Lihao Ge1, Deshui Yu2, Ruichao Su1, Yang Cao1. 1. Department of Orthopedics, the First Affiliated Hospital of Jinzhou Medical University, Jinzhou Liaoning, 121001, P.R.China. 2. Jinzhou Medical University, Jinzhou Liaoning, 121001, P.R.China.ydsfsyy@163.com.
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.
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.
Authors: Qian Li; Olindi Wijesekera; Sussan J Salas; Joanna Y Wang; Mingxin Zhu; Colette Aprhys; Kaisorn L Chaichana; David A Chesler; Hao Zhang; Christopher L Smith; Hugo Guerrero-Cazares; Andre Levchenko; Alfredo Quinones-Hinojosa Journal: Clin Cancer Res Date: 2014-05-01 Impact factor: 12.531
Authors: Bruna R Sousa; Ricardo C Parreira; Emerson A Fonseca; Maria J Amaya; Fernanda M P Tonelli; Samyra M S N Lacerda; Pritesh Lalwani; Anderson K Santos; Katia N Gomes; Henning Ulrich; Alexandre H Kihara; Rodrigo R Resende Journal: Cytometry A Date: 2013-11-25 Impact factor: 4.355