[Generation and Evaluation of Human Umbilical Cord Derived Mesenchymal Stem Cells with Antioxidant Capacity].

Q4 Medicine

中国实验血液学杂志 Pub Date : 2024-12-01 DOI:10.19746/j.cnki.issn.1009-2137.2024.06.039

Xiao-Yu Zhang, Pei-Lin Li, Jie Tang, Zhi-Ling Li, Rui-Cong Hao, Xiao-Tong Li, Wen-Jing Zhang, Shi-Rong Zhao, Li Ding, Wen-Qing Wu, Heng Zhu

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Abstract

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.

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[具有抗氧化能力的人脐带间充质干细胞的制备和评价]。

目的：制备具有抗氧化能力的人脐带间充质干细胞（AO-MSC）并评价其细胞生物学特性。方法：对照组采用0.2%胶原酶II消化人脐带沃顿氏水母组织分离间充质干细胞（MSC），收集释放细胞，在动物无血清培养基中培养。在AO-MSC组，允许不完全胶原酶ii消化的组织碎片粘附在flusk平底上，通过贴壁培养收获AO-MSC。以常规消化培养法为对照。采用成纤维细胞集落形成试验（CFU-F）评价间充质干细胞集落形成能力。CCK-8法检测骨髓间充质干细胞增殖能力。流式细胞术和免疫荧光染色检测间充质干细胞表面标志物。采用体外多分化法评价MSC的成脂成骨能力，采用实时荧光定量PCR （RT-qPCR）检测成脂成骨分化调控基因mRNA表达；RT-qPCR检测MSC中SOD-1、GSH、GAT、NQO1等抗氧化物质的mRNA表达。结果：该方法分离的AO-MSC在18 d左右达到80%-90%的合流，呈旋流生长。流式细胞术和免疫荧光染色结果显示，AO-MSC中CD73、CD29、CD105、CD90高表达，CD31、CD45、HLA-DR低表达。与MSC相比，AO-MSC具有更强的自我更新和分化能力。而对照组MSC的体外成脂成骨能力强于AO-MSC。RT-qPCR检测显示，AO-MSC表达的抗氧化物质mRNA水平高于MSC。结论：在不含动物血清的情况下，成功制备了人脐带AO-MSC。

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