中文摘要：

目的：目前已知端粒酶活性的丧失及其增殖相关基因表达的改变是造成多种成体干细胞体外复制和扩增受限的主要原因，而端粒酶反转录酶对端粒酶活性起关键作用。体外分离培养人胚胎、少儿、成人3种不同皮肤来源的表皮干细胞，对比观察不同发育阶段端粒酶反转录酶表达的差异。 方法：实验于2005—09／2007—04在南昌大学第一附属医院烧伤研究所完成。①对象：因创伤等原因致意外流产的妊娠24-26周龄胎儿皮肤标本，4～12周岁少儿及25～45岁成年人烧伤整形植皮手术剩余皮片标本，分别由南昌大学第一附属医院产科、烧伤科提供，产妇与烧伤患者对治疗及实验均知情同意，实验经医院医学伦理委员会批准。②实验方法：取胎儿、少儿和成年人的全层皮肤，用胰蛋白酶和EDTA联合消化法分离表皮，胶原快速贴附法纯化人表皮干细胞，用未黏附的角质细胞作为对照，以含表皮生长因子、角质细胞无血清培养液组成的表皮干细胞培养基进行体外培养。③实验评估：通过β1整合素、角蛋白19、p63免疫细胞化学染色对培养细胞进行鉴定，计算克隆形成率。以免疫细胞化学染色法和图像定量分析法检测3种不同皮肤来源的表皮干细胞端粒酶反转录酶的表达差异。结果：①细胞生长特征：分离培养的人表皮干细胞呈明显克隆性生长，克隆形成率高于角质细胞对照组（P〈0．05）。②表皮干细胞的鉴定：细胞克隆经免疫细胞化学染色后，β1整合素、角蛋白19、p63均呈阳性表达。③端粒酶反转录酶免疫细胞化学染色及定量表达：不同皮肤来源的表皮干细胞端粒酶反转录酶均呈阳性，但表达强度不同，人胚胎〉少儿〉成人。3种皮肤来源的表皮干细胞平均吸光度值和阳性面积值均随年龄增加而逐渐降低，人胚胎〉少儿〉成人（P〈0．05）。 结论：人胚

英文摘要：

AIM： The main reasons for limiting the replication and expansion ability of the adult stem cells in vitro are the loss of telomerase activity and the changes of related proliferation gene expression, in which telomerase reverse transcriptase plays a key role. This article investigates the isolation, culture and identification of the epidermal stem cells derived from human fetus, child and adult skins, and to compare the telomerase reverse transcriptase expression of the different developmental periods of the epidermal stem cells. METHODS： Experiments were performed at Institute of Burn, First Affiliated Hospital, Nanchang University from September 2005 to April 2007. ① The skin samples of fetus were taken from the accidental abortion （24-26 weeks gestational age） and provided by Department of Maternity, the First Affiliated Hospital of Nanchang University. The skin samples of children （4-12 years） and adults （25-45 years） came from the operation of bum and plastic and provided by Department of Burn, the First Affiliated Hospital of Nanchang University. The use of these samples were obtained with consent from parturients and patients and approved by Medical Ethics Committee of the Hospital. ②The sample was disposed with Trypsin-EDTA respectively and got the epidermis, and then we digested the epidermis and harvested the epidermal cells. The type 1V collagen was used to isolate and purify the human epidermal stem cells, and the epidermal growth factor and the keratinocyte serum free medium were used to culture the cells. The keratinocyte cells were used as the control groups. ③ The β1 integrin, keratin19 and p63 transcription factor were identified by the immunocytochemical stain. The colony forming efficiency was calculated. The expressions of the telomerase reverse transcnptase in the different developmental periods of epidermal stem cells were detected by the immunocytochemistry stain and the image quantitative analysis. RESULTS： ①The cultured cells revealed clonal growth. The clon