摘要
背景:脂肪组织来源干细胞是位于脂肪基质内的一种细胞,具有多向分化潜能,可以定向分化成多种细胞类型。目的:探索兔脂肪组织来源干细胞体外生长特性及胶原酶消化适宜条件。设计、时间及地点:细胞分子生物学实验,2008-05/09在解放军南京军区福州总医院比较医学科及实验科完成。材料:日本大耳白兔5只。Ⅰ型胶原酶为美国Worthington公司产品,低糖DMEM/F12培养液为美国Gibco公司产品。方法:兔颈后部皮下脂肪取材,采用Ⅰ胶原酶消化法获取细胞,按Ⅰ型胶原酶消化质量浓度分为1.0,1.5,2.0g/L3组,每组再按消化时间分为0.5,1,1.5h3个亚组。脂肪组织剪碎混匀后均分为9等分,最后重悬细胞终体积均定为2mL并计数,每个亚组计数6次,共取5次脂肪组织,采用含血清的DMEM培养液体外培养细胞。主要观察指标:细胞体外生长特性并比较不同胶原酶浓度、胶原酶消化时间对兔脂肪来源干细胞体外培养的影响。结果:采用1.0,1.5,2.0g/L三种不同胶原酶浓度进行消化,发现在消化时间为0.5h和1h时,2.0g/L组消化细胞总数最多,细胞总数与其他组比较,差异有显著性意义(P<0.01)。在消化时间为1.5h时,消化细胞总数较0.5h和1h均有增加,同时,1.0,1.5,2.0g/L组消化细胞总数无统计学差异,活细胞比值1.0g/L组最高,但与其他组无统计学差异,观察第3代细胞生长特性,1.0g/L消化1.5h组别具有更好的增殖活性。结论:脂肪来源干细胞具有较强的自我更新能力,对于兔脂肪组织来源干细胞,胶原酶浓度1.0g/L、消化时间1.5h是最佳的获取条件。
BACKGROUND： Adipose tissue-derived stem cells are located in adipose stroma, with multi-directional differentiational potential and can differentiate into various kinds of cells. OBJECTIVE： To investigate the in vitro growth characteristics and suitable condition of collagenase duration for rabbit adipose tissue-derived stem cells （ADSCs）. DESIGN, TIM E AND SETTING： The cell molecular biology study was performed at the Department of Comparative Medicine and Department of Experiment of Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA from May 2008 to September 2008. MATERIALS： Five Japanese rabbits, type Ⅰ collagenase （Worthington, USA）, and low-glucose DMEM/F12 （Gibco, USA） were used in this study. METHODS： The subcutaneous fat tissue was harvested from posterior cervical region of rabbit, and cells were harvested by the type Ⅰ collagenase digestion method. According to the Ⅰ type collagenase digestion density, cells were divided into 1.0, 1.5, 2.0 g/L three groups, each group pressed the digestion time to divide into 0.5, 1 and 1.5 h three subunits. After the fatty tissue cuttings mix uniform, divided equally into 9 divisions, finally suspended cell volume was decided as 2 mL and to count again, each subunit counted 6 times, and up to take 5 fatty tissues. Dulbecco＇s Modified Eagle Medium with fetal bovine serum was used for cell culture. MAIN OUTCOME MEASURES： Ceil growth characteristics in vitro, and effects of collagenase at different concentrations and collagenase digestion period on in vitro culture of ADSCs. RESULTS： Based on the collagenase concentration, cells were divided into three groups, with concentrations of 1.0, 1.5, 2.0 g/L. At a duration of 0.5 hour and 1 hour, 2.0 g/L group had the highest total cell number and the total cell rate was statistically different from the other groups （P 〈 0.01 ）. When the duration was 1.5 hours, total cell number increased in every group. There was no significant differences among the 1.0, 1.5, 2.0 g/L groups. Live cell rate was the highest in the 1.0 g/L group, but there was no significant difference among these groups. To observe the cell growth characteristics of the third passage, with concentrations of 1.0 g/L and take 1.5 hour had the best proliferation capability. CONCLUSION： ADSCs have strong self-renewal capacity. The optimal culture conditions for rabbit ADSCs are collagenase concentration at 1.0 g/mL, with digestion duration of 1.5 hours.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3685-3688,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
