Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (2): 283-288.doi: 10.3969/j.issn.2095-4344.2014.02.020
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Guo Ting-ting, Li Li-xi, Tian Hong
Received:
2013-11-26
Online:
2014-01-08
Published:
2014-01-08
Contact:
Tian Hong, M.D., Associate professor, Department of Medical Genetics, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
About author:
Guo Ting-ting, Studying for master’s degree, Department of Medical Genetics, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Supported by:
the National Natural Science Foundation of China, No. 31271043
CLC Number:
Guo Ting-ting, Li Li-xi, Tian Hong. Adipose-derived stem cells for repair of urinary system injury: current situation and problems in application[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(2): 283-288.
2.1 脂肪干细胞的获取 2.1.1 脂肪干细胞的来源 干细胞最初是在骨髓中分离出来的,随后在脂肪组织、肌肉组织、牙根、胎盘、羊水及脐血中均分离出了干细胞[5-6]。自从2001年Zuk从人脂肪组织中成功分离培养出脂肪源性干细胞,脂肪干细胞以其取材方便的独特优势,逐渐成为人们研究的热点。在人体中脂肪组织可占到正常体质量的10%-29%[7],哺乳动物主要有两种脂肪组织:棕色脂肪组织和白色脂肪组织,虽然二者同属脂肪组织,但却在形态、分布和功能上相差甚远。有研究称棕色脂肪组织仅在婴儿期发挥作用[8],然而最新研究表明在低温环境下成人体内也可产生棕色脂肪细胞,该类细胞可通过高比率燃烧脂肪,保持体温[9]。白色脂肪组织主要分布在腹腔内的网膜、肠、肾周及臀部、大腿、腹部的皮下区域,在人体,白色脂肪组织起储存及释放能量的调节器作用。两类脂肪组织均存在脂肪来源干细胞,然而棕色脂肪组织中干细胞的数量和分化潜能都不如白色脂肪组织中的干细胞[10-11],因此研究重点集中在白色脂肪组织。 脂肪细胞是一种终末分化期细胞,当机体出现细胞坏死或发生细胞凋亡时,脂肪来源干细胞会分化成脂肪细胞补充损失的脂肪细胞[12],研究表明1 g脂肪组织可以提取约5×103个干细胞,是1 g骨髓中提取干细胞数量的100-500倍[13],脂肪干细胞以其易大量获取、提取方法简单且不影响供区功能的优势,成为再生医学理想的种子细胞。 2.1.2 脂肪干细胞的分离培养 脂肪来源干细胞的分离培养在文献中已有很详细描述[14-22],综合多篇文献,以下操作为多见:手术分离脂肪组织,PBS清洗3-5遍,剪碎成2 mm3/小块。用0.1% Ⅰ型胶原酶37 ℃水浴消化1 h,400×g室温离心10 min,弃上清。用PBS重悬洗涤,100 μm滤网滤除未消化的组织,400×g室温离心10 min,弃去上清,培养基重悬并用移液管反复吹打,使之成为单细胞悬液,接种到含体积分数为10%胎牛血清的DMEM培养液中,置 37 ℃、体积分数为5%CO2的饱和湿热培养箱培养。脂肪干细胞是贴壁细胞,原代接种24 h后可见大量细胞贴壁,初呈短梭形或多角形[23],接下来2 d,每天换液1次,并用PBS轻轻洗涤,弃去组织块和未贴壁的细胞,以后每3 d换液1次。48 h后分裂增殖速度加快,细胞体积增大,呈长梭形,形成集落呈涡轮状紧密排列[24]。当细胞达80%- 90%融合时,0.25%胰酶常规消化,进行传代,传代密度为6 700/cm2,传代培养后的细胞增殖速度明显加快。其中值得注意的几个关键因素,如细胞种植密度、血清浓度、氧浓度,它们都能影响到细胞的生长和分化。通常细胞种植密度为(1.0-2.0)×109 L-1,血清体积分数为10%,氧体积分数为21%。 2.2 脂肪干细胞的鉴定 2.2.1 脂肪干细胞的标志物 目前干细胞研究领域认为:脂肪干细胞尚未发现特异性的细胞表面标志物,其既表达其他成体干细胞中广泛表达的标志物如CD44、CD90、CD105,但又不完全和其他成体干细胞相同。研究者们对此做了大量研究,提取的原代脂肪来源干细胞多还混杂有内皮细胞、红细胞和成纤维细胞等细胞群,随着传代次数增加会逐步得到纯化,通常选取传代至第3代的脂肪干细胞进行细胞免疫表型的检测。通过对多篇文献的查阅发现[8, 25-29],脂肪干细胞表达CD9、CD10、CD13、CD29、CD44、CD49d、CD49e、CD54、CD55、CD58、CD59、CD90、CD105、CD146、CD166、SH-3等,其中CD29、CD44、CD90和CD105均高表达[30];不表达CD31、CD34、CD45、HLA-DR,但对CD34和STRO-1的表达尚存在争议[25,31-33]。此外脂肪干细胞所取位置、细胞所处时期、供体的年龄、手术类型、培养环境、细胞密度不同以及供体种的不同都有可能引起细胞表面标志物的差异[32-34],如CD90在人干细胞中高度表达,而在小鼠中不表达,除此之外随着细胞的传代CD90表达也会下降;林立新等[35]研究也发现CD31、CD49d虽然在原代脂肪干细胞呈阳性表达,但在第2代转为阴性表达,CD29、CD105、CD166、Stro-1、Flk-1在第3代脂肪干细胞中的表达量也明显多于原代脂肪干细胞。因此免疫组化和流式细胞仪检测等免疫表型的结果对确认脂肪干细胞也只能起到辅助作用,而鉴定其具有干细胞特性的最好方法是进行诱导分化,并进行相应检测以证明诱导分化成功[36]。 2.2.2 脂肪干细胞的分化潜能 干细胞之所以受到人们的关注,除了其快速增长的特点外,还因为它的增殖分化潜能,在不同细胞因子的诱导下,脂肪干细胞可以分化成脂肪细胞、成骨细胞、软骨细胞、肌细胞等,除此之外有研究发现脂肪干细胞经相应诱导因子的诱导后,检测到神经细胞、心肌细胞标志物的表达[37]。Gentile等[19]对脂肪来源干细胞诱导分化的常见不同细胞系进行了总结。"
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