【作者】 王联群；

【导师】 刘德伍；

【作者基本信息】 南昌大学 ， 外科学， 2010， 博士

【摘要】 表皮干细胞是皮肤组织特异性干细胞,具有强大增殖潜能和自我更新能力,是皮肤及其附属器发生、修复、改建的关键性源泉,有望成为皮肤组织工程理想的种子细胞。.了解表皮干细胞增殖分化调控机制是其应用的重要前提和理论基础。国内外现有研究主要集中在干细胞壁龛、整合素家族、Wnt信号转导通路、Notch信号转导通路、c-Myc和p63、p16基因的表达等途径上,且已取得一定进展。近期研究发现,表皮干细胞离体后其增殖性能与在体有很大差异,即很容易失去其干细胞特性,体外复制较为困难,限制了研究的进一步深入。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。但造成表皮干细胞这种差异的原因是否也是如此,目前尚不十分清楚。端粒酶是一种能延长端粒末端并保持端粒长度的核糖蛋白酶,它能以自身RNA为模板合成端粒DNA并加到染色体末端,补充染色体复制时丢失的端粒DNA以使端粒延长,从而延长细胞寿命甚至使其永生化。人端粒酶由一个互补于端粒DNA的RNA亚基(hTR)和具有逆转录活性的端粒酶逆转录酶(hTERT)及其它相关蛋白(TPI)组成。其中人端粒酶逆转录酶(humantelomerase reverse transcriptase, hTERT)基因的表达和转录是决定端粒酶活性的主要限速步骤。在人体,端粒酶活性存在于干细胞、生殖细胞、部分有再生能力的体细胞及绝大多数恶性肿瘤组织中。它的高活性表达是肿瘤细胞恶性增殖的一项重要条件,但其适量表达又具有延长细胞寿命的作用。随着干细胞分化的进行,端粒酶活性也随之降低,至终末分化细胞已无法测到端粒酶。上调和维持干细胞的端粒酶活性,对提高干细胞体外复制和扩增能力及揭示干细胞衰老机制都将具有重要意义。至今已有不少文献报道应用hTERT基因转染人骨髓间充质干细胞、成纤维细胞、成骨细胞、脐血间质细胞、视网膜色素细胞、内皮细胞等多种细胞来延长细胞增殖寿命的成功尝试,初步结果均表明显著延长了细胞生命周期,并保持正常表型且没有恶性转化倾向。皮肤发育生物学现有研究发现,在富含表皮干细胞的表皮基底层和生长期毛囊中均有端粒酶活性表达,提示端粒酶活性表达很可能与表皮干细胞增殖分化机制之间也存在着重要的内在联系。目前对体外培养的人表皮干细胞端粒酶活性表达特征及其与表皮干细胞增殖分化的关系仍缺乏了解。基于此,本课题采用表皮干细胞体外分离培养方法,观察hTERT基因及其蛋白和端粒酶活性在人表皮干细胞中的表达特征,探讨其对体外培养的人表皮干细胞生物学特性的作用及意义；采用脂质体介导转染法,将构建的携人端粒酶逆转录酶和增强型绿色荧光蛋白双基因的重组真核表达质粒pIRES2-EGFP-hTERT转染体外培养的人表皮干细胞,观察hTERT mRNA转录及其蛋白的表达、端粒酶活性和细胞生物学特性的变化,为其在皮肤组织工程中的应用提供新思路和理论依据。第一部分人表皮干细胞体外培养、鉴定与端粒酶逆转录酶表达目的：观察体外培养的人表皮干细胞hTERT基因及其蛋白和端粒酶活性的表达特征,探讨其对表皮干细胞生物学特性的作用及意义。方法：取因创伤等原因致意外流产的妊娠24～26周龄胎儿背部皮肤标本,由南昌大学第一附属医院产科提供,均得到产妇或家属知情同意,实验经医院医学伦理委员会批准。采用胰蛋白酶和乙二胺四乙酸联合消化法分离表皮、Ⅳ型胶原快速粘附法纯化人表皮干细胞,以含表皮生长因子、角质细胞无血清培养基组成的人表皮干细胞培养液进行体外培养。倒置相差显微镜下观察人表皮干细胞形态学变化,计算其克隆形成率,测定细胞周期,免疫细胞化学染色法行β1整合素、K19、p63和hTERT单抗检测。逆转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹法(Western blot)分别检测人表皮干细胞hTERT mRNA及其蛋白的表达,端粒重复序列扩增法(TRAP)-ELISA检测端粒酶活性。结果：分离培养的人胎儿表皮干细胞呈克隆样生长,其克隆形成率明显高于角质细胞对照组,差异有统计学意义(P<0.01)；细胞周期分析发现,82.64%的表皮干细胞处于G0/G1期；免疫细胞化学染色显示,人表皮干细胞β1整合素、K19、p63和hTERT表达均阳性；RT-PCR和Western blot检测人表皮干细胞hTERT mRNA及其蛋白表达弱阳性；TRAP-ELISA检测人表皮干细胞端粒酶活性表达弱阳性。结论：1、本实验采用胰蛋白酶联合乙二胺四乙酸消化法分离表皮、Ⅳ型胶原快速黏附法纯化培养的人胎儿表皮干细胞呈克隆样生长,大部分细胞处于G0/G1期。表明本培养体系分离培养的人胎儿表皮干细胞具有干细胞生物学特性。2、体外培养的人胎儿表皮干细胞hTERT mRNA及其蛋白表达和端粒酶活性弱阳性,提示人表皮干细胞端粒酶逆转录酶的表达与调控对维持其增殖和自我更新能力可能具有重要意义。第二部分hTERT基因转染人表皮干细胞的实验研究目的：将携人端粒酶逆转录酶和增强型绿色荧光蛋白双基因的重组真核表达质粒pIRES2-EGFP-hTERT转染体外培养的人表皮干细胞,观察hTERT mRNA及其蛋白的表达、端粒酶活性和细胞生物学特性的变化,为其在皮肤组织工程中的应用提供新思路和理论依据。方法：构建与鉴定携人端粒酶逆转录酶和增强型绿色荧光蛋白双基因的重组真核表达质粒pIRES2-EGFP-hTERT,采用脂质体介导转染法将其导入体外培养的人胎儿表皮干细胞,G418筛选法进行抗性克隆筛选。RT-PCR和Western blot分别检测人表皮干细胞hTERT mRNA及其蛋白表达,TRAP-ELISA检测端粒酶活性,倒置相差显微镜下观察人表皮干细胞形态学变化,计算其克隆形成率,MTT法测定生长曲线,流式细胞仪测定细胞周期,并行染色体核型分析。结果：构建扩增重组真核表达质粒pIRES2-EGFP-hTERT,经酶切的片段与理论预期长度相符,并转染体外培养的人胎儿表皮干细胞,24h在荧光显微镜488nm波长处可见到EGFP阳性表达细胞,整个细胞均呈绿色。经G418筛选得到阳性克隆。转入hTERT基因的质粒转染组细胞hTERT mRNA及其蛋白表达和端粒酶活性均为阳性,表达水平强于空载体组和非转染组表皮干细胞(P<0.05),空载体组和非转染组组间差异无统计学意义；质粒转染组细胞形态未发生改变,呈克隆样生长,其克隆形成率高于空载体组和非转染组细胞(P<0.05)；生长曲线显示,质粒转染组细胞较空载体组和非转染组细胞增殖迅速,生长活跃；细胞周期分析显示,转染P5代表皮干细胞仍84.60%处于G0/G1期；染色体核型分析,细胞染色体为正常二倍体核型,未发生畸变。结论：1、采用脂质体介导转染法可成功将hTERT-pIRES2-EGFP重组真核表达质粒转染体外培养的人胎儿表皮干细胞,转染细胞hTERT mRNA及其蛋白表达与端粒酶活性明显增强。3、转染hTERT基因的人表皮干细胞呈克隆样生长,增殖能力增强,生命周期延长,且大部分表皮干细胞仍处于相对静止期(G0/G1),维持了干细胞的特性,染色体核型分析未发生畸变。提示hTERT基因调控的人表皮干细胞可稳定培养,有望成为细胞移植和皮肤组织工程理想的种子细胞。更多还原

