【作者】 甘露；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 硕士

【摘要】 皮肤组织有着很强的再生能力,表皮基底层中的干细胞终身不断自我更新、持续分化以取代终末分化细胞,从而进行组织结构的更新。终末分化细胞的死亡、脱落与基底干细胞的分裂维持一定的平衡,这是维持正常的表皮组织结构和细胞内环境稳定的基本要求[1]。随着分子生物学、细胞生物学、组织工程学及生物工程学等学科的发展,表皮干细胞(Epidermal stem cell, ESC)凭借其特有的生物学优势在基因治疗、细胞治疗中越来越受到重视,成功地分离、培养表皮干细胞对临床上创伤修复、皮肤癌的研究也起着至关重要的作用[2-8]。皮肤创面的修复是一个多细胞与细胞因子参与的复杂过程。创伤早期,创缘周围产生大量渗出液,多种细胞因子可能调节创伤修复过程中的细胞反应,影响细胞增殖、迁移、细胞外基质合成和释放等。间质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)是由间质细胞分泌,并被证实在多种成体组织损伤修复过程中介导成体干细胞向受损区域迁移,参与创伤修复。因此我们推测SDF-1很可能在皮肤组织创伤后也有表达,并进而影响表皮干细胞在创缘的分布变化及数量改变。目的1.利用改良的人胎盘Ⅳ型胶原快速粘附法从人包皮组织获得ESC并进行无血清培养,观察其体外培养的生长特点,寻求一种理想的人ESC体外分离培养技术。2.观察SDF-1对体外培养的人ESC增殖、迁移等生物学活性的影响,并初步探讨其作用机制。3.以ESC为种子细胞构建三维皮肤等价物(three–dimensional skin equivalents, TDSE)全层创伤模型,观察皮肤创面发生过程中创缘SDF-1的表达及ESC的动态变化,分析二者相关性。方法1.按照Dispase II-胰酶两步消化法从人包皮组织获得ESC,以人胎盘Ⅳ型胶原按照5μg/cm2的密度包被培养瓶,将ESC以无血清培养基(defined keratinocyte-serum free medium,DK-SFM)进行体外培养,观察其生长及形态变化、克隆形成,并对原代培养的ESC表面标记物β1-integrin、CK19、PCNA进行细胞免疫组织化学染色,对CD49f和分化标记CD71双染进行免疫荧光鉴定。2.通过MTT实验检测不同浓度SDF-1对ESCs增殖的影响,以及在相同浓度SDF-1作用下另添加不同浓度AMD3100对ESCs增殖的影响;通过划痕实验,检测不同浓度SDF-1对ESCs趋化迁移的作用趋势,及SDF-1促迁移运动的时间效应。3.将成纤维细胞与鼠尾胶原、DMEM培养基、血清按比例混匀制成“真皮等价物”。经过72h培养,原代人角质形成细胞接种在真皮等价物的表面。继续培养1周后,抬高到气-液面,使ESC分化为各层,最终形成“三维皮肤等价物”随后,用液氮冷冻的金属棒冻伤模型,建立离体全层冻伤创面。在冻伤后3、7天、10天取材免疫组化染色观察创缘SDF-1的表达变化;另添加外源性SDF-1和其受体阻断剂AMD3100作用后,在冻伤后3、7天取材免疫组化染色观察创缘ESCs的分布变化。结果1.Ⅳ型胶原分选的粘附细胞在DK-SFM培养基中生长良好,细胞胞体呈圆形,细胞核大,核质比大,培养9天能行成含200个细胞以上的大克隆,呈铺路石样铺满瓶底。β1-integrin、CK19、PCNA均呈阳性表达。免疫荧光法双重标记显示细胞为α6briCD71dim细胞。2. SDF-1α浓度依赖性的促进ESC的增殖、迁移作用。在100ng/ml时促增殖和迁移的作用最显著,AMD3100则能有效阻断上述增殖和迁移的作用。3.利用ESC为种子细胞成功构建了三维皮肤等价物全层冻伤模型。HE染色显示正常未冻伤区细胞分层良好,冻伤创缘区表皮细胞坏死增多,表皮层变薄。免疫组织细胞化学染色显示,冻伤发生后,创缘周围组织中SDF-1表达量不断增加。但是表达量的增加不是持续的,第7天即为高峰,随后表达量迅速减少。至愈合晚期,局部无SDF-1表达。伴随创面愈合进程,ESC的定位分布不象正常皮肤那样呈单层片状、散在分布于表皮基底层,而是向创面迁移聚集的趋势更加明显,且在多层内出现散在β1整合素阳性细胞。组织形态学上表现为新生表皮组织较正常组织明显增厚。结论1.对人ESC的体外无血清培养方法进行观察发现通过Ⅳ型胶原快速粘附法能有效的分离ESC。2.外源性的SDF-1α对ESC增殖和迁移的促进作用,这些影响是通过SDF-1α及其受体CXCR4的相互作用实现的。3. TDSE离体创面愈合过程中,SDF-1在创缘呈可控性表达;SDF-1通过趋化效应引起ESC在创缘的异位分布,并且与增殖效应一起共同作用加速实现创面的上皮化过程。更多还原

【Abstract】 Skin tissue has high ability of regeneration. Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish the worn out or damaged cells in tissue during life. The balance between cell division and cell loss is essential for epidermal homeostasis and must be maintained for life. With the developments of molecular biology、cell biology、tissue engineering and biotechnology, epidermal stem cells(ESCs) are become more and more important in gene therapy and cell therapy because of their distinguished biological characters. How to isolate and culture ESCs is of great importance not only for the study on tissue repair in trauma but also on skin cancer.Many cells and cytokines are involved in the complex process of epidermal wound healing. At the early stage, a great deal of exudate was generated in wound margin. Variety of cytokines play key roles in wound healing process, mainly including regulating various cell reactions, releasing of extracellular matrix, as well as affecting cell proliferation, migration and synthesisSDF-1 was secreted by the stromal cells and could mediate adult stem cells migration toward damaged region in a variety of adult tissue injuries and repair processes. Therefore, we speculated that SDF-1 might be expressed around wound, and had effect on the distribution and number of ESC in this area.Objectives:1. Epidermal stem cells (ESCs) were isolated from foreskins,separated based on collagen type IV adhesiveness, and cultured in serum free medium in vitro. Their growth was observed so we can establish an ideal method of ESC sorting and culture.2. To observe the effects of stromal cell derived factor-1(SDF-1) on ESCs proliferation and migration, and to explore its mechanism.3. To build a three-dimensional equivalent of full thickness skin wound model, observe SDF-1 expression in the process of skin wound healing and then analyze their correlation. Method:1. ESCs were isolated from human foreskins by dispase II-trypsin combined digestion. the culture dishes were coated with human placenta collagen typeⅣ, then ESCs were planted on human placenta collagen typeⅣ-coated dishes and cultured in Defined Keratinocyte Serum Free Medium(DK-SFM). The change on ESCs morphology, colony forming efficiency and the expression of surface markers (CD71,α6-integrin,β1-integrin, CK19 and PCNA) were detected.2. ESCs proliferation under the effect of different concentration of SDF-1 or AMD3100 was measured by MTT assay. ESCs migration and chemotaxis under the effect of different concentration of SDF-1 were measured by wound scrap experiment.3. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a rat tail collagen matrix, were grown on cell culture dishes. After 72 hour, primary human keratinocytes were seeded on this base. One week later, an air-lift transition was performed, leading to the differentiation of the ESCs, which were macroscopically visible as "three-dimensional skin equivalents" after a couple of days. We then used liquid nitrogen freezing metal rod to establish a full-thickness frostbite wounds in vitro. The distribution of SDF-1 on the wound margin, as well as the distribution of ESC under the effect of SDF-1 solely or combined with AMD3100, were detected by immunohistochemical staining on 3rd, 7th, and 10th day of frostbite,.Result:1. ESCs were enriched based on collagen type IV adhesiveness. They exhibited many characteristics predicted of ESCs such as quiescence, small blast-like morphology, and the greatest long-term cell regenerative output in vitro.β1-integrin, CK19 and PCNA expression were positive. Almost all cells from a single colony wereα6briCD71dim staining by immunofluorescence.2. The proliferation and migration of ESCs dependent on SDF-1αconcentration. The promoting effect of SDF-1αwas the most significant in 100ng/ml. AMD3100 was able to effectively block the effect of SDF-1α.3. Using ESCs as seed cells, we successfully constructed a full-thickness skin frostbite model in the three-dimensional skin model. HE staining showed the well developed skin structure in normal area, as well as thinner skin and increased ESCs in wound margin of frostbite. As shown by Immunohistochemical staining, the expression of SDF-1 in the wound margin increased with time and peaked in day 7, followed by a rapid decline. There was on SDF-1 expression in wound area in the late healing stage. Instead of single-sheet and scattered in the basal layer just like in normal skin, the distribution of ESCs was tend to gather around wound. Theβ1-integrin-positive cells appeared in both basal and upper layers of skin. The newborn epidermal tissue was thicker than normal tissue.Conclusion:1. ESCs can be selected and cultured in vitro successfully based on collagen type IV adhesiveness.2. Exogenous SDF-1αcould promote the proliferation and migration of ESCs by interacting with its receptor CXCR4.3. A controllable expression of SDF-1 was detected in wound margin during skin repair on TDSE. SDF-1 could induce the ectopic distribution of ESCs with its chemotaxis, and accelerate skin epithelization with the proliferation promoting and chemotactic effects.更多还原