缺氧对人牙周膜细胞增殖、迁移能力及IGF-1表达的影响
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摘要
目的:探讨缺氧对人牙周膜细胞(human periodontal ligament cells,hPDLCs)生物学活性的影响。方法:选择第5代hPDLCs,在严重缺氧(1%O_2)、轻度缺氧(5%O_2)和常氧(21%O_2)3种环境下培养,用MTT法评价细胞增殖率,用倒置显微镜观察细胞形态变化,细胞划痕试验评价细胞迁移能力,用免疫荧光染色染色研究细胞中低氧诱导因子-1(hypoxia inducible factor-1,HIF-1)的表达及分布情况,用RT-PCR及Western blot检测HIF和IGF-1在不同氧浓度环境下的mRNA及蛋白表达水平。结果:①与正常hPDLCs相比,在1%O_2浓度下,hPDLCs的数量在一个显微镜视野下明显减少,排列稀疏,结构发生改变。②hPDLCs的增长速率随着氧浓度的下降而减少,氧气浓度越低,增殖速率越慢。③在划痕处理后72 h,在3组不同氧浓度下,hPDLCs均向划痕区域迁移,但严重缺氧组的移动速率明显小于轻度缺氧和常氧组(划痕72 h:1%O_2组0.917±0.023,5%O_2组0.742±0.046,21%O_2组0.453±0.039,F=530.237,P=0.002)。④HIF/DAPI染色结果显示在常氧条件下,HIF-1主要表达在细胞质中,随着氧气浓度降低,HIF逐渐转移至细胞核内。结论:缺氧能够抑制人牙周膜细胞的增殖和迁移能力,改变hPDLCs的形态。在缺氧条件下,HIF从细胞质转移到细胞核中,HIF和IGF-1的表达增加,提示缺氧微环境对人牙周膜细胞的生物学活性有一定影响。
Objective:To investigate the influence of hypoxia on the bioactivities of human periodontal ligament fibroblasts(hPDLCs).Methods:The fifth-generation hPDLCs were selected and cultured under the condition of severe hypoxia(1% O_2),mild hypoxia(5%O_2),or normoxia(21% O_2). MTT assay was used to measure the proliferation rate of hPDLCs. An inverted microscope was used to observe cell morphological changes. Wound healing assay was used to observe the migration ability of hPDLCs. Immunofluorescence staining was used to measure the expression and distribution of hypoxia inducible factor-1(HIF-1) in hPDLCs,and RT-PCR and Western blot were used to measure the mRNA and protein expression of HIF-1 and insulin-like growth factor-1(IGF-1) under different levels of O_2. Results:Compared with normal hPDLCs,the hPDLCs cultured in 1% O_2 had a significant reduction in cell number in a microscopic field,with sparse arrangement and a change in structure. The growth rate of hPDLCs decreased with the reduction in oxygen concentration;the lower the oxygen concentration,the slower the proliferation rate. At 72 hours after scratch in the wound healing assay,hPDLCs migrated to the scratched area in all three groups,but the severe hypoxia group had a significantly lower migration rate than the mild hypoxia group and the normoxia group(0.917±0.023 vs. 0.742±0.046/0.453±0.039,F=530.237,P=0.002).HIF/DAPI staining showed that under the condition of normal oxygen,HIF-1 was mainly expressed in the cytoplasm,and with the gradual reduction in oxygen concentration,HIF-1 gradually migrated to the nucleus. Conclusion:Hypoxia can inhibit the proliferation and migration of hPDLCs and change their morphology. Under the hypoxic condition,HIF-1 gradually migrates from the cytoplasm to the nucleus,and there are increases in the expression of HIF-1 and IGF-1,suggesting that hypoxia has certain influence on the bioactivity of hPDLCs.