【Abstract】 As the skin tissue-specific stem cells, epidermal stem cells have strong proliferative potential and self-renewal capacity. They are also the main source for generation, reparation and rebuilding of skin, which make them seed cells in tissue engineering. Proliferation and differentiation of epidermal stem cells to understand regulatory mechanisms is an important prerequisite for their application, and theoretical basis. The research on epidermal stem cells has focus on stem cell niches, integrin family, Wnt signaling pathway, Notch signaling pathway, c-Myc and p63, p16 gene expression and other pathways, which have made progress. Recent study found that after epidermal stem cells in vitro and in vivo the proliferation of performance are very different, that is very easy to lose their stem cell properties, in vitro replication more difficult, limiting further research.. The loss of telomerase activity are known and proliferation related gene expression changes are caused by a variety of adult stem cells in vitro’ replication and the main reason for limited expansion. However, epidermal stem cells caused by the reasons for this difference is also true, is not yet clear.Telomerase is a telomere end can extend and maintain telomere length ribosomal protein, it to its own RNA synthesis of telomeric DNA as a template and added to the chromosome ends, add the missing chromosome telomere DNA replication in order to extend telomeres, thus extending life and even to immortalized cells. Human telomerase includes a telomere DNA complementary to the RNA subunit (hTR) and a reverse transcriptase activity of telomerase reverse transcriptase (hTERT), and other associated protein (TPI). Of the three subunits of telomerase, only human telomerase reverse transcriptase (hTERT) expression is coincide with telomerase activation.In humans, telomerase activity present in stem cells, germ cells, some capacity for regeneration of body cells and the vast majority of malignant tumors. Its expression is highly active malignant proliferation of tumor cells is an important condition, but the amount of expression but also has extended the role of cell life. With the conduct of stem cell differentiation, telomerase activity may also decrease to terminally differentiated cells can no longer measured telomerase. Up and maintenance of telomerase activity in stem cells, stem cells to improve the replication and amplification, and reveals the stem cell aging mechanisms will be important. Number of applications has been reported in the literature hTERT gene into human bone marrow mesenchymal stem cells, fibroblasts, osteogenic cells, umbilical cord blood mesenchymal cells, retinal pigment cells, endothelial cells and other cells to extend the life of the success of cell proliferation try. Preliminary results from both significantly prolong the cell life cycle, and maintain normal phenotype and no tendency to malignant transformation. Developmental biology of skin current study found that epidermal stem cells in the epidermis is rich in the basal layer and hair follicle growth phase in both the expression of telomerase activity, suggesting that telomerase activity may be related mechanism of proliferation and differentiation of epidermal stem cells also exist between important internal links. Present in vitro telomerase activity of human epidermal stem cells and its expression characteristics of epidermal stem cell