体外分离培养兔脂肪干细胞的生物学特性

Biological properties of rabbit adipose-derived stem cells isolated and cultured in vitro

目的:建立兔脂肪干细胞的体外分离培养方法,鉴定并分析其生物学特性。方法:实验于2005-06/2006-03在华中科技大学同济医学院附属同济医院骨科实验室和同济医院中心实验室完成。选用雌性新西兰大白兔,兔龄4个月,无菌取出雌兔双侧腹股沟脂肪垫,贴壁法及Ⅰ型胶原酶法分离出兔脂肪干细胞,并进行体外培养,取5代以后的兔脂肪干细胞观察其形态特征,并绘制细胞生长曲线及倍增时间,同时进行细胞过氧化物酶、苏丹黑B、碱性磷酸酶、α-醋酸萘酚酯酶与糖原化学染色;用流式细胞仪检测其细胞表面标志并进行细胞周期分析。结果:①兔脂肪干细胞原代及传代细胞形态:原代及传代的兔脂肪干细胞均呈长梭形或多边形贴壁生长,生长分化活跃。②兔脂肪干细胞的生长曲线及倍增时间:传代的兔脂肪干细胞生长曲线呈“S”形,群体倍增时间为22h。③兔脂肪干细胞的化学染色特点:兔脂肪干细胞的过氧化物酶、苏丹黑B染色呈阴性,而碱性磷酸酶、α-醋酸萘酚酯酶与糖原染色呈阳性。④兔脂肪干细胞表面标志及细胞周期:流式细胞仪检测结果显示兔脂肪干细胞的表面标志物CD29,CD44,CD90,CD105,CD166表达阳性,而CD31,CD34,CD45和CD106表达阴性。细胞周期分析显示,G0/G1期占80.25%,G2/M期占6.03%,S期占13.72%。结论:本次实验所分离出来的兔脂肪干细胞在体外具有生长稳定,增殖较快的特点。

AIM： To investigate the methods of isolating and culturing rabbit adiposede-rived stem cells （ADSCs）, and to identify and analyze the biological characteristics of rabbit ADSCs. METHODS： The experiment was performed at the Laboratory of Department of Orthopaedics and Central Laboratory of Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from June 2005 to March 2006. Adipose was collected from the inguinal region of 4 months old female News Zealand rabbits and the ADSCs were isolated with adherent method and type Ⅰ collagenase method and cultured in vitro. The morphology of ADSCs after passage 5 was analyzed. The growth curve and doubling time were drawn. At the same time, peroxydase （POX）, Sudan black （SB）, alkaline phosphatase （ALP）, alpha-naphthol acetate esterace （α-NAE） and periodic acid schiff （PAS） chemical staining were performed. The surface antigens and cell cycle were analyzed with flow cytometer （FCM）. RESULTS： ①Formation of primary and passage of ADSCs： ADSCs had spindle and polygon shape adherent growth, and their growth and differentiation were active. ②Growth curve and doubling time of ADSCs： The growth curve was like ＂S＂ shape. The doubling time of ADSCs was about 22 hours. ③Cytochemistry feature of ADSCs： The ADSCs were homogeneously negative for POX, SB and positive for ALP, α-NAE and PAS. ④Surface antigens and cell cycle of ADSCs： Flow cytometer showed that ADSCs expressed CD29, CD44, CD90, CD105, CD166 but did not express CD31, CD34, CD45 and CD106. Cell cycle included G0/G1 phase： 80.25%, G2/M phase： 6.03%, S phase 13.72%. CONCLUSION： The ADSCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro.