人脂肪间充质干细胞的分离培养及冻存前后生物学变化  

Isolation,culture of human adipose-derived mesenchymal stem cells and biological characteristics changes after cryopreservation

作　　者：谢甬淋[1] 戚飞腾[1] 童洋萍[1] 毕涌[1] 张旭[1] 

机构地区：[1]温州医学院附属第一医院神经内科,325000

出　　处：《中国神经免疫学和神经病学杂志》2013年第4期263-266,共4页Chinese Journal of Neuroimmunology and Neurology

基　　金：浙江省自然科学基金(NO.Y2101091)

摘　　要：目的有效地分离并扩增人脂肪间充质干细胞(hADSCs),并观察冻存前后hADSCs的一般生物学特征。方法采用Ⅰ型胶原酶消化的方法从抽脂手术废弃的脂肪组织中分离hADSCs,接种在人脂肪间充质干细胞完全培养基中培养。采用胰酶消化法传代扩增,取处于生长对数期的第3代细胞,以液氮冻存6个月。复苏后以台盼蓝染色检测细胞存活率,MTT法描绘细胞生长曲线;以流式细胞仪检测冻存前后细胞表面分子CD44、CD105、CD29、CD73、CD31、HLA-DR的表达水平。结果细胞大小均等,呈长梭形漩涡状生长。复苏后细胞存活率可达(91.25±3.02)%;冻存前后细胞生长曲线无明显差别,均呈"S"形;经过2～3d潜伏期后进入增殖期,7d后进入平台期。冻存前CD44、CD105双阳性细胞占(99.850±0.001)%,CD29、CD73双阳性细胞占(92.600±0.028)%,CD31阳性细胞占(4.186±0.010)%,HLA-DR阳性细胞占(1.02±0.007)%;冻存后CD44、CD105双阳性细胞占(98.060±0.028)%,CD29、CD73双阳性细胞占(91.224±0.041)%,CD31阳性细胞占(3.832±0.009)%,HLA-DR阳性细胞占(1.514±0.006)%。冻存前后上述6种hADSCs表面分子表达水平差异均无统计学意义(P>0.05)。结论来源于人的脂肪间充质干细胞易于体外培养扩增和冻存,复苏后存活率高,一般生物学特征无明显变化,可用作组织工程的种子细胞。Objective To study efficient methods to isolate and amplify human adipose-derived mesenchymal stem cells（hADSCs）,and to study whether their biological characteristics change after cryopreservation. Methods We used 0.1% collagenase Ⅰ to digest human adipose obtained through suction-assisted lipectomy to isolate hADSCs,then incubate in human adipose-derived mesenchymal stem cells complete medium.hADSCs were passaged and amplified by the trypsin digestion,the third passage of cells in logarithmic growth phase was cryopreserved in liquid nitrogen for six months.After recovery,trypan blue staining was used to test cell viability and MTT method was used to get cell growth curve.Cell surface molecules were examined by flow cytometry. Results hADSCs mainly showed equal cell size,a spindle-shaped morphology and whirlpool-shaped arrangement.The cell survival rate could reach（91.25±3.02）% after six months cryopreservation.The growth curve of ADSCs had no significant changes after cryopreservation.All showed the＂S＂shape： after passed incubation period for two to three days,the cells entered logarithmic growth phase;then reaching the plateau in the 7th day.Before cryopreservation,the proportion of CD44,CD105 double positive cells was（99.850 ± 0.001）%,the proportion of CD29,CD73 double positive cells was（92.600±0.028）%,the proportion of CD31 positive cells was（4.186 ± 0.010）%,the proportion of HLA-DR positive cells was（1.02 ± 0.007）%.After cryopreservation,the proportion of CD44,CD105 double positive cells was（98.060 ± 0.028）%,the proportion of CD29,CD73 double positive cells was（91.224 ± 0.041）%,the proportion of CD31 positive cells was（3.832 ± 0.009）%,the proportion of HLA-DR positive cells was（1.514 ± 0.006）%.The expression of these six cell surface molecules of hADSCs had no significant changes between before and after cryopreservation（P0.05）. Conclusions hADSCs could be cultivated,amplified and cryopreserved in vitro,the cells get high survival rate after recove

关 键 词：人脂肪间充质干细胞 流式细胞仪 冻存 分离 生物学特征 

分 类 号：R394.2[医药卫生—医学遗传学] Q813.11[医药卫生—基础医学]