmented skin. Though there have many methods to treat vitiligo, such as photochemotherapy, drug treatment and topica therapy, etc., most of them have some side effects or individual difference in therapeutic outcoming. Over the past three decades, diverse surgical methods have been developed. Epidermal grafting, minigrafting, thin dermoepidermal grafts, epidermal suspensions, individual hair gafts and in vitro cultured melanocytes either with epidermal membranes or with pure melanocyte suspensions, are the basic procedures published to date, although a few modifications of some techniques have also been described. Each of these methods has been reported with varying degrees of successful repigmentation and also with a few side effects.Cultured pure melanocyte grafting is a successful repigmentation method of vitiligo therapy. The cultured melanocytes are isolated primarily from normal epidermis of vitiligo patients. The contents of melanocytes in epidermis are very limited which account approximately only 2 percent in total epidermal cells. In vitro cultured melanocytes have no reproductive activity and limited growth ability, some growth factors were used usually in
【Key words】 Cre/loxP site-specific recombinase system; Simian virus 40 large T antigen (SV40LTAg); Human melanocytes; Immortalization; Reversible immortalization; Transfection; Gene expression; Cell biological characters; Tumorigenicity; Animal model;