Mutant hypoxia inducible factor 1alpha transfection promotes proliferation of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.25.004

Abstract

Abstract:

BACKGROUND: It has been found that hypoxia inducible 1α (HIF1α) gene can improve the ability of tissues and cells to survive in the ischemic environment, which is of great significance in promoting blood vessel regeneration.
OBJECTIVE: To investigate the effect of the recombinant adenovirus Ad-CMV-HIF1α^mu-IRES-hrGFP-1 on the proliferation of bone marrow mesenchymal stem cells.
METHODS: Passage 3 bone marrow mesenchymal stem cells were cultured and transfected with Ad-CMV-HIF1α^mu-IRES-hrGFP-1 virus (experimental group), with non-mutated gene Ad-CMV-HIF1α-IRES-hrGFP (positive control group), with Ad-CMV-IRES-hrGFP-1 empty virus (negative control group), or with no virus solution (blank control group). After transfection for 24 hours, the expression of HIF1α mRNA and protein was detected. Cell proliferation was detected by MTT assay at 24, 48, 72, 96 and 120 hours after transfection.
RESULTS AND CONCLUSION: (1) Except that the mRNA expression of HIF1α in the experimental group and the positive control group was significantly higher than that in the negative control group and the blank control group (P < 0.05), there was no significant difference between two groups. (2) The expression of HIF1α protein in the experimental group was significantly higher than that in the positive control group, the negative control group and the blank control group (P < 0.05), and there was no significant difference between the latter three groups. (3) Cell proliferation was faster in the experimental group than the positive control group and the blank control group (P < 0.05), and moreover, there was no significant difference between the latter two groups. Our experimental findings indicate that the recombinant adenovirus-mediated three-point mutant HIF1α combined with hrGFP gene transfected bone marrow mesenchymal stem cells could not only express the target protein, HIF1α, under normoxic conditions, but also promote the proliferation of bone marrow mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Mesenchymal Stem Cells, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Zhang Wei-wei, Wang Jue. Mutant hypoxia inducible factor 1alpha transfection promotes proliferation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3956-3963.

Figures/Tables 2

2.1 HIF1α基因测序结果测序显示，供体质粒携带的HIF1α基因与GenBank上登记的HIF1α mRNA序列(NM_001530)完全一致，序列全长为4 082 bp，CDS区序列长度为2 481 bp(405-2 885 bp)，共编码了826个氨基酸。对供体质粒行SmaⅠ单酶切及SmaⅠ/NotⅠ双酶切，与预期结果相符(图1)。对HIF1α基因序列内部的限制性内切酶识别位点分析结果显示，序列内部不含有PacⅠ、PvuⅠ、XhoⅠ及NotⅠ、PmeⅠ、SamⅠ等关键酶切位点。 2.2 HIF1α突变后基因测序结果测序显示，供体质粒中所携带的HIF1α基因CDS区402位氨基酸由CCA突变成GCA，564位氨基酸由CCC突变成GCC，803位氨基酸由ATT突变成AGC，均为丙氨酸(图2)。 2.3 双基因载体构建结果电泳显示，腺病毒穿梭质粒pShuttle-CMV-HIF1αmu-IRES-hrGFP-1及重组腺病毒突变型真核表达载体pAd-HIF1αmu-IRES-hrGFP-1均构建成功(图3，4)。 2.4 HEK293A细胞培养情况 HEK293A细胞从50%汇合到70%汇合约需2 d；显微镜下观察细胞密度大时呈铺路石状并有伪足伸展(图5A)，腺病毒经Lipofectamine 2000转染HEK293A细胞后24 h可见绿色荧光表达，随着时间延长，绿色荧光表达越来越强，可见明显的细胞脱落、变圆等病变效应(图5B-D)。 2.5 骨髓间充质干细胞培养情况骨髓间充质干细胞原代培养5 d首次换液后，在倒置显微镜下见有长条形细胞贴壁生长，培养至第3代80%汇合，细胞纯度较高、状态饱满、贴壁良好(图6A)；最佳MOI值经计算为100，以MOI=100转染腺病毒后24 h即可见绿色荧光表达(图6B)，48 h后荧光细胞显著增多(图6C)。 2.6 腺病毒滴度测定结果重组腺病毒Ad-CMV- HIF1αmu-IRES-hrGFP-1按终点稀释法测得病毒滴度为1.6×108 pfu/mL，阳性及阴性对照的病毒滴度分别为1.3× 108 pfu/mL和2.0×108 pfu/mL，滴度满足后续实验要求。 2.7 RT-PCR检测结果实验组和阳性对照组细胞内HIF1α mRNA表达比较差异无显著性意义(P > 0.05)；阴性对照组和空白对照组细胞内HIF1α mRNA表达量非常低，组间比较差异无显著性意义(P > 0.05)；实验组、阳性对照组细胞内HIF1α mRNA表达量均明显高于阴性对照组、空白对照组(P < 0.05)，见图7。 2.8 Western blot检测结果实验组细胞中HIF1α蛋白表达高于阳性对照组、阴性对照组、空白对照组(P < 0.05)，后3组间比较差异无显著性意义(P > 0.05)，见图8。提示三点突变型HIF1α可在常氧条件下正常且高效表达，有利于促进种子细胞移植后的持续增殖及新血管再生。 2.9 细胞增殖结果测定阳性对照组及空白对照组的骨髓间充质干细胞生长速度均较慢，两组之间相比较差异无显著性意义(P > 0.05)；实验组细胞增殖程度快于阳性对照组及空白对照组(P < 0.05)，见图9，说明三点突变型HIF1α可显著提高骨髓间充质干细胞的增殖。"

