Effect of bone marrow mesenchymal stem cells transfected by Zrt/Irt-like protein 1 on adipogenic differentiation via tumor necrosis factor alpha signaling pathway

doi:10.3969/j.issn.2095-4344.2017.13.004

Abstract

Abstract:

BACKGROUND: Previous studies have confirmed that ethanol can promote adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and up-regulate the expression of PPARγ and aP2 in the tumor necrosis factor α (TNF-α) signaling pathway. As a member of the ZIP protein family, Zrt/Irt-like protein 1 (ZIP1) is closely related to bone metabolism and osteogenic differentiation.
OBJECTIVE: To study the effect of BMSCs transfected by ZIP1 on TNF-α signaling pathway in the process of adipogenic differentiation.
METHODS: The BMSCs from rabbits were isolated and cultured under different concentrations of alcohol (0.03, 0.09, 0.15, 0.21 mol/L), followed by transfection by ZIP1 siRNA and ZIP1 expression vector.
RESULTS AND CONCLUSION: After culture in alcohol, the expression levels of aP2 and PPARγ proteins were both significantly increased (P < 0.05), and the level of triglyceride was increased in all alcohol groups except for 0.03 mol/L alcohol group (P < 0.05). After siRNA transfection, the expression levels of aP2 and PPARγ as well as the level of triglyceride were increased significantly in all the alcohol groups (P < 0.05); however, ZIP1 transfection decreased the expression levels of aP2 and PPARγ proteins (P < 0.05). To conclude, ZIP1 siRNA could promote the adipogenic differentiation of BMSCs through the activation of TNF-α signaling pathway.

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, Femur Head Necrosis, Tissue Engineering

CLC Number:

R394.2

Zhang Xiao-yun, Li Jin-huan, Yuan Zhen-zhong, Chen Yue-ping, Xia Tian, Zhuo Ying-hong, Feng Yang, Lan Jiao, Dong Pan-feng, Zhao Bin. Effect of bone marrow mesenchymal stem cells transfected by Zrt/Irt-like protein 1 on adipogenic differentiation via tumor necrosis factor alpha signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(13): 1986-1991.

Figures/Tables 2

2.1 细胞生长状态将兔骨髓间充质干细胞常规培养1，2，3 d后观察细胞生长情况，可见细胞逐渐增多，形态由梭形变为细长梭形，详见图1。 2.2 油红O染色成脂鉴定结果用定向成脂诱导液诱导30 d后，可见大量被染为红色的较标准的脂滴，表明骨髓间充质干细胞成脂分化能力正常，详见图2。 2.3 ZIP1 siRNA序列筛选结果 ZIP1 siRNA转染骨髓间充质干细胞24 h后，ZIP1 siRNA空序列组，ZIP1 siRNA序列1，2，3组中ZIP1 mRNA表达量分别为(1.00±0.32)，(0.41±0.18)、(1.96±0.08)，(0.11±0.07) mmol/L，与ZIP1 siRNA空序列组比较，序列1组与3组中的ZIP1 mRNA表达量明显降低，序列2组ZIP1 mRNA表达量明显升高(P < 0.05)；ZIP1 siRNA序列3组与ZIP1 siRNA序列1组比较ZIP1 mRNA表达含量有略低，差异无显著性意义(P > 0.05)。结果显示，ZIP1 siRNA序列3为最佳抑制序列，能够明显抑制ZIP1 mRNA的表达。 2.4 不同乙醇浓度下骨髓间充质干细胞中aP2，PPARγ蛋白的相对表达量及三酰甘油水平 0.03，0.09，0.15，0.21 mol/L乙醇组aP2，PPARγ蛋白表达明显升高，与正常对照组比较差异有显著性意义(P < 0.05)；0.03 mol/L乙醇组三酰甘油水平略升高，但与正常对照组比较差异无显著性意义(P > 0.05)；0.09，0.15，0.21 mol/L乙醇组三酰甘油水平明显升高，与正常对照组比较差异有显著性意义(P < 0.05)；详见表1。 2.5 ZIP1 siRNA转染骨髓间充质干细胞在同一乙醇浓度诱导下aP2，PPARγ蛋白的相对表达量及三酰甘油水平0.21 mol/L乙醇组，0.21 mol/L乙醇+ZIP siRNA组中aP2，PPARγ蛋白表达及三酰甘油水平较正常对照组明显升高(P < 0.05)；0.21 mol/L乙醇+ZIP siRNA组中aP2，PPARγ蛋白表达及三酰甘油水平明显升高，与0.21 mol/L乙醇组比较差异有显著性意义(P < 0.05)。详见表1。 2.6 ZIP siRNA和ZIP1转染骨髓间充质干细胞对aP2，PPARγ蛋白的相对表达量的影响 ZIP1表达组aP2，PPARγ蛋白表达明显降低，与正常对照组比较差异有显著性意义(P < 0.05)，ZIP1 siRNA组aP2，PPARγ蛋白表达明显升高，与正常对照组比较差异有显著性意义(P < 0.05)；ZIP1表达组aP2，PPARγ蛋白表达降低，与ZIP1 siRNA组差异有显著性意义(P < 0.05)，详见表2。"

References

[1] Hernigou P, Flouzat-Lachaniette CH, Delambre J, et al. Osteonecrosis repair with bone marrow cell therapies: state of the clinical art. Bone. 2015;70(1):102-109.

[2] Chen Y, Gao H, Yin Q, et al. ER stress activating ATF4/CHOP-TNF-α signaling pathway contributes to alcohol-induced disruption of osteogenic lineage of multipotential mesenchymal stem cell. Cell Physiol Biochem. 2013;32(3):743-754.