Objective:To investigate the influence of hypoxia on the bioactivities of human periodontal ligament fibroblasts(hPDLCs).Methods:The fifth-generation hPDLCs were selected and cultured under the condition of severe hypoxia(1% O_2),mild hypoxia(5%O_2),or normoxia(21% O_2). MTT assay was used to measure the proliferation rate of hPDLCs. An inverted microscope was used to observe cell morphological changes. Wound healing assay was used to observe the migration ability of hPDLCs. Immunofluorescence staining was used to measure the expression and distribution of hypoxia inducible factor-1(HIF-1) in hPDLCs,and RT-PCR and Western blot were used to measure the mRNA and protein expression of HIF-1 and insulin-like growth factor-1(IGF-1) under different levels of O_2. Results:Compared with normal hPDLCs,the hPDLCs cultured in 1% O_2 had a significant reduction in cell number in a microscopic field,with sparse arrangement and a change in structure. The growth rate of hPDLCs decreased with the reduction in oxygen concentration;the lower the oxygen concentration,the slower the proliferation rate. At 72 hours after scratch in the wound healing assay,hPDLCs migrated to the scratched area in all three groups,but the severe hypoxia group had a significantly lower migration rate than the mild hypoxia group and the normoxia group(0.917±0.023 vs. 0.742±0.046/0.453±0.039,F=530.237,P=0.002).HIF/DAPI staining showed that under the condition of normal oxygen,HIF-1 was mainly expressed in the cytoplasm,and with the gradual reduction in oxygen concentration,HIF-1 gradually migrated to the nucleus. Conclusion:Hypoxia can inhibit the proliferation and migration of hPDLCs and change their morphology. Under the hypoxic condition,HIF-1 gradually migrates from the cytoplasm to the nucleus,and there are increases in the expression of HIF-1 and IGF-1,suggesting that hypoxia has certain influence on the bioactivity of hPDLCs.
引文
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[16] Guillemin K,Krasnow MA. The hypoxic response:huffing and HIFing[J]. Cell,1997,89(1):9-12.
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[21] Holzenberger M,Dupont J,Ducos B,et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice[J]. Nature,2003,421(6919):182-187.
[22] Burnol AF,Morzyglod L,Popineau L. Cross-talk between insulin signaling and cell proliferation pathways[J]. Ann Endocrinol(Paris),2013,74(2):74-78.
[23] Quesada A,Lee BY,Micevych PE. PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson’s disease[J]. Dev Neurobiol,2008,68(5):632-644.
[24] Perez-Martin M,Cifuentes M,Grondona JM,et al. Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain:role of endogenous IGF-1 as a survival factor[J]. Eur J Neurosci,2003,17(2):205-211.
[25] Kim Y,Li E,Park S. Insulin-like growth factor-1 inhibits 6-hydroxydopaminemediated endoplasmic reticulum stress-inducedapoptosis via regulation of hemeoxygenase-1 and Nrf2 expression in PC12 cells[J]. Int J Neurosci,2012,122(11):641-649.
[26] Beresewicz M,Majewska M,Makarewicz D,et al. Changes in the expression of insulin-like growth factor 1 variants in the post natal brain development and in neonatal hypoxia-ischaemia[J]. Int J Devneurosci,2010,28(1):91-97.
[27] Kaidi A,Williams AC,Paraskeva C. Interaction between betacatenin and HIF-1 promotes cellular adaptation to hypoxia[J]. Nature Cell Biology,2007,9(2):210-217.
[28] Lim JH,Chun YS,Park JW. Hypoxia-inducible factor-1alpha obstructs a Wnt signaling pathway by inhibiting the h ARD1-mediated activation of beta-catenin[J]. Cancer Research,2008,68(13):5177-5184.
[29] Acker T,Plate KH. A role for hypoxia and hypoxia-inducible transcription factors in tumor physiology[J]. J Mol Med,2002,80(9):562-575.
[30] Slomiany MG,Rosenzweig SA. IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407[J]. Invest Ophthalmol Vis Sci,2004,45(8):2838-2847.
[2] Yu Y,Mu J,Fan Z,et al. Insulin-like growth factor 1 enhances the proliferation and osteogenic mineralization of human peri-odontal ligament stem cells via ERK and JNK MAPK pathways[J]. Histochem Cell Biol,2012,137(4):513-525.
[3] Choe Y,Yu JY,Son YO,et al. Continuously generated H2O2 stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts[J]. J Cell Biochem,2012,113(4):1426-1436.
[4] Konermann A,Stabenow D,Knolle PA,et al. Regulatory role of periodontal ligament fibroblasts for innate immune cell function and differentiation[J]. Innate Immun,2011,18:745-752.
[5]李培勇,辛军,伍佰聪,等. HIF-1α、MMP-9、MVD在肾细胞癌中的表达及临床意义[J].重庆医科大学学报,2013,38(2):151-154.
[6]姜蓉,段江洁,田静,等.胎儿骨髓间充质干细胞EGF受体或IGF受体的表达与其向上皮细胞分化的关系[J].重庆医科大学学报,2012,37(1):39-42.
[7]黄辉祥,汤文成,吴斌,等.基于超弹性模型的牙周膜力学行为数值模拟[J].上海交通大学学报,2014,48(9):1263-1273.
