碱性成纤维细胞因子对皮肤干细胞神经分化影响的研究

发布时间：2018-04-25 02:07

本文选题：表皮干细胞 + 分化　；参考：《福建医科大学》2011年硕士论文

【摘要】：目的:探讨表皮干细胞向神经细胞分化的影响因素。方法: 1.分离3-4天的SD大鼠表皮基底层细胞,利用10分钟贴壁法分离出表皮干细胞。2,用免疫组织化学法测定K15、β1整合素、CD34和巢蛋白(nestin)来鉴定培养出的表皮干细胞。3.加入不同的培养基:K-SFM、NEUROBASALTM-A和DMEM培养分离出的表皮干细胞,并观察不同培养基组细胞形态的变化。4.通过调整不同浓度的bFGF,观察表皮干细胞的形态变化。5.用K-SFM为培养基培养表皮干细胞,以表皮干细胞的细胞密度分组,分为0.5×10~7/ml组、0.3×10~7/ml组、0.1×10~7/ml组、0.5×105/ml,每组均加入20ng/ml的bFGF,观察其细胞形态发生以及细胞标记物的改变。结果: 1.利用十分钟贴壁法成功分离出表皮干细胞。分离出来表皮干细胞细胞密度大,细胞呈圆形或多角形,细胞形态清晰,胞质均匀,可见有核分裂细胞,核质比例大,其特征性标记分子K15及β整合素表达阳性。 2.表皮干细胞在NEUROBASALTM-A和DMEM培养基中均出现细胞体积增大,细胞膜边界不清,细胞核溶解、核碎裂、细胞死亡。 3.表皮干细胞在K-SFM培养基中,加入bFGF的浓度为0ng/ ml、5ng/ml、10ng/ml、15ng/ml时,细胞形态没有明显改变,呈圆形或多角形,细胞形态清晰,胞质均匀,可见有核分裂细胞,核质比例大;当加入的bFGF浓度为20ng/ml时, 24h即可见到细胞开始伸展,呈多角形、圆形或短棒样,分布比较均匀,第3-4天可看到部分细胞呈铺路石样,细胞体积增大,核质比例变小,可见有分裂核细胞,极少部分细胞呈现双极,多极细胞改变,约一周后,双极、多极细胞消失,部分铺路石样细胞形成克隆,细胞90%融合。 4.在0.3×10~7/ml组和0.1×10~7/ml组, 24h-48h可见到细胞开始伸展,呈多角形、圆形或短棒样,分布比较均匀,第5-6天可看到部分细胞呈铺路石样改变,细胞体积大,核质比例变小,可见有分裂核细胞,部分细胞呈出现双极,多极细胞改变,约一周后,双极、多极细胞增多。免疫组织化学nestin、NSE染色阳性,β1整合素、CD34染色阴性; 0.5×10~6/ml组,约3-4天后可见到散在的伸展,呈多角形、圆形或短棒样的细胞,大部分细胞一直未伸展; 0.5×10~7/ml组, 24h即可见到细胞开始伸展,呈多角形、圆形或短棒样,分布比较均匀,第3-4天可看到部分细胞呈铺路石样,细胞体积增大,核质比例变小,可见有分裂核细胞,极少部分细胞呈现双极,多极细胞改变,约一周后,双极、多极细胞消失,部分铺路石样细胞形成克隆,细胞90%融合。结论 1.10分钟快速贴壁法可以培养出表皮干细胞,表皮干细胞的特征性标记分子K15及β1整合素表达阳性,CD34、nestin表达阴性。 3.培养表皮干细胞细胞的最佳培养基为K-SFM。 4.通过bFGF可诱导表皮干细胞向神经系分化,bFGF对表皮干细胞的诱导具有细胞密度依赖性。
[Abstract]:Objective: to investigate the factors influencing the differentiation of epidermal stem cells into neural cells. Methods: 1. Epidermal stem cells (ESCs) were isolated from SD rat epidermal basal layer cells for 3-4 days. The epidermal stem cells (ESCs) were isolated by 10-minute adherent method. K15, 尾 1-integrin CD34 and nestin were determined by immunohistochemistry to identify the cultured epidermal stem cells. The epidermal stem cells (ESCs) were cultured in different culture medium: K- K SFMN NEUROBASALTM-A and DMEM, and the morphological changes of ESCs in different culture medium groups were observed. 4. Morphological changes of epidermal stem cells were observed by adjusting different concentrations of bFGF.5. Epidermal stem cells were cultured on K-SFM medium. The epidermal stem cells were divided into 0.5 脳 10~7/ml group (0. 3 脳 10~7/ml group) and 0. 1 脳 10 ~ 5 脳 10~7/ml group (0. 5 脳 10 ~ 5 / ml). The cell morphogenesis and the changes of cell markers were observed in each group. Results: 1. Epidermal stem cells were successfully isolated by ten minutes adherence method. The epidermal stem cells showed high density, round or polygonal shape, clear morphology, homogeneous cytoplasm, mitotic cells, large nuclear / cytoplasm ratio and positive expression of K15 and 尾 integrin. 2. The epidermal stem cells in both NEUROBASALTM-A and DMEM medium had increased cell volume, unclear cell membrane boundaries, nuclear dissolution, nuclear fragmentation and cell death. 3. When epidermal stem cells were added to K-SFM medium at the concentration of 0ng/ 5ng / ml 10 ng / ml 10 ng / ml 15ng / ml, the cell morphology was round or polygonal, the cell morphology was clear, the cytoplasm was uniform, mitotic cells were found, and the ratio of nucleus to cytoplasm was large. When the concentration of bFGF added was 20ng/ml, the cells began to stretch out at 24 hours. The cells were polygonal, round or short stick, and the distribution was even. On the 3-4 days, some cells showed paving stone, the cell volume increased, and the ratio of nucleus and cytoplasm became smaller. There were mitotic nuclear cells, very few cells showed bipolar, multipolar cell changes, about a week later, bipolar, multipolar cells disappeared, some paving stone like cells formed clone, 90% of the cells fused. 4. In 0. 3 脳 10~7/ml and 0. 1 脳 10~7/ml groups, 24h-48h showed that the cells began to stretch, were polygonal, round or short rod, and distributed evenly. On the 5th to 6th day, some of the cells showed paving stone changes, the cells were large, the ratio of nucleus and cytoplasm became smaller, and mitotic cells could be seen. Some cells showed bipolar, multipolar cell changes, about a week later, bipolar, multipolar cells increased. In the 0.5 脳 10~6/ml group, scattered, polygonal, round or short stick like cells could be seen after 3 to 4 days. In 0. 5 脳 10~7/ml group, the cells began to extend at 24 hours, which were polygonal, round or short stick, and distributed evenly. On the 3-4 days, some cells were found to be paving stone, the cell volume was increasing, and the ratio of nucleus and cytoplasm was smaller. There were mitotic nuclear cells, very few cells showed bipolar, multipolar cell changes, about a week later, bipolar, multipolar cells disappeared, some paving stone like cells formed clone, 90% of the cells fused. Conclusion 1. Epidermal stem cells could be cultured by rapid adherence for 10 minutes. The characteristic marker molecules K15 and 尾 1 integrin were positive and CD34 nestin expression was negative in epidermal stem cells. 3. The best culture medium for epidermal stem cells was K-SFM. 4. BFGF can induce the differentiation of epidermal stem cells into neural system.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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