Effects of hypoxia-inducible factor 1α on hypoxic tolerance of human amniotic mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GE Lihao ¹ ,  YU Deshui ² , SU Ruichao ¹ , CAO Yang ¹

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;

Corresponding?author：

YU Deshui, Email: ydsfsyy@163.com

Keywords：

Hypoxia-inducible factor 1α; human amniotic mesenchymal stem cells; hypoxia; apoptosis

DOI：

10.7507/1002-1892.201710104

Video：

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ObjectiveUnder 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.MethodsThe 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 CoCl₂, 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.ResultsCCK-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).ConclusionOverexpression 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.

Citation： GE Lihao, YU Deshui, SU Ruichao, CAO Yang. Effects of hypoxia-inducible factor 1α on hypoxic tolerance of human amniotic mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(3): 264-269. doi: 10.7507/1002-1892.201710104 Copy

1.	Zhou HL, Zhang XJ, Zhang MY, et al. Transplantation of human amniotic mesenchymal stem cells promotes functional recovery in a rat Model of traumatic spinal cord injury. Neurochem Res, 2016, 41(10): 2708-2718.
2.	Tajiri N, Acosta S, Portillo-Gonzales GS, et al. Therapeutic outcomes of transplantation of amniotic fluid-derived stem cells in experimental ischemic stroke. Front Cell Neurosci, 2014, 8: 227.
3.	Roh DH, Seo MS, Choi HS, et al. Transplantation of human umbilical cord blood or amniotic epithelial stem cells alleviates mechanical allodynia after spinal cord injury in rats. Cell Transplant, 2013, 22(9): 1577-1590.
4.	Lee HJ, Ryu JM, Jung YH, et al. Novel pathway for hypoxia-induced proliferation and migration in human mesenchymal stem cells: involvement of HIF-1α, FASN, and mTORC1. Stem Cells, 2015, 33(7): 2182-2195.
5.	孔宏亮, 孫紅波, 宋麗杰, 等. 缺氧誘導因子-1α 過表達對骨髓間充質干細胞缺氧時生存能力的影響. 中國普外基礎與臨床雜志, 2011, 18(3): 261-266.
6.	Barbati A, Grazia Mameli M, Sidono A, et al. Amniotic membrane: separation of amniotic mesoderm from amniotic epithelium and isolation of their respective mesenchymal stromal and epithelial cells. Curr Protoc Stem Cell Biol, 2012, Chapter 1: Unit 1E.8. doi: 10.1002/9780470151808.sc01e08s20.
7.	Choi SA, Choi HS, Kim KJ, et al. Isolation of canine mesenchymal stem cells from amniotic fluid and differentiation into hepatocyte-like cells. In Vitro Cell Dev Biol Anim, 2013, 49(1): 42-51.
8.	Bartoszewska S, Kochan K, Piotrowski A, et al. The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1a expression in human endothelial cells through a negative feedback loop. FASEB J, 2015, 29(4): 1467-1479.
9.	Sousa BR, Parreira RC, Fonseca EA, et al. Human adult stem cells from diverse origins: an overview from multiparametric immunophenotyping to clinical applications. Cytometry A, 2014, 85(1): 43-77.
10.	Song M, Jue SS, Cho YA, et al. Comparison of the effects of human dental pulp stem cells and human bone marrow-derived mesenchymal stem cells on ischemic human astrocytes in vitro. J Neurosci Res, 2015, 93(6): 973-983.
11.	Sharma HS. Early microvascular reactions and blood-spinal cord barrier disruption are instrumental in pathophysiology of spinal cord injury and repair: novel therapeutic strategies including nanowired drug delivery to enhance neuroprotection. J Neural Transm (Vienna), 2011, 118(1): 155-176.
12.	Li Q, Wijesekera O, Salas SJ, et al. Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival. Clin Cancer Res, 2014, 20(9): 2375-2387.
13.	王立維, 黃文, 趙渝. 低氧培養對間充質干細胞生物學的影響. 中國組織工程研究, 2012, 16(23): 4329-4334.
14.	祁琳, 張曉雷, 毛立群, 等. HIF-1 與低氧性疾病關系的研究進展. 武警后勤學院學報 (醫學版), 2013, 22(4): 330-333.
15.	Frost P, Berlanger E, Mysore V, et al. Mammalian target of rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression and angiogenesis. J Oncol, 2013, 2013: 897025.
16.	Shen F, Fan Y, Su H, et al. Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice. Gene Ther, 2008, 15(1): 30-39.
17.	Reischl S, Li L, Walkinshaw G, et al. Inhibition of HIF prolyl-4-hydroxylases by FG-4497 reduces brain tissue injury and edema formation during ischemic stroke. PLoS One, 2014, 9(1): e84767.
18.	Wang JA, Chen TL, Jiang J, et al. Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta Pharmacol Sin, 2008, 29(1): 74-82.
19.	梁俊清, 徐海波, 陳小娟, 等. 通心絡通過 PI-3K/Akt/HIF 信號通路改善血管內皮細胞缺氧損傷. 中國病理生理雜志, 2012, 28(5): 846-851.
20.	陳萌, 田煥娜, 吳曉光, 等. 半胱氨酸蛋白酶抑制劑 C 對腦缺血再灌注大鼠 Bcl-2、Bax 和 Beclin-1 的表達及細胞凋亡的影響. 解剖學雜志, 2017, 40(4): 417-420.
21.	Li Z, Pang L, Fang F, et al. Resveratrol attenuates brain damage in a rat model of focal cerebral ischemia via up-regulation of hippocampal Bcl-2. Brain Res, 2012, 1450: 116-124.
22.	Li Q, Li Z, Xu XY, et al. Neuroprotective properties of picroside Ⅱ in a rat model of focal cerebral ischemia. Int J Mol Sci, 2010, 11(11): 4580-4590.

