EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUFengxu ^1,2 , CHENYongen ¹ , CHENFeng ³ , XIAJiyi ³ , LIUHongduan ¹ , FUYong ¹ , LIMiaoling ^2,4 ,  LIAOBin ^1,2

1. Department of Cardiothoracic Surgery, the Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P. R. China;
2. Institute of Cardiovascular Research, Ministry of Education of China, Southwest Medical University;
3. Central Laboratory, the Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P. R. China;
4. Key Laboratory of Medical Electrophysiology, Ministry of Education of China, Southwest Medical University;

Corresponding author：

LIAOBin, Email: liaobindr@tom.com

Keywords：

Akt1; Bone marrow mesenchymal stem cells; Recombinant lentivirus; Apoptosis; Hypoxia; Rat

DOI：

10.7507/1002-1892.20160096

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Objective To investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. Method LVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3FLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94%N2, 1%O2, and 5%CO2 for 0, 3, 6, 9, and 12 hours (group B1 and group C1) . The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. Results After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t=17.525, P=0.013) . The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P<0.05) . In group C1, the apoptosis rate and cell death rate decreased temporarily at 3 hours after hypoxia intervention, then increased gradually, and difference was significant (P<0.05) . The apoptosis rate and cell death rate of group C1 were significantly lower than those of group B1 at each time point (P<0.05) except at 0 hour. MTT assay showed that absorbance (A) values of groups B and C were significantly higher than those of groups B1 and C1 at each time point (P<0.05) ; the A value of group B was significantly lower than that of group C at each time point (P<0.05) . The A value of group B1 was significantly lower than that of group C1 at 6, 9, and 12 hours after hypoxia intervention (P<0.05) . Western blot results showed that the Caspase-3 expression of group C1 significantly reduced when compared with group B1 at each time point (P<0.05) . Conclusions Akt1 gene transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.

Citation： YUFengxu, CHENYongen, CHENFeng, XIAJiyi, LIUHongduan, FUYong, LIMiaoling, LIAOBin. EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(4): 479-484. doi: 10.7507/1002-1892.20160096 Copy

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2.	Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature, 2001, 410(6829) :701-705.
3.	Leistner DM, Fischer-Rasokat U, Honold J, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI):final 5-year results suggest long-term safety and efficacy. Clin Res Cardiol, 2011, 100(10) :925-934.
4.	Abeyrathna P, Su Y. The critical role of Akt in cardiovascular function. Vascul Pharmacol, 2015, 74:38-48.
5.	Hemmings BA. Akt signaling:linking membrane events to life and death decisions. Science, 1997, 275(5300) :628-630.
6.	Persad S, Attwell S, Gray V, et al. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase:critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem, 2001, 276(29) :27462-27469.
7.	Ernens I, Goodfellow SJ, Innes F, et al. Hypoxic stress suppresses RNA polymerase Ⅲ recruitment and tRNA gene transcription in cardiomyocytes. Nucleic Acids Res, 2006, 34(1) :286-294.
8.	Bilgimol JC, Ragupathi S, Vengadassalapathy L, et al. Stem cells:An eventual treatment option for heart diseases. World J Stem Cells, 2015, 7(8) :1118-1126.
9.	王晓静, 李贵华. 急性心肌梗死院前延迟的研究进展. 心血管病学进展, 2010, 31(2) :251-253.
10.	Xu X, Liu Y, Cui ZF. Effects of cryopreservation on human mesenchymal stem cells attached to different substrates. J Tissue Eng Regen Med, 2014, 8(8) :664-672.
11.	Perin EC, Dohmann HF, Borojevic R, et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation, 2003, 107(18) :2294-2302.
12.	Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function of introcoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol, 2004, 94(1) :92-95.
13.	Moon HH, Joo MK, Mok H, et al. MSC-based VEGF gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated polyethyleneimine. Biomaterials, 2014, 35(5) :1744-1754.
14.	Mangi AA, Noiseux N, Kong D, et al. Mesenchymal a stem cells modified with Akt prevents remodeling and restores performance of infarcted hearts. Nat Med, 2003, 9(9) :1195-1201.
15.	Visciano C, Liotti F, Prevete N, et al. Mast cells induce epithelial to mesenchymal transition and stem cell features in human thyroid cancer cells through an IL-8-Akt-Slug pathway. Oncogene, 2015, 34(40) :5175-5186.
16.	Choi KH, Park JK, Kim HS, et al. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin. PLoS One, 2013, 8(8) :e72184.
17.	Tiscornia G, Singer O, Verma IM, et al. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc, 2006, 1(1) :234-240.
18.	Fotia C, Massa A, Boriani F, et al. Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells. Cytotechnology, 2015, 67(6) :1073-1084.
19.	Zhang Y, Goodyer C, LeBlanc A. Selective and protracted apoptosis in human primary neurons microinjection with active caspase-3, -6, -7 and -8. J Neurosci, 2000, 20(22) :8384-8389.

1. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A, 2001, 98(18) :10344-10349.
2. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature, 2001, 410(6829) :701-705.
3. Leistner DM, Fischer-Rasokat U, Honold J, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI):final 5-year results suggest long-term safety and efficacy. Clin Res Cardiol, 2011, 100(10) :925-934.
4. Abeyrathna P, Su Y. The critical role of Akt in cardiovascular function. Vascul Pharmacol, 2015, 74:38-48.
5. Hemmings BA. Akt signaling:linking membrane events to life and death decisions. Science, 1997, 275(5300) :628-630.
6. Persad S, Attwell S, Gray V, et al. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase:critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem, 2001, 276(29) :27462-27469.
7. Ernens I, Goodfellow SJ, Innes F, et al. Hypoxic stress suppresses RNA polymerase Ⅲ recruitment and tRNA gene transcription in cardiomyocytes. Nucleic Acids Res, 2006, 34(1) :286-294.
8. Bilgimol JC, Ragupathi S, Vengadassalapathy L, et al. Stem cells:An eventual treatment option for heart diseases. World J Stem Cells, 2015, 7(8) :1118-1126.
9. 王晓静, 李贵华. 急性心肌梗死院前延迟的研究进展. 心血管病学进展, 2010, 31(2) :251-253.
10. Xu X, Liu Y, Cui ZF. Effects of cryopreservation on human mesenchymal stem cells attached to different substrates. J Tissue Eng Regen Med, 2014, 8(8) :664-672.
11. Perin EC, Dohmann HF, Borojevic R, et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation, 2003, 107(18) :2294-2302.
12. Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function of introcoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol, 2004, 94(1) :92-95.
13. Moon HH, Joo MK, Mok H, et al. MSC-based VEGF gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated polyethyleneimine. Biomaterials, 2014, 35(5) :1744-1754.
14. Mangi AA, Noiseux N, Kong D, et al. Mesenchymal a stem cells modified with Akt prevents remodeling and restores performance of infarcted hearts. Nat Med, 2003, 9(9) :1195-1201.
15. Visciano C, Liotti F, Prevete N, et al. Mast cells induce epithelial to mesenchymal transition and stem cell features in human thyroid cancer cells through an IL-8-Akt-Slug pathway. Oncogene, 2015, 34(40) :5175-5186.
16. Choi KH, Park JK, Kim HS, et al. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin. PLoS One, 2013, 8(8) :e72184.
17. Tiscornia G, Singer O, Verma IM, et al. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc, 2006, 1(1) :234-240.
18. Fotia C, Massa A, Boriani F, et al. Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells. Cytotechnology, 2015, 67(6) :1073-1084.
19. Zhang Y, Goodyer C, LeBlanc A. Selective and protracted apoptosis in human primary neurons microinjection with active caspase-3, -6, -7 and -8. J Neurosci, 2000, 20(22) :8384-8389.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF Akt1 GENE TRANSFECTION ON HYPOXIA TOLERANCE OF BONE MARROW MESENCHYMAL STEM CELLS

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