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 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.
题目: 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.
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。.
Keywords: umbilical cord mesenchymal stem cells; Wharton’s jelly; serum-free culture system; collagenase-digested tissue debris culture protocol; antioxidant.