体外早期培养兔脂肪源性间充质细胞的增殖能力

背景：最新研究表明人脂肪源性间充质细胞的表面标志分子及分化能力会随着培养时间的延长而有所改变。 目的：观察体外早期培养的兔脂肪源性间充质细胞形态学特点及集落形成能力。 设计、时间及地点：细胞学体外观察，于2008-01/03在复旦大学附属眼耳鼻喉科医院眼科研究所和中心实验室完成。 材料：3月龄雌性新西兰大白兔9只，由上海市银根养兔室提供。 方法：兔麻醉后取颈背处的皮下脂肪，I型胶原酶消化法获得原代细胞，接种至含体积分数为0.1胎牛血清的DMEM培养液中，细胞达80%融合时传代，取第2，3，4代细胞用于实验。 主要观察指标：倒置显微镜观察细胞形态，流式细胞仪分析细胞表型，计数细胞集落形成率。 结果：第2，3，4代兔脂肪源性间充质细胞均呈成纤维细胞样生长，体外生长迅速，CD29及PCNA均呈阳性表达，集落形成率分别为（8.0±0.6）%，（6.7±0.4）%，（4.6±0.5）%，经方差分析差异有显著性意义（F=12.18，P < 0.05）。 结论：体外早期培养的兔脂肪源性间充质细胞生长增殖旺盛，集落形成能力随着传代次数增加呈显著下降趋势。

Proliferation capacity of rabbit adipose tissues-derived mesenchymal stem cells at early stage in vitro

BACKGROUND: Surface marker molecules and differential ability of human adipose tissues-derived mesenchymal stem cells can change with the prolonged culture time. OBJECTIVE: To analyze the morphological characteristics and colony forming capacity of early cultured rabbit adipose tissues-derived mesenchymal stem cells in vitro. DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Institute of Ophthalmology, and Central Laboratory, Eye and ENT Hospital, Fudan University from January to March 2008. MATERIALS: Nine female New Zealand rabbits aged three months were obtained from the Shanghai Yingenyang Rabbit Room. METHODS: Adipose tissue was harvested from the nape fat pad of the New Zealand rabbits following anesthesia. Primary cultured cells were established using type I collagenase and cell cultures were maintained with DMEM containing 0.1 volume fraction of fetal bovine serum. Cells were passaged when 80% was confluent. The second, third and fourth passages of cells were utilized for the study. MAIN OUTCOME MEASURES: The morphology of adipose tissues-derived mesenchymal stem cells was observed using inverted microscope. Cell phenotype was detected utilizing flow cytometry. The cloning capacity was determined. RESULTS: Adipose tissues-derived mesenchymal stem cells were fibroblast-like and proliferated rapidly in vitro at the second, third and fourth passage. Flow cytometry showed that adipose tissues-derived mesenchymal stem cells expressed CD29 and PCNA. The colony-forming rates were respectively (8.0±0.6)%, (6.7±0.4)% and (4.6±0.5)% (F=12.18, P < 0.05). CONCLUSION: Rabbit adipose tissues-derived mesenchymal stem cells at early stage proliferate rapidly in vitro. The colony forming capacity is gradually decreased obviously with increased passage number.