不同代次人脐带间充质干细胞生物学特性评估  

Evaluation of the biological properties of human umbilical cord mesenchymal stem cells in different passages

作　　者：林惠珠 李翠平[1] 钱师宇 阳莉[1] 陈晓燕[1] 陈文捷[1] 卢建溪[1] Lin Huizhu;Li Cuiping;Qian Shiyu;Yang Li;Chen Xiaoyan;Chen Wenjie;Lu Jianxi(The Third Affiliated Hospital of Sun Yat-sen University,Biotherapy Center;Jinan Universityof Public Health,Guangzhou 510000,China)

机构地区：[1]中山大学附属第三医院生物治疗中心 [2]暨南大学公共卫生学院,广州510000

出　　处：《中国组织化学与细胞化学杂志》2022年第4期341-350,共10页Chinese Journal of Histochemistry and Cytochemistry

摘　　要：目的明确传代扩增代次对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)生物学特性的影响。方法采用组织块法分离HUC-MSCs,体外传代培养后比较P3、P5、P10细胞的生物学特性:采用细胞计数法与流式细胞仪检测细胞增殖活性和表面免疫标志物;染色体核型分析检测基因稳定性;成脂、成骨诱导分化检测多向分化潜能;SA-β-gal活性检测细胞衰老情况;蛋白芯片检测胞内及培养上清细胞因子表达水平。结果3个代次的hUC-MSCs形态特征及生长曲线相似,随着扩增代次的增加,细胞的增殖活性降低。3个代次的hUC-MSCs均高表达CD105、CD90、CD73等表面免疫标志物,不表达或低表达CD11b、CD19、CD34、CD45及HLA-DR。染色体核型分析证实3个代次的hUC-MSCs均为正常二倍体核型,染色体核型未发生结构畸形改变。3个代次的hUC-MSCs成脂和成骨分化潜能均未见显著性差异。SA-β-gal活性检测显示,随着扩增代次的增加,HUC-MSCs开始呈现衰老趋势。hUC-MSCs胞内及培养上清均能检测到IL-6、IL-1β、TNF-α、G-CSF和GM-CSF,胞内IL-6和IL-1β随着扩增代次增加升高;培养上清GM-CSF随着扩增代次增加下降。结论不同代次HUC-MSCs在表面免疫标志物、基因稳定性及其多向分化潜能等基本生物学特性方面均无显著性变化。随着扩增代次增加会分泌SASP,而致细胞老化,增殖活性降低。Objective To clarify the effect of the generation of passage and amplification on the biological properties of human umbilical cord mesenchymal stem cells(hUC-MSCs).Methods hUC-MSCs were isolated by tissue block method.After in vitro cul-tivation,the main biological properties of P3,P5 and P10 cells were compared.Cell proliferation activity and immune cell surface markers were detected by cell counting and flow cytometry.Chromosome analysis(karyotyping)was used to detect genetic stability.In addition,the multi-directional differentiation potential was detected by adipogenic and osteogenic differentiation analysis.And cell senescence was detected by measuring SA-β-Gal activity.The expression levels of cytokines in cells and culture supernatants were detected by protein chip.Results The morphological characteristics and growth curves of hUC-MSCs were similar among cells of three passages.With the increase of amplification passage,the proliferation activity of hUC-MSCs have decreased.The surface markers were highly expressed in CD105,CD90 and CD73,but not in CD11b,CD19,CD34,CD45 and HLA-DR.Karyotype analysis confirmed that hUC-MSCs were normal diploid karyotypes,and there were no structural abnormalities in the karyotypes.There was no significant difference in adipogenic and osteogenic differentiation potential.SA-β-Gal activity detection showed that hUC-MSCs began to show an aging trend with the increase of amplification passage,especially in the 10th passage.IL-6,IL-1β,TNF-α,G-CSF and GM-CSF could be detected in hUC-MSCs and culture supernatant.And the levels of intracellular IL-6 and IL-1βincreased along the amplification passage.GM-CSF level in culture supernatant decreased with the increase of amplification passage.Conclusion The basic biological properties of hUC-MSCs,including surface markers,genetic stability and multi-directional differentiation potential do not change through different passages.With the increase of amplification passage,hUC-MSCs will secrete a variety of aging related factors,result-i

关 键 词：人脐带间充质干细胞 染色体核型分析 Β-半乳糖苷酶活性 细胞因子 

分 类 号：R-331[医药卫生]