利用人毛囊干细胞构建组织工程表皮的实验研究-外科学(整形外科)专业论文.docxVIP

上海交通大学博士学位毕业论文熟，细胞表面的微绒毛较长而密，克隆形成率高于对照组(PO．05)， 上海交通大学博士学位毕业论文 熟，细胞表面的微绒毛较长而密，克隆形成率高于对照组(PO．05)， 克隆形成大，表现为干细胞的特征；毛囊干细胞在饲养层细胞上生长 良好，培养至12代检测，免疫组化染色均表达K15、B卜整合素及 P63，P0至P12均表达K15 mRNA，但克隆形成率逐渐降低，毛囊干 细胞在CGM上生长良好。毛囊干细胞--CGM修复裸鼠后1周切片示创 面有新生上皮覆盖，表皮分化良好，而对照组残留较大未愈创面。修 复后一月和三月可见对照组收缩较实验组显著。 结论：消化法适合毛囊外根鞘细胞的体外培养，为进一步分离纯 化培养毛囊干细胞奠定了基础；利用Ⅳ型胶原差速贴壁法可初步分 选、纯化人毛囊干细胞；通过在饲养层上接种毛囊干细胞，结合传代 过程中中性蛋白酶消化可大量扩增毛囊干细胞；体外构建的毛囊干细 胞一CGM复合物能修复裸鼠皮肤缺损。 关键词毛囊干细胞，组织工程，分选，表皮替代物，皮肤缺损 VI 上海交通大学博士学位毕业论文EXPERIMENTAL 上海交通大学博士学位毕业论文 EXPERIMENTAL STUDY oF HUML▲N HAIR FoLLICLE STEM CELLS IN THE FABRICATION oF TISSUE ENGINEERED EPIDERMIS ABSTRACT The aim of this study is to establish a method for isolation and expansion of human hair follicle stem cells from outer root sheath(ORS) in vitro．To isolate human hair follicle stem cells from ORS cells，type IV collagen coated dishes were utilized to separate cells based on their different adhering potential．After enrichment，the hair follicle stem cells were expanded and characterized．Finally,the hair follicle stem cells were seeded on the chitosan-gelatin membrane(CGM)，and the cell—scaffold construct was utilized to repair the nude mouse skin defects． Single hair follicles were obtained after digestion skin with dispase， then the ORS cells were harvested from enzyme digestion or explants culture．Cells were observed under microscope and cell growth curve was achieved by cell counting；To enrich the stem cells，hair follicle ORS cells were cultivated on type IV collagen coated dishes．After 20 min， Ⅶ 上海交通大学博士学位毕业论文nonadherent 上海交通大学博士学位毕业论文 nonadherent cells were removed to a new plate and keep in culture as control group；adherent ceils were harvested as experimental group·Cell8 were ch眦cterized by immunfluorescent stains and flow cytometry wimmonoclonal antibody against cytokeratin 15(K15)、131-integrin alld inv01ucrin．The microstructure of both cell types was observed under electron“croscope．Colony．forming efficiency(CFE)was measured· The hair follicle stern cells were