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FOXO4通过抑制凋亡维持人脐带间充质干细胞衰老

吴平平¹, 胡文龙^1,*, 殷嫦嫦², 费久旺²

¹九江学院附属医院骨科，九江 332000；²九江学院基础医学院生物化学系，九江 332000

摘要

本文旨在探讨叉头盒O4 (forkhead box O4, FOXO4)在人脐带间充质干细胞(human umbilical cord-derived mesenchymalstem cells, hUC-MSCs)衰老中的作用。采用自然传代法诱导hUC-MSCs衰老，用慢病毒shRNA抑制FOXO4表达，用β-半乳糖苷酶染色法检测细胞衰老情况，用CCK-8法检测细胞活性，用流式细胞术检测细胞凋亡，用qPCR和Western blot检测细胞Bcl-2、Bax、FOXO4、白介素6 (interleukin 6, IL-6)和cleaved Caspase-3的表达情况，用免疫荧光染色法检测细胞FOXO4表达情况，用ELISA检测细胞IL-6分泌量。结果显示，相比第一代hUC-MSCs，衰老hUC-MSCs中FOXO4和Bax表达水平上调，Bcl-2和cleaved Caspase-3表达水平下调，IL-6 mRNA表达水平上调、分泌量增加。抑制FOXO4的表达后，衰老hUC-MSCs凋亡增加，细胞活力下降，IL-6 mRNA表达水平下调，分泌量降低。上述结果提示，FOXO4能通过抑制凋亡来维持衰老hUCMSCs活力和功能，加速整个细胞集落衰老。

关键词： 人脐带间充质干细胞; 叉头盒O4; 细胞衰老; 凋亡

分类号：R339.3+8; Q255

FOXO4 maintains senescence in human umbilical cord mesenchymal stem cells by repressing apoptosis

WU Ping-Ping¹, HU Wen-Long^1,*, YIN Chang-Chang², FEI Jiu-Wang²

¹Department of Orthopaedics, Jiujiang University Affiliated Hospital, Jiujiang 332000, China；²Department of Biochemistry, College of Basic Medicine, Jiujiang University, Jiujiang 332000, China

Abstract

The purpose of the present study was to investigate the effects of forkhead box O4 (FOXO4) on the senescence of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). The hUC-MSCs were induced to senescence by natural passage, and FOXO4 expression was inhibited by lentiviral shRNA transfection. The hallmark of cell senescence was analyzed by β-galactosidase staining, and the cell viability was assayed by CCK-8 method. Flow cytometry was used to investigate the apoptosis of hUC-MSCs. The expression levels of Bcl-2, Bax, FOXO4, interleukin 6 (IL-6) and cleaved Caspase-3 were detected by qPCR and Western blot. Immunofluorescence staining was used to detect FOXO4 expression. The amount of IL-6 secreted by hUC-MSCs was detected by ELISA. The results showed that, compared with the passage 1, senescent hUC-MSCs showed up-regulated expression levels of Bax and FOXO4, down-regulated expression levels of Bcl-2 and cleaved Caspase-3, and increased IL-6 mRNA expression and secretion. FOXO4 inhibition in senescent hUC-MSCs promoted cell apoptosis, reduced cell viability, and inhibited the mRNA expression and secretion of IL-6. These results suggest that FOXO4 maintains viability and function of senescent hUC-MSCs by repressing their apoptosis response, thus accelerating senescence of the whole cell colony.

Key words: human umbilical cord-derived mesenchymal stem cells; forkhead box O4; cellular senescence; apoptosis

收稿日期：2019-08-25　　录用日期：2019-12-25

通讯作者：胡文龙　　E-mail: huwenlong1988@126.com

DOI: 10.13294/j.aps.2020.0045

引用本文：

吴平平, 胡文龙, 殷嫦嫦, 费久旺. FOXO4通过抑制凋亡维持人脐带间充质干细胞衰老[J]. 生理学报 2020; 72 (4): 426-432.

WU Ping-Ping, HU Wen-Long, YIN Chang-Chang, FEI Jiu-Wang. FOXO4 maintains senescence in human umbilical cord mesenchymal stem cells by repressing apoptosis. Acta Physiol Sin 2020; 72 (4): 426-432 (in Chinese with English abstract).