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目的探讨低氧对人脐带间充质干细胞(h UC-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h UC-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h UC-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h UC-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h UC-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h UC-MSCs增殖的调控。
Objective To investigate the effect of hypoxia on the expression of Sirtuin (SIRT) protein in human umbilical cord mesenchymal stem cells (h UC-MSCs) and its relationship with cell proliferation. Methods The hUC-MSCs were isolated and cultured by tissue block method. The cell surface markers were identified by flow cytometry. The proliferation of UC-MSCs was detected by CCK-8 assay at different time points under hypoxia and normoxia. Flow Cytometry The changes of cell cycle in hypoxia and normoxia were detected. The localization and expression of SIRT protein in hypoxia and normoxia were detected by immunofluorescence and Western blotting. Results The hUC-MSCs were successfully isolated. The expression of CD90, CD105, CD29 and CD44 were highly expressed, while the expressions of CD14, CD19, CD34, CD45 and HLA-DR were not expressed. (P <0.05). The percentage of S phase cells cultured in hypoxia for 24 and 48 h was higher than that in normoxia culture (all P <0.05). SIRT1 and 6 in nuclei were detected by immunofluorescence and SIRT2 Positioning in the cytoplasm, SIRT3,4,5 located in mitochondria. The protein expression of SIRT1 in hypoxia for 24h and 48h was higher than that in normoxia (all P <0.05) by Western blotting, and the expression of SIRT1 was higher in hypoxia for 72 h than that of 24 h and 48 h (P <0.05) ; SIRT2 expression was higher than that of normoxic (P <0.05) at 24 h of hypoxia, lower than that of normoxia at 48 h and higher than that of normoxia at 72 h (P <0.05) (P <0.05). The expression of SIRT5 in both normoxia and hypoxia cells was higher than that of normoxia (P <0.05) after 48 h and 72 h, respectively. Conclusion Hypoxia treatment can promote the proliferation of h UC-MSCs 24, 48 and 72 h. Hypoxia-induced changes in the expression of SIRT1, 2 and 5 suggest that SIRT1,2,5 may be involved in the regulation of hUC-MSCs proliferation by hypoxia.