Zip1转染骨髓间充质干细胞对Wnt1/β-catenin信号成骨通路的影响
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摘要
目的观察ZIP1 siRNA转染的骨髓间充质干细胞对Wnt1/β-catenin信号成骨通路的影响。方法从成年雄性新西兰兔股骨骨髓中提取骨髓间充质干细胞并培养传代,然后使用0.03 mol/L,0.09 mol/L,0.15 mol/L,0.21 mol/L酒精进行培养,经ZIP1 siRNA及ZIP1表达转染后,检测各组骨髓间充质干细胞中ALP、I型胶原、OCN及Runx2蛋白的表达量。结果与空白对照组相比,不同酒精组的ALP、I型胶原、OCN及Runx2蛋白表达量均有所下降,随着浓度升高,其表达量逐渐下降,但0.03 mol/L组中ALP及OCN下降不明显,统计学无明显意义(P>0.05),其他各组均有明显的统计学意义(P<0.05),不同酒精组中的I型胶原及Runx2与正常组之间差异均有明显统计学意义(P<0.05)。与空白对照组对比,ZIP1表达组ALP、I型胶原及OCN表达量明显升高,差异具有明显的统计学意义(P<0.05),ZIP1siRNA组ALP、OCN表达量明显降低,差异具有明显的统计学意义(P<0.05),而I型胶原表达量下降不明显,差异无统计学意义(P>0.05);与ZIP1siRNA组相比,ZIP1表达组ALP、I型胶原及OCN表达量明显升高,差异有统计学意义(P<0.05)。结论 ZIP1能促使骨髓间质干细胞向成骨分化,从而促进骨修复及骨代谢。沉默ZIP1表达能抑制骨髓间充质干细胞的WNT1/β-catenin信号成骨通路的表达,从而抑制促进细胞的成骨分化能力。
Objective To study the effect of BMSCs transfected by ZIP1( Zrt/Irt-like Protein 1) on Wnt1/β-catenin signaling pathway in the process of osteogenic differentiation. Methods BMSCs were extracted from bone marrow of the femurs of adult male New Zealand rabbits and cultured under different concentrations of alcohol( 0. 03 mol/L,0. 09 mol/L,0. 15 mol/L,and 0. 21 mol/L,respectively). Cells were transfected by ZIP1 siRNA and ZIP1. The expression ALP,collagen type I,OCN,and Runx2 were detected in BMSCs. Results Compared with the control group,the protein expression of ALP,collagen type I,OCN,and Runx2 decreased in different alcohol groups. With the increase of alcohol concentration,the expression decreased gradually. The decrease of ALP and OCN was not significant in 0. 03 mol/L group( P > 0. 05). The difference was statistically significant( P <0. 05) in other groups. The differences of the expression of collagen type I and Runx2 between different alcohol groups and normalgroup were statistically significant( P < 0. 05). Compared with the control group,the expression of ALP,collagen type I and OCN increased significantly in the ZIP1 group,and the difference was significant( P < 0. 05). The expression of ALP and OCN,except the type I collagen,decreased significantly in the ZIP1 siRNA group,and the difference was significant( P < 0. 05). Compared with ZIP1 siRNA group,the expression of ALP,collagen type I,and OCN increased significantly in the ZIP1 group,and the difference was statistically significant( P < 0. 05). Conclusion ZIP1 promotes the osteogenic differentiation of BMSCs and promotes bone repair and bone metabolism. ZIP1 siRNA suppresses the osteogenic differentiation of BMSCs by activating Wnt1/β-catenin signaling pathway.
Objective To study the effect of BMSCs transfected by ZIP1( Zrt/Irt-like Protein 1) on Wnt1/β-catenin signaling pathway in the process of osteogenic differentiation. Methods BMSCs were extracted from bone marrow of the femurs of adult male New Zealand rabbits and cultured under different concentrations of alcohol( 0. 03 mol/L,0. 09 mol/L,0. 15 mol/L,and 0. 21 mol/L,respectively). Cells were transfected by ZIP1 siRNA and ZIP1. The expression ALP,collagen type I,OCN,and Runx2 were detected in BMSCs. Results Compared with the control group,the protein expression of ALP,collagen type I,OCN,and Runx2 decreased in different alcohol groups. With the increase of alcohol concentration,the expression decreased gradually. The decrease of ALP and OCN was not significant in 0. 03 mol/L group( P > 0. 05). The difference was statistically significant( P <0. 05) in other groups. The differences of the expression of collagen type I and Runx2 between different alcohol groups and normalgroup were statistically significant( P < 0. 05). Compared with the control group,the expression of ALP,collagen type I and OCN increased significantly in the ZIP1 group,and the difference was significant( P < 0. 05). The expression of ALP and OCN,except the type I collagen,decreased significantly in the ZIP1 siRNA group,and the difference was significant( P < 0. 05). Compared with ZIP1 siRNA group,the expression of ALP,collagen type I,and OCN increased significantly in the ZIP1 group,and the difference was statistically significant( P < 0. 05). Conclusion ZIP1 promotes the osteogenic differentiation of BMSCs and promotes bone repair and bone metabolism. ZIP1 siRNA suppresses the osteogenic differentiation of BMSCs by activating Wnt1/β-catenin signaling pathway.
