【作者】 李孝建；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 生物医学工程， 2009， 博士

【摘要】 第一章鼠表皮干细胞的分离、培养及鉴定目的:建立简单、可靠的分选鼠表皮干细胞的方法,为建立体外调控表皮干细胞增殖、分化的培养体系和以表皮干细胞为种子细胞构建组织工程皮肤的研究奠定基础。方法:清洁级1-3d龄大鼠20只,采用酶消化法获取单个表皮细胞悬液,Ⅳ型胶原快速粘附法分选表皮干细胞,加入含胎牛血清、0.05mmol/L CaCl2以及表皮细胞生长因子、腺苷、胰岛素、氢化可的松、霍乱毒素等成分的复合DMEM培养基进行培养。分离细胞时,胎牛血清体积百分数为20%以维持细胞活性,第2次换液时改为10%。光镜下观察细胞生长情况;免疫组化法检测分选细胞角蛋白19、角蛋白15和P63的表达;流式细胞仪检测β1整合素和CD71、CD34的表达;细胞周期及生长曲线观察分选细胞增殖能力。结果:Ⅳ型胶原分选的粘附细胞在复合DMEM培养基中生长良好,细胞活性＞98%;培养6d后形成含100-200个细胞的大克隆,呈鹅卵石样铺满瓶底。角蛋白19、角蛋白15和P63均呈阳性表达。粘附细胞的β1整合素表达率为98.59%,CD71表达率为9.05%。94.8%的粘附细胞处于G0/G1期,细胞呈指数增长。结论:两步酶消化+Ⅳ型胶原快速粘附法可成功分选稳定生长的鼠表皮干细胞。含霍乱毒素、腺苷、胰岛素等因子的复合DMEM培养基可维持表皮干细胞的增殖能力,其中合适的血清浓度及Ca²⁺浓度至关重要。第二章不同培养体系对鼠表皮干细胞增殖、分化的影响目的:探讨不同培养体系对表皮干细胞增殖、分化的影响,建立理想的调控表皮干细胞增殖、分化的培养体系。方法:酶消化和Ⅳ型胶原快速粘附法获取鼠表皮干细胞,分别在普通培养皿、与几丁质膜生物支架材料及以几丁质膜材料作为载体植入裸鼠体内等不同培养体系下培养,观察表皮干细胞生长情况;普通培养和几丁质膜共培养4w后,表皮干细胞克隆形成率的差异;免疫组织化学染色观察表皮干细胞以几丁质膜为载体植入裸鼠体内后4w,表皮干细胞的增殖、分化情况等。结果:表皮干细胞在普通培养皿培养3d左右,细胞开始克隆增殖,12d左右融合成片,传代培养后增殖能力逐渐减低,融合成片时间逐渐延长,传代培养3-4代后细胞终末分化,失去增殖能力;几丁质膜培养表皮干细胞,2w后呈棋盘式集落生长,4w后几丁质膜上有大量的表皮干细胞小集落,集落上有大量的增殖细胞附着生长,扫描电镜下见几丁质膜纤维直径约10μm,以纤维为主,上下两层呈纵横排列成十字孔,孔间有大量表皮干细胞集落。几丁质膜支架材料培养表皮干细胞4w后,其克隆形成率明显高于普通培养皿培养（P＜0.05）。表皮干细胞几丁质膜支架植入裸体内培养4w后,细胞大量增殖形成“巢状”排列,在“表皮巢”周围,可见皮肤附件结构形成。结论:表皮干细胞在体外普通培养皿培养,可增殖生长,但维持增殖时间较短;与几丁质膜材料培养,可较长时间地维持表皮干细胞的增殖特性;植入体内后表皮干细胞大量增殖,并可分化形成毛囊等皮肤附件结构。第三章鼠表皮干细胞与角质形成细胞蛋白质组差异表达的初步研究目的:研究鼠表皮干细胞和角质形成细胞的蛋白质组学差异,为进一步研究调控表皮干细胞的增殖、分化提供参考信息。方法:酶消化和Ⅳ型胶原快速粘附法分选鼠表皮干细胞和角质形成细胞,提取其中总蛋白质,Bradford法测定蛋白质浓度;双向凝胶电泳技术展示蛋白质分子的表达,Image ScannerⅡ透射扫描仪及Labscan扫描软件进行扫描获取图像,利用ImageMaster 2D Elite 5.0分析软件对图像进行强度校正、点检测、背景消减、匹配,以获取两种细胞的差异蛋白点;切取差异蛋白点,MOLDI-TOF-TOF质谱仪进行质谱分析,利用软件Mascot distiller过滤基线峰、识别信号峰,Mascot软件搜索NCBInr数据库（http://www.matrixscience.com）,寻找匹配的相关蛋白质,同时查询其功能。结果两种细胞的2-DE蛋白表达谱具有良好的重复性和可比性,表皮干细胞与角质形成细胞的电泳图谱平均蛋白质点数分别为（930±15）、（982±18）个,匹配点数分别为（798±11）、（850±13）个,匹配率分别为85.81%、86.56%;两种细胞蛋白质间匹配点数（868±8）个,匹配率是76.98%;找到差异蛋白质点11个,其中只在表皮干细胞表达或高表达的有8个,只在角质形成细胞表达或高表达的有3个。差异蛋白质点分别进行MALDI-TOF/TOF-MS质谱分析,鉴定出11个有意义的蛋白质。其中表皮干细胞表达或表达明显增高的8个蛋白质包括RhoGDP解离抑制蛋白a（dissociation inhibitor（GDI））)、翻译延长因子1（Translationelongation factor-1）、增殖细胞核抗原（Proliferating cell nuclear antigen,PCNA）、谷胱苷肽S-转移酶（Glutathione S-transferase Mu 2,GSTM-2）、醛糖还原酶（Aldosereductase,AR）等;角质形成细胞表达或表达明显增高的3个蛋白质,包括Nitrilasehomolog 2（Nit 2）、膜联蛋白A5（Annexin A5）和Ubiquitin-protein hydrolase等。结论:利用双向电泳和质谱分析技术,成功分离和鉴定出鼠表皮干细胞和角质形成细胞差异表达的蛋白质。这些差异蛋白质可能与表皮干细胞和角质形成细胞体外增殖、分化特性的差异有一定关系,为进一步研究调控表皮干细胞增殖、分化的培养体系奠定了基础。第四章以表皮干细胞为种子细胞构建组织工程皮肤修复裸鼠全层皮肤缺损创面目的:探讨应用表皮干细胞作为种子细胞构建组织工程皮肤,并修复全层皮肤缺损创面的可行性。方法:以鼠尾腱纤维为原料,酸溶解法提取Ⅰ型胶原,与6-硫酸软骨素按一定比例混合后冷冻抽干成膜,0.25%戊二醛溶液交联制成“人工真皮”。“人工真皮”包被Ⅳ型胶原后,表面接种Ⅳ型胶原快速粘附法分选获得的表皮干细胞构建组织工程皮肤,并将其移植于裸鼠全层皮肤缺损创面。术后每天观察伤口愈合情况,并分别于术后1、2、4、6、8、10w取创面组织HE染色检查。结果:构建的“人工真皮”为半透明的薄膜,厚度约为0.2mm,具有一定的抗张力强度。扫描电镜检测,其内部为比较均匀的网状结构,孔径大小在50-100um左右。构建的组织工程皮肤移植裸鼠皮肤全层缺损创面后3d,与创面贴合紧密,第7d,移植物与创面进一步融合,第14d创面基本愈合。10w后,移植创面愈合平整、有弹性,接近正常皮肤。组织学观察:移植后7d,表皮干细胞开始增殖、分化,真皮内有大量的成纤维细胞及少量炎性细胞,可见新生毛细血管。移植后第2w,移植创面完全上皮化,新生上皮含4-5层细胞;移植后第6w,移植创面已形成8-10层细胞,且真皮层有丰富的毛细血管增生。结论:在胶原+6-硫酸软骨素制成的胶原海绵“人工真皮”上接种表皮干细胞,可构建具有较强增殖能力的组织工程皮肤,并可覆盖全层皮肤缺损创面。且愈合创面平整,收缩少,组织学结构接近正常皮肤。提示以表皮干细胞为种子细胞构建组织工程皮肤,有可能解决传统方法存在的种子细胞老化问题,可望成为一种较为理想的组织工程皮肤。更多还原

