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Title: [Effects of hypoxia-inducible factor 1α on hypoxic tolerance of human amniotic mesenchymal stem cells].
Author: Ge L, Yu D, Su R, Cao Y.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2018 Mar 15; 32(3):264-269. PubMed ID: 29806273.
Abstract:
OBJECTIVE: Under hypoxic conditions, the survival and apoptosis of human amniotic mesenchymal stem cells (hAMSCs) were observed by transient transfection of hypoxia-inducible factor 1α (HIF-1α) gene, to investigate the effect of HIF-1α on hypoxic tolerance of hAMSCs. METHODS: The hAMSCs were isolated and cultured from amniotic membrane tissue from voluntary donors who were treated with cesarean section. And the morphological observation by inverted phase contrast microscope and immunofluorescence detection of the expressions of stem cell markers OCT-4 and NANOG were performed to identify the cultured cells. The third generation hAMSCs were treated with 200 μmol/L CoCl ₂, and transient transfection of plasmids were added according to the following grouping: group A was hAMSCs blank group; group B was pcDNA3.1 negative control group; group C was short hairpin RNA (shRNA) negative control group; group D was shRNA-HIF-1α interference group; group E was pcDNA3.1-HIF-1α over expression group. Cell survival rate of each group was measured by cell counting kit 8 (CCK-8) at 12, 24, 48 hours after hypoxia treatment. Flow cytometry was used to detect apoptosis rate of each group at 24 hours after hypoxia treatment. The expression levels of HIF-1α, vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), Bax, and cleaved Caspase-3 (C-Caspase-3) proteins were detected by Western blot at 24 hours after hypoxia treatment. RESULTS: CCK-8 assay showed that the cell survival rate of group D was significantly lower than those of groups A and C at all time points after hypoxia treatment; while the cell survival rate in group E was significantly increased than those in groups A and B, and the diffrences at 24 hours were significant ( P<0.05). In group E, the cell survival rate at 24 hours was significantly higher than those at 12 and 48 hours ( P<0.05). The results of flow cytometry showed that the apoptosis rate in group D was significantly higher than those in groups A and C ( P<0.05), and the apoptosis rate in group E was significantly lower than those in groups A and B ( P<0.05). Western blot showed that the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group D were significantly decreased when compared with those in groups A and C, and the expressions of Bax and C-Caspase-3 proteins were significantly increased ( P<0.05). On the contrary, the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group E were significantly higher than those in groups A and B, and the expressions of Bax and C-Caspase-3 proteins were significantly decreased ( P<0.05). CONCLUSION: Overexpression of HIF-1α gene can significantly improve hAMSCs tolerance to hypoxia, the mechanism may be related to up-regulation of VEGF and Bcl-2 expressions, and down-regulation of Bax and C-Caspase-3 expressions. 目的: 通过瞬时转染缺氧诱导因子 1α（hypoxia-inducible factor 1α，HIF-1α）基因，观察缺氧处理后人羊膜间充质干细胞（human amniotic mesenchymal stem cells，hAMSCs）成活及凋亡情况，探讨 HIF-1α 对 hAMSCs 耐受缺氧能力的影响。. 方法: 取健康剖宫产产妇自愿捐赠的羊膜组织分离、培养 hAMSCs，通过倒置相差显微镜观察细胞形态及免疫荧光法检测干细胞标志物 OCT-4、NANOG 表达，对培养细胞进行鉴定。取第 3 代 hAMSCs 进行缺氧处理（200 μmol/L CoCl ₂）后，按以下分组瞬时转染对应质粒：A 组为 hAMSCs 空白组，B 组为 pcDNA3.1 阴性对照组，C 组为 shRNA 阴性对照组，D 组为 shRNA-HIF-1α 干扰组，E 组为 pcDNA3.1-HIF-1α 过表达组。缺氧处理后 12、24、48 h 采用细胞计数试剂盒 8（cell counting kit 8，CCK-8）检测各组细胞成活率；24 h 采用流式细胞仪检测各组细胞凋亡率，并采用 Western blot 检测 HIF-1α、VEGF、B 细胞淋巴瘤 2（B-cell lymphoma 2，Bcl-2）、Bax、活化型半胱天冬酶-3（cleaved Caspase-3，C-Caspase-3）蛋白表达。. 结果: CCK-8 法检测示，缺氧处理后各时间点与 A、C 组比较，D 组细胞成活率明显降低（ P<0.05）；与 A、B 组比较，E 组细胞成活率明显升高，其中 24 h 组间比较差异有统计学意义（ P<0.05）；E 组内 24 h 时细胞成活率明显高于 12、48 h 时（ P<0.05）。流式细胞仪检测示，与 A、C 组比较，D 组细胞凋亡率明显升高（ P<0.05）；与 A、B 组比较，E 组细胞凋亡率明显降低（ P<0.05）。Western blot 检测示，与 A、C 组比较，D 组细胞中 HIF-1α、VEGF、Bcl-2 蛋白表达明显减少，Bax、C-Caspase-3 蛋白表达明显增加，差异有统计学意义（ P<0.05）；而 E 组结果相反，与 A、B 组比较，E 组细胞中 HIF-1α、VEGF、Bcl-2 蛋白表达明显增加，Bax、C-Caspase-3 蛋白表达明显减少，差异有统计学意义（ P<0.05）。. 结论: HIF-1α 基因过表达能够明显改善 hAMSCs 耐受缺氧能力，其机制可能与上调 VEGF 和 Bcl-2 表达及下调 Bax 和 C-Caspase-3 表达作用相关。.

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