[8] Seo BM,Miura M,Gronthos S,et al. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet,2004,364(9429):149-155.
[9] Murakami Y,Kojima T,Nagasawa T,et al. Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts[J]. J Peri,2003,74(6):780-786.
[10] Kaneda T,Miyauchi M,Takekoshi T,et al. Characteristics of periodontal ligament subpopulations obtained by sequential enzymatic digestion of rat molar periodontal ligament[J]. Bone,2006,38(3):420-426.
[11] Salim A,Nacamuli RP,Morgan EF,et al. Transient changes in oxygen tension inhibit osteogenic differentiation and Rux2 expression in osteoblast[J]. J Biol Chem,2004,279(38):40007-40016.
[12] Mylonis I,Sembongi H,Befani C,et al. Hypoxia causes triglyceride accumulation via HIF-1-mediated stimulation of lipin 1 expression[J]. J Cell Sci,2012,125(Pt14):3485-3493.
[13]蔡秀红,黄饴涛,张子平,等.缺氧诱导因-I(HIF-1)及其在水生动物中的研究进展[J].农业生物技术学报,2014,22(1):119-132.
[14] Pichon A,Zhenzhong B,Favret F,et al. Long-term ventilatory adaptation and ventilatory response to hypoxia in plateau pika(Ochotona curzoniae):role of nNOS and dopamine[J]. Am J Physiol Regul Integr Comp Physiol,2009,297(4):978-987.
[15] Rowland KJ,Yao J,Wang L,et al. Up-regulation of hypoxia-inducible factor 1 alpha and hemodynamic responses following massive small bowel resection[J]. J Pediatr Surg,2013,48(6):1330-1339.
[16] Guillemin K,Krasnow MA. The hypoxic response:huffing and HIFing[J]. Cell,1997,89(1):9-12.
[17] Hisada T,Ayaori M,Ohrui N,et al. Statin inhibits hypoxia-induced endothelin-1 via accelerated degradation of HIF-1αin vascular smooth muscle cells[J]. Cardiovasc Res,2012,95(2):251-259.
[18] Bento CF,Pereira P. Regulation of hypoxia-inducible factor l and the loss of the cellular response to hypoxia in diabetes[J]. Diabetologia,2011,54(8):1946-1956.
[19] Rey S,Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling[J]. Cardiovasc Res,2010,86(2):236-242.
[20]唐开亮,于西佼,杜毅.乏氧诱导因子-1α在人牙周膜细胞的表达[J].中国医学创新,2013,8(13):6-7.
[21] Holzenberger M,Dupont J,Ducos B,et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice[J]. Nature,2003,421(6919):182-187.
[22] Burnol AF,Morzyglod L,Popineau L. Cross-talk between insulin signaling and cell proliferation pathways[J]. Ann Endocrinol(Paris),2013,74(2):74-78.
[23] Quesada A,Lee BY,Micevych PE. PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson’s disease[J]. Dev Neurobiol,2008,68(5):632-644.
[24] Perez-Martin M,Cifuentes M,Grondona JM,et al. Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain:role of endogenous IGF-1 as a survival factor[J]. Eur J Neurosci,2003,17(2):205-211.
[25] Kim Y,Li E,Park S. Insulin-like growth factor-1 inhibits 6-hydroxydopaminemediated endoplasmic reticulum stress-inducedapoptosis via regulation of hemeoxygenase-1 and Nrf2 expression in PC12 cells[J]. Int J Neurosci,2012,122(11):641-649.
[26] Beresewicz M,Majewska M,Makarewicz D,et al. Changes in the expression of insulin-like growth factor 1 variants in the post natal brain development and in neonatal hypoxia-ischaemia[J]. Int J Devneurosci,2010,28(1):91-97.
[27] Kaidi A,Williams AC,Paraskeva C. Interaction between betacatenin and HIF-1 promotes cellular adaptation to hypoxia[J]. Nature Cell Biology,2007,9(2):210-217.
[28] Lim JH,Chun YS,Park JW. Hypoxia-inducible factor-1alpha obstructs a Wnt signaling pathway by inhibiting the h ARD1-mediated activation of beta-catenin[J]. Cancer Research,2008,68(13):5177-5184.
[29] Acker T,Plate KH. A role for hypoxia and hypoxia-inducible transcription factors in tumor physiology[J]. J Mol Med,2002,80(9):562-575.
[30] Slomiany MG,Rosenzweig SA. IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407[J]. Invest Ophthalmol Vis Sci,2004,45(8):2838-2847.