兔羊膜成体干细胞培养及体外定向诱导神经细胞分化  

Differentiation of rabbit amnion-derived stem cells induced into neural cells

作　　者：徐蔚[1] 王震[1] 曲申[1] 刘思维[1] 王培军[1] 

机构地区：[1]同济大学附属同济医院眼科,上海200065

出　　处：《中华眼底病杂志》2013年第3期296-299,共4页Chinese Journal of Ocular Fundus Diseases

基　　金：上海市科委自然科学基金（07ZR14123）;上海市卫生局面上科研项目（2007011）

摘　　要：目的观察体外分离、培养的兔羊膜成体干细胞（ADsc）定向诱导神经细胞的分化结果。方法新西兰雌兔羊膜分离、培养ADSC。实时定量聚合酶链反应（PCR）检测羊膜ADSC的纤连蛋白（Fibronectin）、巢蛋白（Nestin）、波形蛋白（Vimentin）mRNA的表达水平。单克隆形成实验证实羊膜ADSC是否具有自我复制能力。培养成功的ADSC中加入神经分化诱导培养基诱导神经细胞分化。诱导分化5d后，免疫荧光染色检测伊微管蛋白（tubulin）和胶质纤维酸性蛋白（GFAP）的表达。结果培养的ADSC在24h后逐渐从羊膜组织中移行出；72h可见明显的多角形细胞富集在羊膜组织周边；120h后多角形细胞致密地分布于羊膜四周。分裂后的子代细胞形态同父代细胞。单克隆形成实验结果证实羊膜ADSC具有自我复制能力。实时定量PCR检测结果显示，羊膜ADSC的Nestin、Vimentin、FibronectinmRNA分别是脾脏细胞同种分子表达量的15．79、1．91、7．65倍，两者差异有统计学意义（Z=-5．243、-3．972、-2．524，P〈0．05）。免疫荧光染色结果显示，β-tubulin在大部分细胞的胞质中表达；GFAP在部分细胞的细胞质中表达。结论羊膜ADSC具有自我复制能力，在合适条件下能够分化成神经元和神经胶质细胞。Objective To observe the differentiation effect of rabbit amnion-derived stem cells （ADSC） induced into neural cells. Methods ADSC of New Zealand female rabbits were isolated and cultured. Its mRNA level of Fibronectin, Nestin and Vimentin were detected by real-time quantitative polymerase chain reaction. The self-replication ability of ADSC was confirmed by monoclonal formation experiments. These ADSC were further induced into neural ceils in vitro. Five days after induced differentiation, the expression of β-tubulin and glial fibrillary acidic protein （GFAP） were detected by immunofluorescent staining. Results ADSC were separated from amnion tissue gradually after 24 hours. There were polygonal cells gathered around the amnion tissue at 72 hours, and were distributed compactly around the amnion at 120 hours. The morphology of cleavage daughter cells was basically the same as parent cells. ADSC has the ability of self-replication. The Nestin, Vimentin, Fibronectin mRNA expressions in ADSC were 15.79, 1.91, 7.65 times those in spleen ceils. The differences were statistically significant （Z=-5. 243, -3. 972, -2. 524; P〈0.05）. The β-tubulin expression was found in cytoplasm of most cells. The GFAP expression was found in cytoplasm in some cells. Conclusions ADSC has self-replication ability. It can be induced into neurons and neurogliaI cells under the right conditions.

关 键 词：成体干细胞 细胞学 细胞分化 免疫学 细胞培养技术 方法 羊膜 

分 类 号：R3[医药卫生—基础医学]