【Abstract】 Chapter 1-Isolation,culture and identification of rat epidermal stem cells in vitroObjective:To establish a simple and reliable method for separating rat epidermal stem cells from skin in vitro and provide more information for study proliferation and differentiation of epidermal stem cell.Methods:Twenty 1-3 days-old rats of clean grade were selected to prepare epidermal stem cells.Single epidermal stem cell suspension were obtained by enzyme digestion. The target cells were harvested by rapidly adherence on typeⅣcollagen plate and were cultured in complex DMEM containing fetal bovine serum（FBS）,0.05 mmol/L CaCl2,epidermal cell growth factor,adenosine,insulin,hydrocortisonum and choleratoxin,ect.Volume percentage of FBS was 20%to maintain call activity when cells were primarily isolated and was 10%when the medium was changed at the second time.Cell growth was observed under light microscope.Keratin 19,Keratin 15 and P63 integrin of separated cells were detected by immunohistochemistry.β1 integrin and CD71,CD34were measured by flow cytometry.Proliferation activity was examined by cell cycle and growth curve.Results:The typeⅣcollagen screened cells grew well in the complex DMEM, resulting in a cell activity of over 98%.Six days later,a clone containing 100-200 cells was detected,showing cobble-stone-like.The rapidly adherent cells were positive for P63,keratin 19and keratin 15.The expression rates ofβ1 intergrin and CD71 were respectively 98.59%and 9.05%.Cell cycles showed that about 94.8% cells were in resting state/pre-DNA-synthetic gap（G0/G1 phase）.The growth-curve showed that the rapidly adherent cells presented exponential growth.Conclusion:Rat epidermal stem cells were successfully collected by enzymatic digestion and rapidly adherence on typeⅣcollagen.Complex DMEM containing choleratoxin,adenosine and insulin can maintain proliferation activity of epidermal stem cells.Suitable serum and Ca²⁺ concentrations are also important.Chapter 2-Effects on proliferation and differentiation of rat epidermal stem cells cultured in different system conditionsObjective:To explore the effects on proliferation and differentiation of epidermal stem cells cultured in different system and establish a culture system which can regulate and control the proliferation and differentiation of epidermal stem cells.Methods:The rat epidermis stem cells isolated based on rapidly adherent on typeⅣcollagen cultured in three different system conditions as follows,cultured in common glass dish,cultured with chitin membrane,seeded on the chitin membrane functioning as a cell carrier and implanted subcutaneously into nude mice.Cell colonies