具有抗氧化能力的人脐带间充质干细胞制备及评价策略研究

Generation and Evaluation of Human Umbilical Cord Derived Mesenchymal Stem Cellswith Antioxidant Capacity

目的:从人脐带中分离制备一种具有抗氧化能力的间充质干细胞(antioxidant mesenchymal stem cell,AO-MSC)并评价其细胞生物学特性。方法:采用无血清培养体系,结合胶原酶消化培养法以及胶原酶消化组织块培养法,从人脐带华通氏胶组织分离培养MSC。使用0.2%Ⅱ型胶原酶消化,未完全消化的组织块贴壁培养收获AO-MSC。以常规消化培养法为对照。成纤维细胞集落形成实验检测MSC集落形成能力;CCK-8检测MSC增殖能力;流式细胞术及免疫荧光染色检测MSC表面标志物;体外诱导分化评价MSC成脂成骨能力,实时荧光定量PCR(RT-qPCR)检测成骨和成脂关键转录因子的表达差异;RT-qPCR检测MSC抗氧化还原物质SOD-1、GSH、GAT、NQO1的表达情况。结果:本策略分离的AO-MSC在18 d汇合率为80%-90%,细胞呈漩涡状生长。流式细胞术及免疫荧光染色检测结果显示,AO-MSC高表达CD73、CD29、CD105、CD90,低表达CD31、CD45、HLA-DR;胶原酶消化组织块培养法收获的AO-MSC自我更新及分化能力强于对照组MSC;对照组MSC体外成脂成骨能力强于胶原酶消化组织块培养法所得AO-MSC;RT-qPCR检测结果显示,胶原酶消化组织块培养法收获的AO-MSC其表达的抗氧化还原物质水平高于对照组。结论:成功制备了基于无血清体系的具备抗氧化能力的人脐带MSC。

Objective:To prepare mesenchymal stem cells with antioxidant capacity(AO-MSC)from human umbilical cords and evaluate its cell biological properties.Methods:In control group,mesenchymal stem cells(MSC)were isolated by digesting human umbilical cord Wharton’s Jelly tissues with 0.2%collagenase II,and the released cells were collected and cultured in an animal serum-free culture medium.In AO-MSC group,incompletely collagenase II-digested tissue debris were allowed to adhere to flusk flat bottoms and the AO-MSC was harvested by adherent culture.The conventional digestion and culture method was used as control.MSC colony forming ability was evaluated by fibroblast colony forming assay(CFU-F).MSC proliferative capacity was evaluated by CCK-8 assay.The MSC surface markers were detected by using flow cytometry and immunofluorescence staining.The adipogenic and osteogenic 2023-10-19收稿;2023-11-22接受capacity of MSC was evaluated by multi-differentiation in vitro,and the mRNA expression of genes that control adipogenic and osteogenic differentiation was detected by real-time fluorescence quantitative PCR(RT-qPCR);Moreover,the mRNA expression of antioxidant substances such as SOD-1,GSH,GAT,and NQO1 in MSC was also evaluated by RT-qPCR.Results:The AO-MSC isolated by this strategy reached a confluence of 80%-90%at around 18 days and grew in a swirling pattern.Flow cytometry and immunofluorescence staining assays showed that CD73,CD29,CD105,CD90 were highly expressed and CD31,CD45,HLA-DR were scarcely expressed in AO-MSC.AO-MSC exhibited stronger self-renewal and differentiation ability compared to MSC.However,the in vitro adipogenic-osteogenic capacity of MSC in the control group was stronger than that of AO-MSC.RT-qPCR assay showed that AO-MSC expressed higher mRNA levels of antioxidant substances compared to MSC.Conclusion:Human AO-MSC is successfully prepared from human umbilical cord without animal serum.