References

[1] Verseijden F,Posthumus-van Sluijs SJ,van Neck JW,et al. Comparing scaffold-free and fibrin-based adipose-derived stromal cell constructs for adipose tissue engineering: an in vitro and in vivo study. Cell Transplant.2012;21(10):2283-2297.

[2] 伞光,宋佳.血管内皮生长因子165转染可促进脂肪间充质干细胞增殖[J].中国组织工程研究, 2015,19(36):5782-5788.

[3] Joo HH,Jo HJ,Jung TD,et al.Adipose-derived stem cells on the healing of ischemic colitis: a therapeutic effect by angiogenesis.Int J Colorectal Dis.2012;27(11): 1437-1443.

[4] Chua KH,Raduan F,Wan Safwani WK,et al.Effects of serum reduction and VEGF supplementation on angiogenic potential of human adipose stromal cells in vitro.Cell Prolif.2013;46(3): 300-311.

[5] 潘玮敏,刘民,杨建昌,等. LIM矿化蛋白1/低氧诱导因子1α慢病毒载体转染脂肪源性干细胞的成骨分化[J].中国组织工程研究, 2015,19(32):5140-5147.

[6] Ide C,Nakano N,Kanekiyo K.Cell transplantation for the treatment of spinal cord injury - bone marrow stromal cells and choroid plexus epithelial cells.Neural Regen Res.2016; 11(9):1385-1388.

[7] 李沙,李树仁,张倩辉.缺氧诱导因子1a基因转染心脏干细胞修复坏死心肌:应用前景[J].中国组织工程研究,2015,19(23):3750- 3754.

[8] Fiorenzo P,Mongiardi MP,Dimitri D,et al. HIF1-positive and HIF1-negative glioblastoma cells compete in vitro but cooperate in tumor growth in vivo.Int J Oncol. 2010;36(4): 785-791.

[9] Hao C,Wang Y,Shao L,et al.Local Injection of Bone Mesenchymal Stem Cells and Fibrin Glue Promotes the Repair of Bone Atrophic Nonunion In Vivo.Adv Ther.2016; 33(5):824-833.

[10] Zhang W,Chang Q,Xu L,et al.Graphene Oxide-Copper Nanocomposite-Coated Porous CaP Scaffold for Vascularized Bone Regeneration via Activation of Hif-1α.Adv Healthc Mater. 2016;5(11):1299-1309.

[11] 王钰,朱志图,陈峻江.血管内皮生长因子165基因促进人脂肪间充质干细胞的增殖[J].中国组织工程研究,2015,19(28):4485-4492.

[12] 张馨,周琳,张晓雷.重组腺病毒介导突变型低氧诱导因子1α基因转染脂肪间充质干细胞并促进其增殖[J].中国组织工程研究, 2016,20(23):3386-3393.

[13] Ou M,Sun X,Liang J,et al.A polysaccharide from Sargassum thunbergii inhibits angiogenesis via downregulating MMP-2 activity and VEGF/HIF-1α signaling.Int J Biol Macromol.2016; 94(Pt A):451-458.