were examined under the inverted microscope and the growth of the stem cells on the chitin was observed by the fluorescence microscope and the scanning electron microscope.The colony forming efficiency（CFE） after cultured 4 weeks in these two culture system were measured,the proliferation ability of the epidermal stem cells implanted into mice were evaluated by immunohistochemistry.Furthermore,the influence of the bionic chitin membrane leaching solution to cells was also detected.Results:The separated epidermal stem cells grew well cultured in vitro.The cell began to clonal expansion 3^rd day and confluent dish in 12 days.The proliferation ability gradually decreased during serial subculture and lost after 5-time passage culture in common glass culture dish.Cultured in chitin membrane.,checkerboard cell colonies were visualized on the chitin membrane in 2-4 weeks and massive stem cell colony multiplication was observed under the fluorescence microscope and the scanning electron microscope.The colony forming efficiency after cultured 4 weeks in chitin membrane was also higher than that in culture dish.The stem cell proliferated large amount and formed "epidermal nest" after implanted subcutaneously into nude mice.The chitin membrane leaching solution showed slight cell proliferation at 1:8-1:512 of leaching diluted solution.F=0.781,P＞0.05.The statistics had showed non-significance difference.Conclusion:Epidermal stem cells could have good proliferation ability for a long time cultured with chitin membrane and could be used as seed cells for tissue engineering-skin.It provides evidence for further more animal experiments.Chapter 3-Comparative proteomic analysis of rat epiidermal stem cells and keratinocytesObjective:To study the differential proteomics expression of rat epidermal stem cells and keratinocytes and provide clues for regulating the proliferation and differentiation of epidermal stem cells in vitro.Methods:The single epidermal cells suspension were obtained by enzyme digestion and the epidermal stem cells or keratinocytes were harvested based on rapidly adherence on typeⅣcollagen.Cell total protein were extracted and the concentration were measured according Bradford method.The protein expression maps were presented by two-dimensional electrophoresis（2-DE） and the differential expressed protein spots were analyzed by Image Master 2D Elite 5.0 software.The different-expressed protein spots were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF-MS） after in-gel protein digestion,Proteins were identified by searching the peptide mass profiles in a public available NCBInr databases（http://www.matrixscience.com）.Results:There were good reproducibility and comparability between two cell lines from the 2-DE protein expression maps,Average protein spots were 982±18 in keratinocYtes and 930±15 in epidermal stem cells,the