小鼠附睾脂肪干细胞的分离培养及鉴定张鉴清1，季佳霖2，崔新明2，张祺3，李艳茹21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，吉林大学基础医学院病理学系，吉林省长春市130021；3吉林大学，吉林省长春市130021 Isolation, culture and identification of adipose-derived stem cells from mouse epididymisZhang Jian-qing1, Ji Jia-lin2, Cui Xin-ming2, Zhang Qi3, Li Yan-ru21 the Fourth Hospital of Jilin University, Changchun 130011, Jilin Province, China;2 the Key Laboratory of Pathobiology of Ministry of Education, Department of Pathology, School of Basic Medical Sciences, Jilin University, Changchun 130021, Jilin Province, China;3 Jilin University, Changchun 130021, Jilin Province, China 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。
中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：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；Abstract：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 aseptically cutting. The tissue was digested using collagenase. Adipose-derived stem cells were separated and purified by using one digestion, multiple collection 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 ofadipose-derived stem cells was identified by flow cytometry. Adipose-derived stem cells were induced to differentiate into adipocytes and osteocytes using cell inductors. Compatibility of adipose-derived stem cells and collagen 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 scroll 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 microvilli on the cell surface. The nuclei were big in size. Some organelles 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 small lipid droplets were seen in the cytoplasm of adipose-derived stem cells. The small 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 refractive granular material deposits at that field after dyeing with alizarin red. Scanning electron microscope revealed that adipose-derived stem cells were spread on the collagen scaffold. Results suggested that adipose-derived stem cells isolated by this method could amplify in vitro and stably subcultured. Under a certain inducing condition, adipose-derived stem cells could differentiate into osteoblasts and adipocytes, which showed a good compatibility with collagen scaffold.中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：Key words：stem cells; adipose tissue; epididymis; mice, inbred BALB C; cells, cultured;中图分类号:R394.2基金资助: 吉林省科技发展计划青年科研基金资助项目(20090192)通讯作者:李艳茹，副教授，硕士生导师，吉林大学病理生物学教育部重点实验室，吉林大学基础医学院病理学系，吉林省长春市130021作者简介: 张鉴清，男，1972年生，吉林省舒兰市人，汉族，副主任医师，主要从事手足外科的临床研究工作。
引用本文:张鉴清,季佳霖,崔新明等. 小鼠附睾脂肪干细胞的分离培养及鉴定[J]. 中国组织工程研究, 2014,18(28): 4535-4541.Zhang Jian-qing,Ji Jia-lin,Cui Xin-ming et al. Isolation, culture and identification of adipose-derived stem cells from mouse epididymis [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(28): 4535-4541.。