1. Zhou HL, Zhang XJ, Zhang MY, et al. Transplantation of human amniotic mesenchymal stem cells promotes functional recovery in a rat Model of traumatic spinal cord injury. Neurochem Res, 2016, 41(10): 2708-2718.
2. Tajiri N, Acosta S, Portillo-Gonzales GS, et al. Therapeutic outcomes of transplantation of amniotic fluid-derived stem cells in experimental ischemic stroke. Front Cell Neurosci, 2014, 8: 227.
3. Roh DH, Seo MS, Choi HS, et al. Transplantation of human umbilical cord blood or amniotic epithelial stem cells alleviates mechanical allodynia after spinal cord injury in rats. Cell Transplant, 2013, 22(9): 1577-1590.
4. Lee HJ, Ryu JM, Jung YH, et al. Novel pathway for hypoxia-induced proliferation and migration in human mesenchymal stem cells: involvement of HIF-1α, FASN, and mTORC1. Stem Cells, 2015, 33(7): 2182-2195.
5. 孔宏亮, 孫紅波, 宋麗杰, 等. 缺氧誘導因子-1α 過表達對骨髓間充質干細胞缺氧時生存能力的影響. 中國普外基礎與臨床雜志, 2011, 18(3): 261-266.
6. Barbati A, Grazia Mameli M, Sidono A, et al. Amniotic membrane: separation of amniotic mesoderm from amniotic epithelium and isolation of their respective mesenchymal stromal and epithelial cells. Curr Protoc Stem Cell Biol, 2012, Chapter 1: Unit 1E.8. doi: 10.1002/9780470151808.sc01e08s20.
7. Choi SA, Choi HS, Kim KJ, et al. Isolation of canine mesenchymal stem cells from amniotic fluid and differentiation into hepatocyte-like cells. In Vitro Cell Dev Biol Anim, 2013, 49(1): 42-51.
8. Bartoszewska S, Kochan K, Piotrowski A, et al. The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1a expression in human endothelial cells through a negative feedback loop. FASEB J, 2015, 29(4): 1467-1479.
9. Sousa BR, Parreira RC, Fonseca EA, et al. Human adult stem cells from diverse origins: an overview from multiparametric immunophenotyping to clinical applications. Cytometry A, 2014, 85(1): 43-77.
10. Song M, Jue SS, Cho YA, et al. Comparison of the effects of human dental pulp stem cells and human bone marrow-derived mesenchymal stem cells on ischemic human astrocytes in vitro. J Neurosci Res, 2015, 93(6): 973-983.
11. Sharma HS. Early microvascular reactions and blood-spinal cord barrier disruption are instrumental in pathophysiology of spinal cord injury and repair: novel therapeutic strategies including nanowired drug delivery to enhance neuroprotection. J Neural Transm (Vienna), 2011, 118(1): 155-176.
12. Li Q, Wijesekera O, Salas SJ, et al. Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival. Clin Cancer Res, 2014, 20(9): 2375-2387.
13. 王立維, 黃文, 趙渝. 低氧培養對間充質干細胞生物學的影響. 中國組織工程研究, 2012, 16(23): 4329-4334.
14. 祁琳, 張曉雷, 毛立群, 等. HIF-1 與低氧性疾病關系的研究進展. 武警后勤學院學報 (醫學版), 2013, 22(4): 330-333.
15. Frost P, Berlanger E, Mysore V, et al. Mammalian target of rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression and angiogenesis. J Oncol, 2013, 2013: 897025.
16. Shen F, Fan Y, Su H, et al. Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice. Gene Ther, 2008, 15(1): 30-39.
17. Reischl S, Li L, Walkinshaw G, et al. Inhibition of HIF prolyl-4-hydroxylases by FG-4497 reduces brain tissue injury and edema formation during ischemic stroke. PLoS One, 2014, 9(1): e84767.
18. Wang JA, Chen TL, Jiang J, et al. Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta Pharmacol Sin, 2008, 29(1): 74-82.
19. 梁俊清, 徐海波, 陳小娟, 等. 通心絡通過 PI-3K/Akt/HIF 信號通路改善血管內皮細胞缺氧損傷. 中國病理生理雜志, 2012, 28(5): 846-851.
20. 陳萌, 田煥娜, 吳曉光, 等. 半胱氨酸蛋白酶抑制劑 C 對腦缺血再灌注大鼠 Bcl-2、Bax 和 Beclin-1 的表達及細胞凋亡的影響. 解剖學雜志, 2017, 40(4): 417-420.
21. Li Z, Pang L, Fang F, et al. Resveratrol attenuates brain damage in a rat model of focal cerebral ischemia via up-regulation of hippocampal Bcl-2. Brain Res, 2012, 1450: 116-124.
22. Li Q, Li Z, Xu XY, et al. Neuroprotective properties of picroside Ⅱ in a rat model of focal cerebral ischemia. Int J Mol Sci, 2010, 11(11): 4580-4590.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of hypoxia-inducible factor 1α on hypoxic tolerance of human amniotic mesenchymal stem cells

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