【作者】 蒋洁；

【导师】 张建湘；

【作者基本信息】 中南大学 ， 人体解剖学与组织胚胎学， 2010， 硕士

【摘要】 目的：建立一种简便、快捷、经济、高效脐带沃顿胶间充质干细胞的获取方法。方法：脐带剔除血管和外膜后,余下的胶冻状组织,即沃顿胶(Wharton’s jelly, WJ)剪碎至1-2mm3大小,按一定比例均匀放置于含FasGrow培养基的6孔板中,37℃、5%的CO2培养箱内培养。待细胞游出后,用胰蛋白酶消化传代培养并入液氮冻存。取第5代细胞行HE、Gimesa染色,观察细胞形态。结果：沃顿胶组织静置培养7天左右,部分细胞从组织块中游出,多为双突起的长梭形、短棒状或扁平形的成纤维样细胞；14天左右,细胞形态变为均一的纺锤形,细胞生长达到单位面积的70%-80%。传代后可维持未分化状态并稳定增殖,细胞倍增时间为3d左右,体外增殖达20代以上；P3代以后,细胞形态较单一。复苏后的细胞能很好生长,细胞形态,生长特点与冻存前基本一致。结论：种植法简单、快捷、经济、高效,能从脐带沃顿胶中分离出大量成纤维样细胞。目的：判断从脐带沃顿胶中分离出的成纤维样细胞是否是脐带沃顿胶间充质干细胞,并确定其分化潜能。方法：取第5代细胞进行免疫组化,检测其免疫表型。用含地塞米松、维生素C、β-甘油磷酸钠的FasGrow培养基诱导该成纤维样细胞向成骨细胞分化,并进行碱性磷酸酶染色、Von Kossa染色及四环素染色鉴定成骨细胞特性；用含地塞米松、维生素C、异丁基甲基黄嘌呤的FasGrow培养基诱导该成纤维样细胞向成脂细胞分化,并进行油红O染色鉴定成脂细胞特性；用含β-巯基乙醇的FasGrow培养基诱导该成纤维样细胞向神经样细胞分化,并通过特异性抗体鉴定其分化能力。结果：表面标记分析显示：CD44、CD105、CD133、MHC-Ⅰ呈阳性,CD34、CD45呈阴性,从脐带沃顿胶中分离出的成纤维样细胞为间充质干细胞；体外诱导实验表明：该细胞具有体外成脂肪、成骨和成神经样细胞分化的能力。结论：从脐带沃顿胶中分离出的大量成纤维样细胞表达间充质干细胞的表面标记,并具有多向分化潜能,可作为组织工程中种子细胞的重要来源。更多还原

【Abstract】 Objective:To establish a simple, fast, economic, and efficient procurement method for mesenchymal stem cells from Wharton jelly of human umbilical cord.Methods:remove blood vessels and the adventitia from umbilical cord, shred the remaining jelly organization (Wharton’s jelly, WJ) to the size of 1-2mm3, and then place them evenly in 6-well plate with FasGrow culture medium according to a certain percentage. Culture in incubator under 37℃, 5% CO2. After cells swim out, digest with trypsin and then subculture and cryopreservate with liquid nitrogen. Carry on HE and Gimesa staining with the 5th generation of cells, observe morphology.Results: The Wharton jelly organization kept still for seven days or so. After that, some cells swam out from the tissue block. Mostly of them were double-protruding long spindle-shaped, short rod-like or flat-shaped fibroblast-like cells; 14 days or so, the cells presented in a uniform spindle morphology shape, and reached 70%-80% confluence. The cells can maintain undifferentiated state and stability of proliferation after passage, and can expand to at least 20 passages in vitro, with a cell doubling time of about 3d. After P3 generation, cells presented in uniform morphology. The recovered cells grew well, and were basically the same as those before cryopreservation on morphology and growth characteristics.Conclusion:The planting method is simple, fast, economic and efficient, with which, it is able to isolate a large number of fibroblast-like cells from the Wharton jelly of human umbilical cord. Objective:To decide whether the isolated fibroblast-like cells from Wharton jelly of human umbilical cord are mesenchymal stem cells, and to prove their differentiation capability.Methods:carry on immunohistochemistry for the 5th generation cells to detect the immune phenotype. Induced differentiate the fibroblast-like cells into bone cells with FasGrow culture medium containing dexamethasone, vitamin C,β-glycerophosphate, and identify the bone characteristics by alkaline phosphatase staining, Von Kossa staining and tetracycline staining; Induced differentiate the fibroblast-like cells into fat cells with FasGrow culture medium containing dexamethasone, vitamin C, isobutyl-methyl xanthine, and identify the adipogenic characteristics by oil red O staining; Induced differentiate the fibroblast-like cells into neural-like cells with FasGrow culture medium containingβ-mercaptoethanol, and identify the characteristics by check specific antibodies.Results:The surface phenotype analysis showed:CD44, CD 105, CD133, MHC-Ⅰwere positive, CD34, CD45 were negative, and the isolated fibroblast-like cells were MSCs; Culture experiment proved that:the cells were capable of differentiating into fat, bone, and nerve-like cells in vitro.Conclusion: The fibroblast-like cells isolated from Wharton jelly of human umbilical cord expresses mesenchymal stem cell surface phenotype, and has great differentiation capability, which can be used as an important source of seed cells in tissue engineering.更多还原