{"title":"[具有抗氧化能力的人脐带间充质干细胞的制备和评价]。","authors":"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","doi":"10.19746/j.cnki.issn.1009-2137.2024.06.039","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC) from human umbilical cords and evaluate its cell biological properties.</p><p><strong>Methods: </strong>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 <i>in vitro</i>, 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 <i>SOD-1, GSH, GAT</i>, and <i>NQO1</i> in MSC was also evaluated by RT-qPCR.</p><p><strong>Results: </strong>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 <i>in vitro</i> 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.</p><p><strong>Conclusion: </strong>Human AO-MSC is successfully prepared from human umbilical cord without animal serum.</p>","PeriodicalId":35777,"journal":{"name":"中国实验血液学杂志","volume":"32 6","pages":"1888-1895"},"PeriodicalIF":0.0000,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"[Generation and Evaluation of Human Umbilical Cord Derived Mesenchymal Stem Cells with Antioxidant Capacity].\",\"authors\":\"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\",\"doi\":\"10.19746/j.cnki.issn.1009-2137.2024.06.039\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objective: </strong>To prepare mesenchymal stem cells with antioxidant capacity (AO-MSC) from human umbilical cords and evaluate its cell biological properties.</p><p><strong>Methods: </strong>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 <i>in vitro</i>, 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 <i>SOD-1, GSH, GAT</i>, and <i>NQO1</i> in MSC was also evaluated by RT-qPCR.</p><p><strong>Results: </strong>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 <i>in vitro</i> 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.</p><p><strong>Conclusion: </strong>Human AO-MSC is successfully prepared from human umbilical cord without animal serum.</p>\",\"PeriodicalId\":35777,\"journal\":{\"name\":\"中国实验血液学杂志\",\"volume\":\"32 6\",\"pages\":\"1888-1895\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"中国实验血液学杂志\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.19746/j.cnki.issn.1009-2137.2024.06.039\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"Medicine\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"中国实验血液学杂志","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.19746/j.cnki.issn.1009-2137.2024.06.039","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Medicine","Score":null,"Total":0}
[Generation and Evaluation of Human Umbilical Cord Derived Mesenchymal Stem Cells with Antioxidant Capacity].
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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