人皮下脂肪干细胞的成骨、成脂分化诱导及鉴定
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中国组织工程研究 第19卷 第32期 2015–08–06出版
Chinese Journal of Tissue Engineering Research August 6, 2015 Vol.19, No.32
ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 5155
www.
CRTER .org
肖建红,女,1980年生,四川省绵阳市人,羌族,2012年中山大学毕业,博士,主治医师,主要从事造血干细胞抑制、干细胞与组织工程,以及免疫性血小板减少性紫癜的临床研究和基础研究。
通讯作者:张阳春,硕士,主治医师,中山大学附属第一医院东院下肢骨科,广东省广州市 510700
中图分类号:R394.2 文献标识码:A 文章编号:2095-4344 (2015)32-05155-07 稿件接受:2015-07-01
Xiao Jian-hong, M.D., Attending physician, Department of Internal Medicine, Eastern Branch of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 51000, Guangdong Province, China
Corresponding author: Zhang Yang-chun, Master, Attending physician, Department of Lower limbs Orthopedics, Eastern Branch of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 51000, Guangdong Province, China
Accepted: 2015-07-01
人皮下脂肪干细胞的成骨、成脂分化诱导及鉴定
肖建红1,张阳春2,张常然1,杨 兴2(中山大学附属第一医院东院,1普通内科,2下肢骨科,广东省广州市 510700)
文章亮点:
1 实验通过密度梯度离心和贴壁筛选法相结合,对人脂肪干细胞进行分离、体外培养及扩增。
2 实验发现第5代脂肪干细胞比较适合进行成脂及成骨诱导分化,以确定其多向分化潜能。
3 DAPI 是一种简单、有效的标记人脂肪干细胞的方法,DAPI 不损伤细胞活性,对细胞标记率高。 关键词:
干细胞;脂肪干细胞;人来源的脂肪干细胞;细胞分化;脂肪组织;细胞培养;细胞鉴定;国家自然科学基金
主题词:
干细胞;皮下脂肪;细胞分化;细胞,培养的
基金资助:
国家自然科学基金青年项目(81500091);广东省自然科学基金博士启动项目(2015A030310022);广州市黄埔区科技计划项目(201444-02)
摘要
背景:脂肪干细胞是从脂肪组织中分离得到的一种具有多向分化潜能的间充质干细胞,在组织工程中比骨髓间充质干细胞具有更多优势,是当今的研究热点之一。
目的:在体外建立一种分离纯化人皮下脂肪来源脂肪干细胞的方法,进行体外培养扩增及诱导其向成骨、成脂细胞分化。
方法:通过密度梯度离心和贴壁筛选法相结合,对人脂肪干细胞进行分离、体外培养及扩增。倒置显微镜下观察人脂肪干细胞的细胞形态和生长特性。选取第2代及第5代细胞,用CCK-8法检测细胞活性,绘制细胞生长曲线,用流式细胞仪检测细胞表面抗原标记。选取第5代细胞,分别进行成脂及成骨诱导分化,以确定其多向分化潜能。
结果与结论:①采用密度梯度离心和贴壁培养相结合方法,可成功从人脂肪组织中获得纯度较高的人脂肪干细胞。②人脂肪干细胞经历了生长滞缓期、对数增殖期和生长平台期,符合正常细胞的生长规律,且其生长具有对数生长期,其倍增时间较短。③人脂肪干细胞表达干细胞表面抗原标记,具有低免疫原性,且具有成脂成骨多向分化潜能。④DAPI 是一种简单、有效的标记人脂肪干细胞的方法,DAPI 不损伤细胞活性,对细胞标记率高。
肖建红,张阳春,张常然,杨兴. 人皮下脂肪干细胞的成骨、成脂分化诱导及鉴定[J].中国组织工程研究,2015,19(32):5155-5161.
doi:10.3969/j.issn.2095-4344.2015.32.013
Human subcutaneous adipose-derived stem cells: osteoblastic/adipogenic differentiation and identification
Xiao Jian-hong 1, Zhang Yang-chun 2, Zhang Chang-ran 1, Yang Xing 2 (1Department of Internal
Medicine, 2Department of Lower limbs Orthopedics, Eastern Branch of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 51000, Guangdong Province, China)
Abstract
BACKGROUND: Adipose-derived stem cells are a kind of mesenchyam stem cells with multipotent differentiation capacity, which have more advantages than bone marrow mesenchymal stem cells in tissue engineering research. OBJECTIVE: To establish a method to isolate and purify adipose-derived stem cells from human subcutaneous adipose tissues followed by in vitro amplification and osteoblastic/adipogenic differentiation.
METHODS: Adipose-derived stem cells were isolated from human subcutaneous adipose tissue and cultured by density gradient centrifugation and adherent culture. Cell morphology and growth features were observed under inverted microscope. Adipose-derived stem cells at passages 2 and 5 were selected for viability measurement using cell counting kit-8 method, and then cell growth curves were drawn. The immunophenotype identification was analyzed by flow cytometry. Passage 5 cells underwent osteoblastic/adipogenic induction to confirm the multi-differentiation potential.
RESULTS AND CONCLUSION: (1) Using density gradient centrifugation and adherent culture method,
high-purity human adipose-derived stem cells can be successfully isolated from human adipose tissues. (2) The growth process of human adipose-derived stem cells includes stagnant phase, logarithmic phase and plateau phase, which meets the growth rhythm of normal cells. Moreover, the population doubling time is shorter. (3).