proliferation and differentiation is still a lack of understanding of the relationship.In this topic, we observed the hTERT gene and its protein and telomerase activity in cultured epidermal stem cells in vitro. To investigate its effects on cultured human epidermal stem cell biological characteristics of the role and significance. To construct the recombinant plasmid pIRES2-EGFP-hTERT encoding human telomerase reverse transcriptase and enhanced green fluorescent protein and transfect into cultured human fetal epidermal stem cells by liposome-mediated transfection. The expression of hTERT mRNA transcription and protein, telomerase activity and biological characteristic were observed, which may offer new ideas and theories in skin tissue engineering.PartⅠIsolation, cultivation and expression of human telomerase reverse transcriptase in human epidermal stem cellsObjectives:To observe the hTERT gene and protein expression of telomerase activity characteristics of cultured human skin stem cells. To explore the biological characteristics of epidermal stem cell function and significance.Method:The skin samples of fetus were taken from the accidental abortion (24-26 weeks gestational age) which provided by the Department of Maternity, 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. The sample was disposed with Trypsin-EDTA respectively and got the epidermis, and then we digested the epidermis and harvested the epidermal cells. The type IV 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. To observe the human epidermal stem cells under inverted phase contrast microscope morphology. Theβ1 integrin, keratin 19 and p63 transcription factor were identified by the immunocytochemical stain. The colony forming efficiency were calculated. The proliferation and cycle of human epidermal stem cells were detected with flow cytometry. The expression of hTERT mRNA and hTERT protein levels were respectively measured by reserve transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The telomerase activity was detected by TRAP-ELISA.Results:The cultured cells revealed colony growth. The cloning efficiency was significantly higher than the control group keratinocytes, the difference was statistically significant. (P< 0.05). The cells instage G0/G1 accounted for 82.64% by cell cycle analysis. The (31 integrin, k19 and p63 had positive expression in the epidermal stem cells but not the keratinocyte cells. The hTERT was expressed weakly at mRNA level and protein levels in the human fetal epidermal stem cells. The telomerase activity was weakly positive.Conclusion:1. In the study, the cultured cells isolated by trypsin-EDTA digestion andⅣrapid adhesion revealed colony growth. Most cells were in the G0/G1 phase, which shows that the cultured human fetal epidermal stem cells have the stem cell biological characteristics.2. The hTERT was expressed weakly at mRNA level and protein levels in the human fetal epidermal stem cells. The telomerase activity was weakly positive. The expression and regulation of telomerase reverse transcriptase in human epidermal stem cells may be of great significance to maintain of their self-renewal capacity.PartⅡTransfection of human telomerase reverse transcriptase gene into human epidermal stem