摘要
为进一步研究脂肪组织来源的干细胞增殖和多向分化潜能,用改进的方法分离脂肪组织来源的干细胞,通过cck-8检测细胞的增殖能力、流式细胞仪检测干细胞相关表面标记的表达、RT-PCR对干细胞相关基因的表达进行分析;并对分离得到的细胞向脂肪、软骨、骨及心肌细胞诱导,观察其多向分化能力.结果显示:采用改进的方法,从400～600mg脂肪组织可收获约5×105个脂肪组织来源的干细胞,并且细胞可以重叠生长1个月以上,期间细胞表现出几个对数增殖期;所有增殖的细胞其干细胞相关表面标记都呈阳性表达;转录因子Nanog、Oct-4、Sox-2和Rex-1也呈强阳性表达;ADSCs向脂肪、骨、软骨和心肌细胞诱导分化后能够分别表达脂滴、碱性磷酸酶和矿化结节、富含黏多糖的软骨细胞外基质,以及少量心肌特异性连接蛋白Connexin-43,表明ADSCs具有向多个胚层细胞分化的能力.此外,为获得更多具有强增殖能力的细胞,根据生长曲线,对细胞进行每14d传代而非传统的5d传代,发现所得到的细胞仍保持强的增殖能力、干细胞表型以及更强的多向分化潜能.
To further study the proliferation and multi-differentiation potentials of adipose-derived stem cells（ADSCs）,the cells were isolated with the improved methods and their growth curves were achieved with cck-8.Surface protein expression was analyzed by flow cytometry to characterize the cell phenotype by PT-PCR.The multi-lineage potential of ADSCs was testified by differentiating cells with adipogenic,chondrogenic,osteogenic and myogenic inducers.The results show that about 5× 105 stem cells can be obtained from 400-600mg adipose tissue.And ADSCs can be continuously cultured in vitro for up to 1month without passage and they have several logarithmic growth phases during the culture period.The relative surface marker expressions of all proliferated cells are positive.Meanwhile,stem cell related transcription factors,Nanog,Oct-4,Sox-2and Rex-1,are strongly positively expressed in ADSCs.After induced differentiation toward adipogenic,osteogenic,chondrogenic and myogenic cells,the differentiated ADSCs can respectively express intracellular lipid,alkaline and calcified nodules,mucopolysaccharide-rich extracellular matrix,and a little Connexin-43,which illustrates the ability of multi-lineage differentiation of ADSCs.In order to obtain more stem cells,ADSCs are subcultured every 14dinstead of normal 5daccording to growth curve.ADSCs still keep strong proliferation ability,maintain their phenotypes,and have stronger multi-differentiation potential.
出处
《大连理工大学学报》
EI
CAS
CSCD
北大核心
2010年第5期649-655,共7页
Journal of Dalian University of Technology
基金
国家自然科学基金资助项目(30670525)
大连理工大学青年教师培养基金资助项目(893228)
关键词
脂肪
干细胞
增殖
倍增时间
多向分化潜能
adipose tissue
stem cells
proliferation
doubling time
multi-differentiation potential
