Sirtuin在低氧诱导人脐带间充质干细胞增殖中的作用

doi:10.6040/j.issn.1671-7554.0.2016.1682

摘要/Abstract

摘要： 目的探讨低氧对人脐带间充质干细胞(hUC-MSCs)中Sirtuin(SIRT)蛋白表达影响及与细胞增殖的关系。方法组织块贴壁法分离和培养hUC-MSCs;流式细胞仪鉴定细胞表面标志物; CCK-8法测定低氧与常氧下不同时间点hUC-MSCs的增殖能力;流式细胞仪检测低氧与常氧下细胞周期的变化;免疫荧光与Western blotting测定低氧与常氧下细胞SIRT蛋白定位与表达。结果成功分离hUC-MSCs;细胞CD90、CD105、CD29、CD44高表达,CD14、CD19、CD34、CD45、HLA-DR无表达;CCK-8测定低氧培养24、48、72 h细胞的增殖能力均高于常氧培养(P均<0.05);在低氧下培养24、48 h后S期细胞比率高于常氧培养(P均<0.05);免疫荧光检测SIRT1、6位于细胞核内,SIRT2定位于胞浆,SIRT3、4、5定位于线粒体。Western blotting测定SIRT1在低氧培养24、48 h蛋白表达量均高于常氧条件(P均<0.05),常氧培养72 h的SIRT1表达量高于24 h及48 h(P均<0.05);SIRT2表达量在低氧培养24 h高于常氧培养(P<0.05),48 h后低于常氧培养,72 h后高于常氧培养(P<0.05);低氧培养24、48、72 h后SIRT5的表达量均高于常氧处理(P均<0.05),常氧与低氧培养细胞48 h和72 h后均高于24 h时SIRT5的表达(P均<0.05)。结论低氧处理24、48、72 h均可以促进hUC-MSCs的增殖。低氧诱导SIRT1、2与5表达改变提示SIRT1、2、5可能参与了低氧对hUC-MSCs增殖的调控。

关键词: 低氧, Sirtuin, 增殖, 人脐带间充质干细胞

Abstract: Objective To explore the effect of hypoxia on Sirtuin(SIRT)protein expression in human umbilical cord-derived mesenchymal stem cells(hUC-MSCs). Methods hUC-MSCs were isolated and cultured by explant technique. The cells were further expanded and assessed for their morphology and phenotype by flow cytometry. The proliferation of hUC-MSCs under normoxia(21%O₂)and hypoxia(5%O₂)was evaluated by CCK-8; Cell cycle was analyzed by flow cytometry. Immunofluorescence assay was used to detect the location of SIRT(1-6)under normoxia and hypoxia 山东大学学报 (医学版)55卷7期 -王凯民,等.Sirtuin在低氧诱导人脐带间充质干细胞增殖中的作用 \=-conditions. Expressions of SIRT(1,2,5)under normoxia and hypoxia conditions for different times(24, 48, 72 h )were detected by Western blotting. Results hUC-MSCs were successfully isolated from umbilical cord. Flow cytometry showed that CD105, CD90, CD44 and CD29 were positive, while CD14, CD19, CD34, CD45 and HLA-DR were negative on hUC-MSCs. The proliferation of hUC-MSCs was enhanced under hypoxia compared with normoxia(all P<0.05). The cells fraction at S phase was also increased under hypoxia compared with normoxia condition after being cultured for 24 h and 48 h(all P <0.05). Immunofluorescence showed that no matter under hypoxia or nomoxia conditions, SIRT1 and SIRT6 were localized in the nucleus, SIRT2 was localized in cytoplasm, and SIRT3, SIRT4, SIRT5 were localized in mitochondria. Western blotting showed the expression of SIRT1 in hUC-MSCs increased under hypoxia after being cultured for 24 h and 48 h(all P<0.05). Similarly, the expression of SIRT5 was increased under hypoxia at all three time points(all P<0.05). The expression of SIRT2 in hypoxia was increased after being cultured for 24 h and 72 h compared with normoxic cells(all P<0.05), however, the expression of SIRT2 under hypoxia condition was lower than that under normoxia condition for 48 h(P<0.05). Conclusion Hypoxia can increase hUC-MSCs’ proliferation. The expressions of SIRT1, SIRT2, SIRT5 were affected by hypoxia, which implies that SIRT1, SIRT2, SIRT5 may be involved in the regulation of hypoxia induced proliferation of hUC-MSCs.

Key words: Human umbilical cord-derived mesenchymal stem cell, Sirtuin, Proliferation, Hypoxia

中图分类号:

R329.2

王凯民,谭娟娟,严志强,李志强. Sirtuin在低氧诱导人脐带间充质干细胞增殖中的作用[J]. 山东大学学报（医学版）, 2017, 55(7): 23-30.

WANG Kaimin, TAN Juanjuan, YAN Zhiqiang, LI Zhiqiang. Effects of Sirtuin on hypoxia-induced proliferation of human umbilical cord-derived mesenchymal stem cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2017, 55(7): 23-30.

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