[14] Song B,Zhang Q,Yu M,et al.Ursolic acid sensitizes radioresistant NSCLC cells expressing HIF-1α through reducing endogenous GSH and inhibiting HIF-1α.Oncol Lett. 2017;13(2):754-762.

[15] Zhang G,Zhao C,Wang Q,et al.Identification of HIF-1 signaling pathway in Pelteobagrus vachelli using RNA-Seq: effects of acute hypoxia and reoxygenation on oxygen sensors, respiratory metabolism, and hematology indices.J Comp Physiol B. 2017.doi: 10.1007/s00360-017-1083-8.[Epub ahead of print]

[16] Liu H,Zhang Z,Xiong W,et al.HIF-1α promotes cells migration and invasion by upregulating autophagy in endometriosis. Reproduction. 2017. pii: REP-16-0643. doi: 10.1530/REP-16-0643.[Epub ahead of print]

[17] Xiang GL,Zhu XH,Lin CZ,et al.125I seed irradiation induces apoptosis and inhibits angiogenesis by decreasing HIF-1α and VEGF expression in lung carcinoma xenografts.Oncol Rep.2017.doi:10.3892/or.2017.5521.[Epub ahead of print]

[18] Zhao H,Jiang H,Li Z,et al.2-Methoxyestradiol enhances radiosensitivity in radioresistant melanoma MDA-MB-435R cells by regulating glycolysis via HIF-1α/PDK1 axis.Int J Oncol.2017.doi: 10.3892/ijo.2017.3924.[Epub ahead of print]

[19] Tanaka Y,Hosoyama T,Mikamo A,et al.Hypoxic preconditioning of human cardiosphere-derived cell sheets enhances cellular functions via activation of the PI3K/Akt/mTOR/HIF-1α pathway.Am J Transl Res.2017; 9(2):664-673.

[20] Wu W,Hu Q,Nie E,et al.Hypoxia induces H19 expression through direct and indirect Hif-1α activity, promoting oncogenic effects in glioblastoma.Sci Rep.2017;7:45029.

[21] Xue L,Chen H,Lu K,et al.Clinical significance of changes in serum neuroglobin and HIF-1α concentrations during the early-phase of acute ischemic stroke.J Neurol Sci. 2017;375: 52-57.

[22] He F,Qi Q,Li X,et al.Association of Indoor Air Pollution, Single Nucleotide Polymorphism of HIF-1α Gene with Susceptibility to Lung Cancer in Han Population in Fujian Province. Zhongguo Fei Ai Za Zhi.2017;20(3):149-156.

[23] Yang SJ,Park YS,Cho JH,et al.Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1α protein stability.EMBO J. 2017. pii: e201694408. doi: 10.15252/embj.201694408.[Epub ahead of print]

[24] Alexander-Shani R,Mreisat A,Smeir E,et al.Long term HIF-1α transcriptional activation is essential for heat-acclimation mediated cross-tolerance: Mitochondrial target genes.Am J Physiol Regul Integr Comp Physiol. 2017:ajpregu.00461.2016. doi: 10.1152/ ajpregu.00461.2016. [Epub ahead of print]

[25] Luo QQ,Qian ZM,Zhou YF,et al.Expression of Iron Regulatory Protein 1 Is Regulated not only by HIF-1 but also pCREB under Hypoxia.Int J Biol Sci.2016;12(10):1191-1202.

[26] 孟祥超,刘卓超,王君,等. MicroRNAs在缺氧诱导因子1α缺失椎间盘组织中的差异性表达[J].中国组织工程研究,2016,20(7): 940-946.

[27] Choi SH,Park JY,Kang W,et al.Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.Apoptosis. 2016; 21(1):85-95.

[28] Jiang YZ,Li Y,Wang K,et al.Distinct roles of HIF1A in endothelial adaptations to physiological and ambient oxygen.Mol Cell Endocrinol.2014;391(1-2):60-67.

[29] 刘竹影,陈颖,刘倩,等.血管内皮祖细胞改善骨质疏松大鼠骨髓间充质干细胞的增殖及凋亡[J].中国组织工程研究,2016,20(14): 1999-2006.

[30] Christoph M,Ibrahim K,Hesse K,et al.Local inhibition of hypoxia-inducible factor reduces neointima formation after arterial injury in ApoE-/- mice.Atherosclerosis.2014; 233(2): 641-647.

[31] 杨超,张雷,周利武,等.低氧诱导因子2α调控骨关节炎:机制研究与转化应用[J].中国组织工程研究,2016,20(24):3634-3641.