引文
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[14]赵刚,刘微微,高伟玮,等.不同组织来源间充质干细胞体外成骨分化能力的比较研究.中国骨质疏松杂志,2017,23(5):561-566,584.Zhao Gang,Liu Weiwei,Gao Weiwei,et al.Comparison of the osteogenesis capacity of mesenchymal stem cells from different tissues.Chin J Osteoporos,2017,23(5):561-566,584.
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[18]Yin J,Zhuang G,Zhu Y,et al.MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.Cell Biol Int,2017,41(7):779-786.
[19]Chen Y,Chen L,Yin Q,et al.Reciprocal interferences of TNF-αand Wnt1/β-catenin signaling axes shift bone marrow-derived stem cells towards osteoblast lineage after ethanol exposure.Cell Physiol Biochem,2013,32(3):755-65.
[20]Tao K1,Xiao D,Weng J,et al.Berberine promotes bone marrowderived mesenchymal stem cells osteogenic differentiation via canonical Wnt/β-catenin signaling pathway.Toxicol Lett,2016,240(1):68-80.
[21]Jiang HJ,Tian XG,Huang SB,et al.Tenuigenin promotes the osteogenic differentiation of bone mesenchymal stem cells in vitro and in vivo.Cell Tissue Res,2017,367(2):257-267.
[22]Ying X,Chen X,Feng Y,et al.Myricetin enhances osteogenic differentiation through the activation of canonical Wnt/β-catenin signaling in human bone marrow stromal cells.Eur J Pharmacol,2014,738(1):22-30.
[23]Liang GS,Chen WC,Yin CC,et al.Effect of Total Ravonoids of Herba Epimedium on BMP-2/Run X2/OSX Signaling Pathway during Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.Zhongguo Zhong Xi Yi Jie He Za Zhi,2016,36(5):614-8.
[24]Lawson R,Maret W,Hogstrand C.Expression of the ZIP/SLC39A transporters inβ-cells:a systematic review and integration of multiple datasets.BMC Genomics,2017,18(1):719.
[25]Yu Y,Wang HD.Zinc transporters:critical regulators of zinc homeostasis.Chinese Journal of Cell Biology,2013,32(2):176.
[26]Tyszka-Czochara M,Grzywacz A,Gdula-Argasińska J,et al.The role of zinc in the pathogenesis and treatment of central nervous system(CNS)diseases.Implications of zinc homeostasis for proper CNS function.Acta Pol Pharm,2014,71(3):369-77.
[27]Shihong Xu,Yongliang Yang,Shumei Han,et al.ZIP1 and zinc inhibits fluoride-induced apoptosis in MC3T3-E1 cells.Biol Trace Elem Res,2014,159(3):399-409.
[2]石金明,赵刚,阮蔷.金属离子Cu2+、Fe3+对水凝胶RADA16-NBD培养条件下成骨细胞生长与分化的影响.中国组织工程研究,2016,20(16):2347-2353.Shi JM,Zhao G,Ruan Q.Effect of Cu2+and Fe3+on osteoblast growth and differentiation in hydrogel RADA16-NBD.Zhongguo Zuzhi Gongcheng Yanjiu.2016,20(16):2347-2353.
[3]Kumar SD,Vijaya M,Samy RP,et al.Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice.Free Radic Biol Med,2012,53(8):1595-606.
[4]Mendivil Perez M,Velez Pardo C,Jimenez-Del-Rio M.TPEN induces apoptosis independently of zinc chelator activity in a model of acute lymphoblastic leukemia and ex vivo acute leukemia cells through oxidative stress and mitochondria caspase-3-and AIF-dependent pathways.Oxid Med Cell Longev,2012,2012:313275.
[5]Kambe T,Hashimoto A,Fujimoto S.Current understanding of ZIP and Zn T zinc transporters in human health and diseases.Cell Mol Life Sci,2014,71(17):3281-95.
[6]Kimura T,Kambe T.The Functions of Metallothionein and ZIP and Zn T Transporters:An Overview and Perspective.Int J Mol Sci,2016,17(3):336.
[7]Kambe T,Tsuji T,Hashimoto A,et al.The Physiological,Biochemical,and Molecular Roles of Zinc Transporters in Zinc Homeostasis and Metabolism.Physiol Rev,2015,95(3):749-84.
[8]Kambe T,Takeda TA,Nishito Y.Activation of zinc-requiring ectoenzymes by Zn T transporters during the secretory process:Biochemical and molecular aspects.Arch Biochem Biophys,2016,611(1):37-42.
[9]Franklin RB,Chellaiah M,Zou J,et al.Evidence that osteoblasts are specialized citrate-producing cells that provide the citrate for incorporation into the structure of bone.Open Bone J,2014,6(1):1-7.
