小鼠附睾脂肪干细胞的分离培养及鉴定  

Isolation,culture and identification of adipose-derived stem cells from mouse epididymis

作　　者：张鉴清[1] 季佳霖 崔新明[2,3] 张祺[4] 李艳茹[2,3] 

机构地区：[1]吉林大学第四医院,吉林省长春市130011 [2]吉林大学病理生物学教育部重点实验室 [3]吉林大学基础医学院病理学系,吉林省长春市130021 [4]吉林大学,吉林省长春市130021

出　　处：《中国组织工程研究》2014年第28期4535-4541,共7页Chinese Journal of Tissue Engineering Research

基　　金：吉林省科技发展计划青年科研基金资助项目(20090192)~~

摘　　要：背景:脂肪干细胞作为一种新的成体干细胞,具有来源丰富、取材容易、增殖能力强等优点,受到人们越来越多地关注。目的:体外分离培养小鼠附睾脂肪组织中的脂肪干细胞,并鉴定其生物学特性。方法:无菌切取小鼠附睾处脂肪组织,采用胶原酶进行消化,利用1次消化多次收集与差速贴壁法分离纯化脂肪干细胞。倒置显微镜及透射电子显微镜观察脂肪干细胞的形态。绘制脂肪干细胞的生长曲线。流式细胞术检测脂肪干细胞的免疫表型。加入细胞诱导剂对脂肪干细胞进行成骨及成脂肪的诱导分化。扫描电子显微镜观察脂肪干细胞与胶原支架材料的相容性。结果与结论:倒置显微镜下可见脂肪干细胞呈长梭形或成纤维细胞样,密集排列成漩涡状或成束状交织,可在体外稳定传至第9代。透射电子显微镜下可见脂肪干细胞表面有丰富的微绒毛结构,细胞核体积大,细胞质内可见线粒体、粗面内质网等细胞器。脂肪干细胞表达CD44、CD29,不表达CD34。经成脂肪诱导剂诱导后,多数脂肪干细胞胞质内可见小脂滴,油红O染色可将小脂滴染成红色。经成骨诱导剂诱导后,在脂肪干细胞生长密集区域内的细胞结构模糊、细胞间界限不清楚。经茜素红染色后,可见较多大小不一的折光率较强颗粒状结构沉积。扫描电子显微镜观察到脂肪干细胞呈铺展状生长于胶原支架材料上。结果表明该方法分离出的脂肪干细胞能在体外扩增并稳定传代,在一定诱导条件下,能够分化为成骨细胞和脂肪细胞并且与胶原支架具有良好的相容性。BACKGROUND：As a new kind of adult stem cells, adipose-derived stem cells get more and more attention, because of rich source, drawing materials easily and powerful proliferation. OBJECTIVE：To isolate and culture adipose-derived stem cells from the epididymal adipose tissue in mice, and to identify their biological characteristics. METHODS：Adipose tissue was obtained from epididymis in mice by aseptical y cutting. The tissue was digested using col agenase. Adipose-derived stem cells were separated and purified by using one digestion, multiple col ection method and differential adhesion method. The morphology of adipose-derived stem cells was observed using inverted microscopy and transmission electron microscopy. Growth curve of adipose-derived stem cells was drawn. Immunophenotype of adipose-derived stem cells was identified by flow cytometry. Adipose-derived stem cells were induced to differentiate into adipocytes and osteocytes using cellinductors. Compatibility of adipose-derived stem cells and col agen scaffold material was observed using scanning electron microscope. RESULTS AND CONCLUSION：Adipose-derived stem cells exhibited long spindle-like or fibroblast-like appearance, grew intensively and arranged in scrol and fascicular shape. In vitro, adipose-derived stem cells could be passaged to passage 9 under the inverted microscope. Under the transmission electron microscope, adipose-derived stem cells showed abundant microvil i on the cellsurface. The nuclei were big in size. Some organel es were seen in cytoplasma, such as mitochondria and rough endoplasmic reticulum. Adipose-derived stem cells expressed CD44 and CD29, did not express CD34. After inducing by inductor, many smal lipid droplets were seen in the cytoplasm of adipose-derived stem cells. The smal lipid droplets were dyed red with oil red O. After induction of osteogenic inductor, the boundary line among adipose-derived stem cells was not clear and the structure of cells was fuzzy in the growth-intensive areas. There were many strong refra

关 键 词：干细胞 脂肪干细胞 小鼠 附睾 脂肪组织 差速贴壁 培养 

分 类 号：R394.2[医药卫生—医学遗传学]