中文摘要：

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。

英文摘要：

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% 02 ） and hypoxia（5% 02 ） 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 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 un.bilical cord. Flow cytome- try showed that CD105, CDg0, 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 condi- tions, 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 hypox- ia after being cultured for 24 h and 48 h （ all P 〈 0.05 ）. Similarly, the expression of SIRT5 was increased under hypox- ia 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 w