体外诱导犬骨髓间充质干细胞向上皮样细胞分化的初步研究

发布时间：2018-06-03 05:56

本文选题：组织工程 + 干细胞　；参考：《第二军医大学》2006年硕士论文

【摘要】：目的探讨体外诱导犬骨髓间充质干细胞(Bone marrow mesenchymal stemcells,BMSCs)向上皮样细胞分化的可能性和基本条件。方法采用密度梯度离心和贴壁培养相结合的方法分离提纯犬骨髓间充质干细胞,观察其生物学特性,体外培养扩增后,用上皮细胞诱导培养基诱导培养,并与犬食管上皮细胞(Esophageal epithelial cells,EECs)直接接触共培养,每日在相差显微镜下观察细胞形态的变化,培养第7、14天,通过免疫荧光检测、免疫细胞化学染色和流式细胞仪鉴定上皮细胞特异性标志物广谱细胞角蛋白(Pan-cytokeratin,CK)和上皮膜抗原(Epithelial Membrane Antigen,EMA)的表达。结果获得的犬BMSCs体外增殖速度快,具有多向分化潜能,细胞纯度高、数量多。含20ng/ml表皮生长因子(Epidermal growth factor,EGF)的无血清角化细胞培养基(Serum-free Keratinocyte Medium,K-SFM)能够在体外诱导犬BMSCs向上皮样细胞分化,分化程度与EGF的浓度及诱导时间有关。与犬EECs直接接触共培养,犬BMSCs可以分化为上皮样细胞。结论本实验提示犬BMSCs在体外能够向上皮样细胞分化,有望成为组织工程食管的种子细胞。
[Abstract]:Objective to investigate the possibility and basic conditions of inducing the differentiation of canine bone marrow mesenchymal stem cells (BMSCs) into epithelioid cells in vitro. Methods Canine bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation and adherent culture. The cells were cultured directly with esophageal epithelial cells (Esophageal epithelial cells). The morphological changes of the cells were observed under phase contrast microscope every day. The cells were cultured for 714 days and were detected by immunofluorescence. Immunocytochemical staining and flow cytometry were used to identify the expression of Pan-cytokeratin CKand Epithelial Membrane antigenase (EMA), a specific marker of epithelial cells. Results the canine BMSCs had the advantages of high proliferation rate, multidirectional differentiation potential, high cell purity and large number of cells. Serum-free Keratinocyte Medium-K-SFM, a serum-free Keratinocyte medium containing 20ng/ml epidermal growth factor (EGF), could induce canine BMSCs to differentiate into epithelioid cells in vitro, and the differentiation degree was related to the concentration and time of EGF. Canine BMSCs can differentiate into epithelioid cells by direct contact with canine EECs. Conclusion this study suggests that canine BMSCs can differentiate into epithelioid cells in vitro and may become seed cells of tissue engineering esophagus.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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