低温冻存对人脂肪间充质干细胞生物学特性的影响

Effect of cryopreservation on biological characteristics of human adipose-derived mesenchymal stem cells

目的:比较人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells,hADSCs)冻存前和复苏后的生物学特性,为今后建立低温干细胞库提供实验支持。方法:采用0.1%Ⅰ型胶原酶消化法从腹部大网膜脂肪组织中分离hADSCs,体外培养扩增,将处于均一稳定生长状态的第3代hADSCs置于液氮中分别冻存1、3和6个月,复苏后台盼蓝染色检测细胞存活率,采用MTT法绘制细胞生长曲线,流式细胞术检测细胞表面抗原,成脂成骨诱导后鉴定多向分化能力,RT-PCR检测成脂特异性基因过氧化物酶体增殖物激活受体γ-2(peroxisome proliferator-activated receptorγ-2,PPARγ-2)和成骨特异性基因骨钙素(osteocalcin,OC)的表达情况。结果:随冻存时间的延长细胞存活率略有下降,但差异无统计学意义(P>0.05)。MTT结果显示冻存前后细胞增殖能力均很强。流式细胞检测结果显示冻存前后细胞均高表达CD29、CD44、CD73、CD90、CD105和CD166,低表达CD45和HLA-DR。冻存前后hADSCs均具有向脂肪细胞和骨细胞分化潜能,且RT-PCR结果显示,PPARγ-2和骨钙素mRNA的表达冻存前后差异均无统计学意义(P>0.05)。结论:经液氮冻存后的hADSCs具有高存活率,生物学特性保持稳定且仍具有多向分化潜能。

Objective:To provide the experimental support for establishing a low-temperature stem cell bank for future use,freeze-thawed human adipose-derived mesenchymal stem cells (hADSCs) were compared with fresh hADSCs in vitro.Methods:The hADSCs were isolated from abdomen epiploica adipose tissue by collagenase digestion and subcultured in vitro.The third passage of hADSCs was cryopreserved in liquid nitrogen for 1,3,and 6 months.Cell viability,proliferation potential,expression of cellular markers and multipotential differentiation were studied after thawing using trypan blue staining,MTT method,flow cytometry analysis,Oil Red O staining,von Kossa,and RT-PCR.Results:After thawing,cell survival rate was found to descend as the time of cryopreservation prolonged.MTT revealed high capability for the proliferation.Phenotypic studies revealed that hADSCs were positive for CD29,CD44,CD73,CD90,CD105,CD166 and negative for CD45,and HLA-DR.Functional analysis demonstrated these cells could be differentiated into adipocytes and osteoblasts in lineage-specific medium.RT-PCR results proved that adipogenic and osteogenic related genes could be expressed.No significant difference (P ＞ 0.05) was noted before and after the cryopreservation in cell viability,proliferation potential,expression of cellular markers and multipotential differentiation.Conclusions:The hADSCs could maintain acceptable viability,biological characteristics,and multipotential differentiation after cryopreservation.