大鼠附睾脂肪垫脂肪源性干细胞的分离培养及分化潜能  

Isolation,culture and differentiational potential of adipose-derived stem cells from rat epididymal fat pads

作　　者：张丽华[1,2] 汤银娟[1,3] 刘杰明[4] 杨林[1] 董为人[1] 郭家松[1] 王海红[1] 戴翔[1] 宋建武[1] 陈英华[1] 肖应庆[1] 

机构地区：[1]南方医科大学基础医学院组胚教研室,广东省广州市510515 [2]佛山科学技术学院组胚教研室,广东省佛山市528000 [3]湘南学院组胚教研室,湖南省郴州市418000 [4]顺德桂洲医院神经外科,广东省顺德市528305

出　　处：《中国组织工程研究与临床康复》2009年第14期2685-2690,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省自然科学基金(05004719)~~

摘　　要：背景:相对于骨髓基质干细胞,脂肪源性干细胞来源丰富,取材方便,干细胞丰度高,极易培养,具有多向分化潜能,有望成为组织工程、细胞治疗以及基因转染良好的源细胞,但目前对脂肪源性干细胞的研究尚处于初级阶段。目的:拟在体外分离培养大鼠脂肪源性干细胞,并探讨其分化潜能。设计、时间及地点:细胞学体外观察,于2008-06/2009-01在南方医科大学组胚教研室和南方医院临床医学实验中心完成。材料:SPF级成年雄性SD大鼠1只,由南方医科大学实验动物中心提供。方法:无菌条件下切取大鼠双侧附睾尾脂肪垫,胶原酶消化法分离培养脂肪源性干细胞,胰蛋白酶消化法传代扩增。取第4代脂肪源性干细胞接种到96孔板,5×104个细胞/皿,待细胞贴壁后分成3组:成骨诱导组加入成骨培养基,成脂诱导组加入成脂培养基,对照组仍用基础培养基,培养14d。主要观察指标:脂肪源性干细胞的形态变化、体外增殖能力、细胞免疫化学鉴定结果。脂肪源性干细胞成骨和成脂分化能力。结果:体外培养的脂肪源性干细胞易扩增,传代后以长梭形细胞为主,呈旋涡状排列,克隆样生长;经多次传代仍能保持较强的增殖能力,细胞生长曲线呈"S"形;细胞表面标志CD44和CD49d呈阳性表达,CD106呈阴性表达。脂肪源性干细胞经成骨诱导7d后,碱性磷酸酶染色呈阳性;成脂诱导3d后,油红O染色示胞浆内有脂滴出现;对照组细胞碱性磷酸酶染色呈阴性,无脂滴存在。结论:从大鼠附睾脂肪垫内分离的脂肪源性干细胞易于培养和传代扩增,在特殊条件下可分化为成骨细胞和脂肪细胞。BACKGROUND： Compared with bone marrow stromal stem cells, adipose-derived stem cells （ADSCs） are easily and abundantly collected and cultured. With multi-directional differentiation potential, ADSCs are good sources for tissue engineering, cell therapy and gene transfection. However, studies concerning ADSCs are in eady stage. OBJECTIVE： To isolate, culture and identify rat ADSCs in vitro, and to investigate its differentiation. DESIGN, TIME AND SETTING： The cytology, in vitro study was performed at the Department of Histology and Embryology of Southern Medical University, and Medical Experiment Center of Nanfang Hospital from June 2008 to January 2009. MATERIALS： One adult male SPF Sprague Dawley rat was provided by Animal Experimental Center, Southern Medical University. METHODS： Collagenase digestion was used to isolate ADSCs from rat epididymal fat pads under sterile condition. ADSCs were passaged and amplified by the trypsin digestion. At the fourth passage, ADSCs were incubated in a 96-well plate, 5× 10^4 cells per dish. After cell adherence, ADSCs were assigned into three groups. ADSCs in the osteoblast induction group were incubated in the osteogenic medium. ADSCs in the adipocyte induction group were incubated in the adipogenic medium. ADSCs in the control group were incubated in the basic medium. The incubation time was 14 days. MAIN OUTCOME MEASURES： The following parameters were measured： morphologic changes and in vitro proliferation of ADSCs, results of immunocytochemical staining, osteogenic and adipogenic differentiation of ADSCs. RESULTS： In vitro cultured ADSCs were easily amplified, and mainly spindle-shaped, whirlpool-shaped arranged, and clone-like growth after passage. Following many passages, ADSCs could maintain strong proliferative capacity. Cell growth showed ＂S＂ shape. ADSCs were positive for CD44 and CD49d, but negative for CD106. Following 7-days osteogenic induction, ADSCs showed a positive reaction for alkaline phosphatase staining. Following 3-days adipogen

关 键 词：脂肪源性干细胞 培养 诱导 免疫细胞化学 

分 类 号：R394.2[医药卫生—医学遗传学]