[3] Chen Y, Chen L,Yin Q, et al. Reciprocal interferences of TNF-α and Wnt1/β-catenin signaling axes shift bone marrow-derived stem cells towards osteoblast lineage after ethanol exposure. Cell Physiol Biochem. 2013;32(3): 755-765.

[4] 侯婧瑛,汪蕾,钟婷婷,等. Apelin干预骨髓间充质干细胞在缺血缺氧条件下的生存和血管再生[J].中国组织工程研究,2017, 21(1):6-12.

[5] Mohty M, Gaugler B. Inflammatory cytokines and dendritic cells in acute graft-versushost disease after allogeneic stem cell transplantation. Cytokine Growth Factor Rev. 2008; 19(1):53-63.

[6] Berg JM, Shi Y. The galvanization of biology: a growing appreciation for the roles of zinc. Science. 1996;271(5252): 1081-1085.

[7] 徐娟,王冬梅,曾力群.锌离子作为信号分子对心功能的影响[J].贵州医药,2016,40(8):885-888.

[8] Kimura T, Kambe T. The Functions of metallothionein and zip and znt transporters: an overview and perspective. Int J Mol Sci. 2016;17(3):336.

[9] Milner MJ, Seamon J, Craft E, et al. Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis. J Exp Boil. 2013;64(1):369-381.

[10] 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;6:1-7.

[11] 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.

[12] Liu Y, Yan F, Yang WL, et al. Effects of zinc transporter on differentiation of bone marrow mesenchymal stem cells to osteoblasts. Biol Trace Elem Res. 2013;154(2):234-243.

[13] Ma XW, Cui DP, Zhao DW. Vascular endothelial growth factor/bone morphogenetic protein-2 bone marrow combined modification of the mesenchymal stem cells to repair the avascular necrosis of the femoral head. Int J Clin Exp Med. 2015;8(9):15528-15534.

[14] Mont MA, Cherian JJ, Sierra RJ, et al. Nontraumatic osteonecrosis of the femoral head: where do we stand today? A Ten-Year Update. J Bone Joint Surg Am. 2015;97(19): 1604-1627.

[15] Li C, Shen L, Yang Y, et al. Plasma ghrelin and von Willebrand Factor levels in patients with non-traumatic osteonecrosis of the femoral head. Hip Int. 2015;25(1):76-81.

[16] 卫穗转,张小磊,谢斌,等.乙醇性股骨头坏死与血脂代谢的关系[J].中国临床研究,2016,29(12):1597-1600.

[17] Kang H, Hata A. The role of microRNAs in cell fate determination of mesenchymal stem cells: balancing adipogenesis and osteogenesis. BMB Rep. 2015;48(6): 319-323.

[18] Meyer SU, Thirion C, Polesskaya A, et al. TNF-α and IGF1 modify the microRNA signature in skeletal muscle cell differentiation. Cell Commun Signal. 2015;29;13:4.

[19] Meyer SU, Krebs S, Thirion C, et al. Tumor necrosis factor alpha and insulin-like growth factor 1 induced modifications of the gene expression kinetics of differentiating skeletal muscle cells. PLoS One. 2015;8;10(10):e0139520.

[20] Meyer SU, Sass S, Mueller NS, et al. Integrative analysis of microrna and mrna data reveals an orchestrated function of micrornas in skeletal myocyte differentiation in response to TNF-α or IGF1. PLoS One. 2015;10(8):e0135284.

[21] Zhuang H, Zhang X, Zhu C, et al. Molecular mechanisms of ppar-γ governing msc osteogenic and adipogenic differentiation. Curr Stem Cell Res Ther. 2016;11(3):255-264.

[22] 田健健,池颖,宋宝全,等.不同成脂条件下人骨髓间充质干细胞的早期成脂特性[J].中国组织工程研究,2016,20(23):3366-3373.

[23] 陈轶霞,骆学农,王红宁.脂肪酸结合蛋白及其生物学功能[J].生命的化学,2012,32(6):502-505.

[24] Ertunc ME, Sikkeland J, Fenaroli F, et al. Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity. J Lipid Res. 2015;56(2):423-434.

[25] 吴洁,邹瑾,钟敏.RNA干扰技术沉默脂肪细胞型脂肪酸结合蛋白基因对脂肪细胞合成三酰甘油及分泌内脂素的影响[J].中国动脉硬化杂志,2011,19(12):973-978.

[26] Suzuki T, Katsumata S, Matsuzaki H. A short-term zinc-deficient diet decreases bone formation through down-regulated BMP2 in rat bone. Biosci Biotechnol Biochem. 2016;80(7):1433-1435.

[27] 王新玲,李桂财,张华,等.乌鲁木齐市维吾尔族青年与老年女性血清微量元素及骨密度的对比[J].中国组织工程研究,2015,19(33): 5264-5268.

[28] Kessels JE, Wessels I, Haase H, et al. Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins. J Trace Elem Med Biol. 2016;37:125-133.

[29] Noh H, Paik HY, Kim J, et al. The alteration of zinc transporter gene expression is associated with inflammatory markers in obese women. Biol Trace Elem Res. 2014;158(1):1-8.

[30] 张岗,李依民,李标等.铁皮石斛锌铁调控转运蛋白基因的克隆与表达分析[J].中国中药杂志,2015,40(1):42-47.

[31] Hennigar SR, Kelley AM, McClung JP. Metallothionein and zinc transporter expression in circulating human blood cells as biomarkers of zinc status: a systematic review. Adv Nutr. 2016;7(4):735-746.