Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells
Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells
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DOI：
10.1007/s11626-014-9782-x
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发表时间：
2014-10-01
影响因子：
2.1
通讯作者：
de Almeida, Fernando Goncalves
中科院分区：
文献类型：
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作者：
de Sa Barretto, Leticia Siqueira;Lessio, Camila;de Almeida, Fernando Goncalves
人类脂肪组织已被描述为干细胞的潜在替代储存库。虽然已经在兔中使用自体脂肪源性干细胞（ADSC）进行了研究，但这些细胞尚未得到很好的表征。本研究的主要目的是证明存在从兔腹股沟脂肪垫分离的脂肪源性干细胞，并通过成骨和成脂体外分化和脂质指纹分析来表征它们。次要目的是通过生长动力学、细胞活力和DNA完整性评价细胞行为。分离兔ADSC以测定体外生长动力学和细胞活力。通过碱性彗星试验评估第0代和第5代的DNA完整性。采用Von Kossa法评价成骨分化，茜素红S染色法评价成骨分化，油红O染色法评价成脂分化。通过MALDI-TOF/MS进行对照、成脂和成骨分化细胞的脂质指纹分析。我们证明兔ADSC在早期传代时具有恒定的生长速率，在第5代或之后增加DNA片段化。第2代和第5代的兔ADSC活力相似（分别为90.7%和86.6%），但第3代后细胞生长速率有下降的趋势。以CD 45（0.77%）作为阴性对照，ADSC的特征在于表面标志物如CD 29（67.4%）和CD 44（89.4%）的表达。成功地从兔腹股沟区分离出ADSC。这些细胞能够分化为成骨和成脂组织时，他们被放置在诱导培养基。每次传代后，细胞生长有下降的趋势。另一方面，DNA片段化在每次传代时增加。ADSC在对照、成脂或成骨培养基中具有不同的脂质分布。
Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.