matched spots were 850±13 and 798±11 in these two types cell,and the average matching rate were 86.56%and 85.81%.There were 886±8 matching protein spots between the two types cell,and the average matching rate was 76.98%.Eleven differential protein spots were identified between two type cell.Eight protein spots were only or higher expressed in epidermal stem cells and three protein spots were only or higher expressed in keratinocytes. There were 11 significant proteins successfully identified by MALDI-TOF-MS. Among the 11 proteins,8 proteins were only or higher expressed in epidermal stem cell,including Rho GDP dissociation inhibitor（GDI）) alpha,Translation elongation factor-1,PCNA,GSTM-2,Aldose reductase,ect,whereas 3 proteins only or higher expressed in keratinocyte,including Nitrilase homolog 2,Annexin A5 and Ubiquitin,protein hydrolase,ect.Conclusion:There were some differences in protein expression between epidermal stem cell and keratinocyte,these different protein may be related to the ability of proliferation and differentiation of two type cell.Chapter 4-Repair of full-thickness skin defect wounds in nude mice with engineering-skin composite of epidermal stem cells as seed cells Objective:To study the feasibility of constructing the tissue-engineering skin composite of epidermal stem cells as seed cell and repairing the full-thickness skin defect wounds in nude mice.Methods:Type I collagen were extracted from rat tail tendon with acetic acid and then mixed with chondroitin-6-sulfate to form a collagen-chondrointin-6-sulfate membrane and also demonstrated the membrane "artificial dermis".The tissue-engineering skin was constructed by seeding epidermal stem cells isolated by rapidly adherence on typeⅣcollagen on the artificial dermis substrate and then grafted On the full-thickness skin defect wounds in nude mice.The grafted wounds were observed daily and the specimens were harvested on the 1 to 10 weeks after grafting for histological examination.Results:The artificial dermis was a semitransparent membrane and with a network structure,the pore size was about 50 to 100um.The tissue-engineering skin achieved good adherence to full-thickness defect wounds on the 3 rd day of grafting and better on the 7rd day.The wounds healed about 14 days after grafting and had a good contour of skin with less contraction and scar formation after 10 weeks of grafting. Histological examination showed the epidermal stem cells proliferated promptly and formed 4-5 cell layers after grafting of 14 days and 8-10 cell layers of 6 weeks,large amount of fibroblasts,capillary vessels and little inflammation were observed in the dermis.Conclusions:Tissue-engineering skin composite of epidermal stem cells as seed cell and collagen-6-sulfate had potential prospects in repairing full-thickness skin defect wounds with advantage of good proliferation.更多还原