cellsObjectives:To transfect the recombinant plasmid pIRES2-EGFP-hTERT encoding human telomerase reverse transcriptase and enhanced green fluorescent protein into cultured human fetal epidermal stem cells, and to observe the hTERT gene and protein expression and telomerase activity of cultured human skin stem cells. To explore the biological characteristics of epidermal stem cell function and significance, which it offer new ideas and theories for the skin tissue engineering. Method:To construct the recombinant plasmid pIRES2-EGFP-hTERT encoding human telomerase reverse transcriptase and enhanced green fluorescent protein and transfect into cultured human fetal epidermal stem cells by liposome-mediated transfection. The positive cells were selected with G418. The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT was transfected into cultured human fetal epidermal stem cells by liposome-mediated transfection. The positive cells were selected with G418. The expression of hTERT mRNA and hTERT protein levels were respectively measured by reserve transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The telomerase activity was detected by TRAP-ELISA. The colony forming efficiency was calculated. The cell cycle of human epidermal stem cells was detected with flow cytometry. The growth curve was detected with MTT. The karyotype analysis in parallel was detected.Results:The plasmid pIRES2-EGFP-hTERT was successfully amplified and constructed recombinant eukaryotic expression. The restriction fragment length consistent with the theoretical expectations. The recombinant plasmid was transfected into cultured human fetal epidermal stem cells. The expression of EGFP-positive cells was observed at 488nm wavelength fluorescence microscope after 24h. The whole cells showed green. The cells selected with G418. The expression of hTERT mRNA and protein and telomerase activity was positive. The expression was higher than the non-transfected group and empty vector group (P<0.05). The difference was statistically significant between non-transfected group and empty vector group. The cells in transfected group did not change shape, revealed colony growth. The cloning efficiency was significantly higher than non-transfected group and empty vector group (P<0.05). Compared with non-transfected group and empty vector group, transfected group cells grew rapidly by growth curve. Cell cycle analysis showed that the cells instage G0/G1 accounted for 84.60%. The chromosomal morphology of cells was normal, diploid karyotypea, without distortion.Conclusion:1. The recombinant plasmid pIRES2-EGFP-hTERT can be successfully transfected into cultured human fetal epidermal stem cells by liposome-mediated transfection method. The expression of hTERT mRNA and protein and telomerase activity was significantly increased.2. The human epidermal stem cells transfed hTERT gene revealed colony growth, enhanced proliferation and life cycle extension. And most of the epidermal stem cells remains relatively quiescent (G0/G1), maintaining the characteristics of stem cells. The karyotype analysis of cells has no distortion. The human epidermal stem cells of hTERT gene regulation can be stably cultured, which may become ideal seed cells of tissue engineering.更多还原