【作者】 雷霆华；

【导师】 安晓荣； 侯健；

【作者基本信息】 中国农业大学 ， 生理学， 2005， 硕士

【摘要】 近年来,对于成体干细胞的研究越来越深入,其中皮肤干细胞对于皮肤损伤治疗等细胞治疗以及转基因核移植等方面有重要的意义。本文对于绵羊皮肤类干细胞的分离,培养和鉴定进行了探索,旨在寻找成功分离和培养皮肤类干细胞的方法,为体细胞核移植提供一个高效和稳定的供体细胞来源。同时对于从皮肤组织分离细胞和进行培养进行了一定研究,并成功分离和培养出成纤维样细胞,建立了稳定的绵羊皮肤来源的成纤维细胞培养体系。 实验选用3月龄绵羊耳尖皮肤组织和90日龄绵羊胎儿皮肤组织作为材料,用胰蛋白酶消化培养和组织块贴壁培养两种方法分离出原代细胞,然后采用条件培养液(含有10ng/ml表皮生长因子,5μg/ml的胰岛素,0.5μg/ml氢化考的松,1.8×10^-4mol/L腺嘌呤,10%标准胎牛血清的高糖DMEM)和饲养层细胞（绵羊胎儿成纤维细胞）培养,成功分离到了呈集落化生长的细胞克隆,并传至10代,细胞倍增次数达到70—90次,说明此细胞克隆具有类似干细胞的增殖能力。 碱性磷酸激酶（AKP）的染色结果显示这些细胞克隆呈红褐色,而周围细胞和饲养层细胞未着色或着色很浅,说明这些细胞克隆的内源性碱性磷酸酶活性较高,从而表明这些细胞克隆处于未分化或低分化状态。利用成纤维细胞和上皮细胞特异性抗体免疫组化染色,结果表明:这些细胞克隆中没有成纤维细胞特征,同时也不具备上皮细胞的特征;而同期传代的非克隆状细胞呈现成纤维细胞特异性染色。 进一步利用β1整合素抗体进行免疫组化检测,这些类干细胞克隆染色呈阴性,说明这些类干细胞不是表皮干细胞。是否属于其它种类的干细胞需要进一步鉴定。 总之,本论文研究结果表明,从绵羊皮肤组织中可以分离出呈集落化生长的类干细胞克隆,并且初步检测显示可能是一类分化程度低的干细胞,但完全的鉴定仍需要进一步试验确证。更多还原

【Abstract】 Recent years, More and more rearches have been done in adult stem cells （ASCs） .Among various ASCs, Stem cells in skin play a important role in cell therapy and transgenic nulear transfer. The focus of this dissertation work is to investigate the isolation, culture and evaluation of stem-like cells in sheep, and to search for a new source of the somatic nuclear transfer donor cells . Meanwhile, the work also has researched the isolation and culture of cells derived from sheep skin.We get the fibroblast cells from skin tissue and established a stable culture system.3 months sheep’s ear tips and 90 days sheep fetal skin tissues have been used as the source of the stem like cells. The method of 0.25% trpysin digest and the tissues adherence were used to get primary cells.Condition medium(Additional 10 ng/ml EGF,5μg/ml Insulin,0. 5μg/ml HICE,l. 8 ×10^-4 mol/L Adenine, 10%FCS in high glucose DMEM medium) and feeder cells have been used to culture 3D-colony cells. The 3D-colony can be passaged 10 times, and the cell doubling time is about 70-90. The result has shown that the 3D-colony cells might have high multiplivation capability as stem cells..The result of AKP test showed that the 3D-colony cells taken the russety colour .The other cells including feeder cells and around cells have not been dyed. It suggests that the 3D-colony cells should have a high activity of AKP, might be in the non-differentiation or low differentiation state. Immunostaining in 3D-colony cells showed neither labeling for vimentin nor labeling for cytokeratin. Immunostaining in the cells from the same passage of the 3D-colony cells showed uniform labeling for vimentin and no labeling for cytokeratin, suggesting that these cells were fibroblast cells.Monoclonal anti- β -1 Intrgrin has been used to lable the 3D-colony cells, and none positive can be observed.This avidence indicate that the 3D-colony cells should not be ESCs. Advanced tests should be done to identify which kind of stem cell it is.In conclusion, this dissertation study showed that it should be viable to isolated the 3D-colony cells which is characteristic to stem cell .And preliminary tests have showed that the 3D-colony cells might be a kind of stem cell. But, the completely identification need more tests to prove.更多还原