[10]Costello LC,Franklin RB.A review of the important central role of altered citrate metabolism during the process of stem cell differentiation.J Regen Med Tissue Eng,2013,2(1):1.
[11]章晓云,黎金焕,袁振中,等.转运蛋白1 siRNA转染骨髓间充质干细胞对肿瘤坏死因子α信号成脂通路的影响.中国组织工程研究,2017,21(13):1986-1991.Zhang XY,Li JH,Yuan ZZ,et al.Effect of bone marrow mesenchymal stem cells transfected by Zrt/Irt-like protein 1 on adipogenic differentiation via tumor necrosis factor alpha signaling pathway.Zhongguo Zuzhi Gongcheng Yanjiu,2017,21(13):1986-1991.
[12]Mayer U,Benditz A,Grssel S.miR-29b regulates expression of collagens I and III in chondrogenically differentiating BMSC in an osteoarthritic environment.Sci Rep,2017,7(1):13297.
[13]Yiyao Wang,Xiaohui Zhang,Jun Shao,et al.Adiponectin regulates BMSC osteogenic differentiation and osteogenesis through the Wnt/β-catenin pathway.Sci Rep,2017,7(2):3652.
[14]赵刚,刘微微,高伟玮,等.不同组织来源间充质干细胞体外成骨分化能力的比较研究.中国骨质疏松杂志,2017,23(5):561-566,584.Zhao Gang,Liu Weiwei,Gao Weiwei,et al.Comparison of the osteogenesis capacity of mesenchymal stem cells from different tissues.Chin J Osteoporos,2017,23(5):561-566,584.
[15]高翔,荆凯鹏,黄瑞,等.miR-137靶向作用Runx2调控MC3T3-E1细胞成骨分化.中国骨质疏松杂志,2016,22(11):1440-1445.Gao Xiang,Jing Kaipeng,Huang Rui,etal.Targe ting of RU NX2by miR-137 and its re gulation on MC3T3-E1 ce lls oste oge nic diffe re ntiation.Chin J Osteoporos,2016,22(11):1440-1445.
[16]Li Z,Wang W,Xu H,et al.Effects of altered CXCL12/CXCR4axis on BMP2/Smad/Runx2/Osterix axis and osteogenic gene expressions during osteogenic differentiation of MSCs.Am J Transl Res,2017,9(4):1680-1693.
[17]Marupanthorn K,Tantrawatpan C,Kheolamai P,et al.Bone morphogenetic protein-2 enhances the osteogenic differentiation capacity of mesenchymal stromal cells derived from human bone marrow and umbilical cord.Int J Mol Med,2017,39(3):654-662.
[18]Yin J,Zhuang G,Zhu Y,et al.MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.Cell Biol Int,2017,41(7):779-786.
[19]Chen Y,Chen L,Yin Q,et al.Reciprocal interferences of TNF-αand Wnt1/β-catenin signaling axes shift bone marrow-derived stem cells towards osteoblast lineage after ethanol exposure.Cell Physiol Biochem,2013,32(3):755-65.
[20]Tao K1,Xiao D,Weng J,et al.Berberine promotes bone marrowderived mesenchymal stem cells osteogenic differentiation via canonical Wnt/β-catenin signaling pathway.Toxicol Lett,2016,240(1):68-80.
[21]Jiang HJ,Tian XG,Huang SB,et al.Tenuigenin promotes the osteogenic differentiation of bone mesenchymal stem cells in vitro and in vivo.Cell Tissue Res,2017,367(2):257-267.
[22]Ying X,Chen X,Feng Y,et al.Myricetin enhances osteogenic differentiation through the activation of canonical Wnt/β-catenin signaling in human bone marrow stromal cells.Eur J Pharmacol,2014,738(1):22-30.
[23]Liang GS,Chen WC,Yin CC,et al.Effect of Total Ravonoids of Herba Epimedium on BMP-2/Run X2/OSX Signaling Pathway during Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.Zhongguo Zhong Xi Yi Jie He Za Zhi,2016,36(5):614-8.
[24]Lawson R,Maret W,Hogstrand C.Expression of the ZIP/SLC39A transporters inβ-cells:a systematic review and integration of multiple datasets.BMC Genomics,2017,18(1):719.
[25]Yu Y,Wang HD.Zinc transporters:critical regulators of zinc homeostasis.Chinese Journal of Cell Biology,2013,32(2):176.
[26]Tyszka-Czochara M,Grzywacz A,Gdula-Argasińska J,et al.The role of zinc in the pathogenesis and treatment of central nervous system(CNS)diseases.Implications of zinc homeostasis for proper CNS function.Acta Pol Pharm,2014,71(3):369-77.
[27]Shihong Xu,Yongliang Yang,Shumei Han,et al.ZIP1 and zinc inhibits fluoride-induced apoptosis in MC3T3-E1 cells.Biol Trace Elem Res,2014,159(3):399-409.