摘要
探讨不饱和脂肪酸对人脂肪间充质干细胞(adipose-derived stem cells, ADSCs)的形态、增殖、免疫表型及分化潜能的影响。采用0.15%Ⅷ型胶原酶消化法从皮下脂肪组织中分离获得ADSCs,使用添加了油酸或亚麻酸的培养基对ADSCs进行传代培养,倒置显微镜下观察细胞形态,计数法绘制细胞生长曲线,流式细胞术检测细胞表面抗原,同时使用特定的诱导培养基对细胞进行成脂、成骨、成软骨诱导培养和染色鉴定,并采用实时定量PCR检测三系分化诱导培养9 d后成脂、成骨及成软骨相关标志基因m RNA的表达水平。结果显示,0.15%Ⅷ型胶原酶消化法能获得形态均一的ADSCs;培养相同的时间后,与未添加脂肪酸的空白对照组相比,含有20μmol/L油酸或亚麻酸的培养体系所收获ADSCs的细胞数量增加了20%左右;各组细胞CD13、CD29、CD44、CD73、CD90、CD105呈阳性表达, CD31、CD45呈阴性表达;使用20μmol/L油酸或亚麻酸进行传代培养的细胞经成脂、成骨、成软骨诱导后,油红O、茜素红、阿利新蓝染色呈阳性反应,同时细胞中的成脂标志基因PPAR-γ、成软骨标志基因SOX9和COL2A1及成骨标志基因ALP、OCN、RUNX2的m RNA表达水平较对照组显著增加。实验结果表明,油酸及亚麻酸能够促进ADSCs增殖,维持细胞的免疫表型,并且提高细胞的三系分化潜能。
To investigate the effects of unsaturated fatty acids on the morphology,proliferation,immunophenotype and differentiation potential of human adipose mesenchymal stem cells(ADSCs),ADSCs were isolated from subcutaneous adipose tissue by digestion with 0.15%typeⅧcollagenase.After subculture,the cells were cultured with oleic acid and linolenic acid,respectively,and were observed by inverted microscope.The cell growth curve was generated by cell-counting.The flow cytometry was used to examine the expression of the surface markers.After being cultured with the differentiation medium,the cells were stained by oil red O for adipogenesis,alizarin red for osteogenesis and alcian blue for chondrogenesis.Meanwhile,quantitative PCR analysis was used to determine the relative mRNA expression levels of stem cell-related genes 9 days after induction.The results showed that ADSCs with uniform morphology could be obtained by using 0.15%typeⅧcollagenase.Compared with the control group,the groups cultured with 20μmol/L oleic acid or linolenic acid had about 20%increase in the number of ADSCs within the same amount of time.The flow cytometry examination showed that expressions of CD13,CD29,CD44,CD73,CD90 and CD105 were positive,and those of CD31 and CD45 were negative.The cells successfully differentiated into adipogenic,osteogenic and chondrogenic lineages after cultured with 20μmol/L oleic acid or linolenic acid,and were stained positively with oil red O,alizarin red and alcian blue.The expression levels of adipogenic gene PPAR-γ,chondrogenic genes SOX9,COL2A1,and osteogenic genes ALP,OCN,RUNX2 were significantly higher compared with those in the control group.The results showed that oleic acid and linolenic acid could promote ADSC proliferation,maintain their immunophenotype and improve their differentiation ability.
作者
李锦灵
林立龙
李治寰
LI Jin-ling;LIN Li-long;LI Zhi-huan(Dongguan Enlife Stem Cell Biotechnology Institute,Dongguan 523000,Guangdong,China)
出处
《生命科学研究》
CAS
CSCD
2021年第1期15-23,52,共10页
Life Science Research
基金
东莞市引进创新创业领军人才计划项目。
