氯化锂促进骨髓间充质干细胞增殖分化的作用途径
|
摘要
背景:寻找促进骨髓间充质干细胞增殖及向成骨细胞分化的方法,可以为治疗骨质疏松类疾病提供治疗思路。目的:探讨氯化锂调控大鼠骨髓间充质干细胞增殖分化的机制。方法:实验采用全骨髓细胞接种法和贴壁纯化培养大鼠骨髓间充质干细胞,分别给予0,2,5 nmol/L氯化锂进行干预;采用CCK-8法检测大鼠骨髓间充质干细胞的增殖情况,茜素红染色法检测钙化结节形成情况,PNPP法测定碱性磷酸酶活性,Western blot法检测大鼠骨髓间充质干细胞中GSK3β,p-GSK3β,β-catenin蛋白含量变化。结果与结论:①2,5 nmol/L氯化锂均能促进大鼠骨髓间充质干细胞增殖,5 nmol/L氯化锂促增殖能力最强;②2,5 nmol/L氯化锂组矿化结节与碱性磷酸酶活性均比0 nmol/L氯化锂组明显,且5 nmol/L氯化锂促成骨分化更为显著;③2,5 nmol/L氯化锂能够显著增加p-GSK3β、β-catenin蛋白表达,而各组GSK3β蛋白表达差异无显著性意义;④结果表明,氯化锂能够促进大鼠骨髓间充质干细胞的增殖及向成骨细胞分化,其可能机制是通过调控Wnt/β-catenin信号通路相关蛋白表达实现的。
BACKGROUND: Seeking proper methods to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells can provide therapeutic ideas for the treatment of osteoporosis.OBJECTIVE: To study the mechanism of lithium chloride on the proliferation and differentiation of rat bone marrow mesenchymal stem cells.METHODS: Rat bone marrow mesenchymal stem cells were cultured with the whole bone marrow cell inoculation and adherent purification, followed by intervention with 0, 2, 5 nmol/L lithium chloride. Cell counting kit-8 was used to detect the effect of lithium chloride on the proliferation of bone marrow mesenchymal stem cells. Alizarin red staining was used to determine calcified nodules.PNPP method was applied to measure alkaline phosphatase activity. The contents of glycogen synthase kinase 3β, phosphorylated glycogen synthase kinase 3β and β-catenin in rat bone marrow mesenchymal stem cells were determined by western blot.RESULTS AND CONCLUSION: Lithium chloride, 2 and 5 nmol/L, promoted the proliferation of bone marrow mesenchymal stem cells and5 nmol/L lithium chloride had the strongest proliferative effect. Compared with 0 nmol/L group, more calcified nodules and higher alkaline phosphatase activity were observed in the 2 and 5 nmol/L groups, especially in the 5 nmol/L group. Both 2 and 5 nmol/L lithium chloride upregulated the expression of phosphorylated glycogen synthase kinase 3β and β-catenin proteins, but showed no difference in the expression of glycogen synthase kinase 3β. In conclusion, lithium chloride can promote the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells, and the possible mechanism is through the regulation of Wnt/β-catenin signaling pathway-related protein expression.
BACKGROUND: Seeking proper methods to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells can provide therapeutic ideas for the treatment of osteoporosis.OBJECTIVE: To study the mechanism of lithium chloride on the proliferation and differentiation of rat bone marrow mesenchymal stem cells.METHODS: Rat bone marrow mesenchymal stem cells were cultured with the whole bone marrow cell inoculation and adherent purification, followed by intervention with 0, 2, 5 nmol/L lithium chloride. Cell counting kit-8 was used to detect the effect of lithium chloride on the proliferation of bone marrow mesenchymal stem cells. Alizarin red staining was used to determine calcified nodules.PNPP method was applied to measure alkaline phosphatase activity. The contents of glycogen synthase kinase 3β, phosphorylated glycogen synthase kinase 3β and β-catenin in rat bone marrow mesenchymal stem cells were determined by western blot.RESULTS AND CONCLUSION: Lithium chloride, 2 and 5 nmol/L, promoted the proliferation of bone marrow mesenchymal stem cells and5 nmol/L lithium chloride had the strongest proliferative effect. Compared with 0 nmol/L group, more calcified nodules and higher alkaline phosphatase activity were observed in the 2 and 5 nmol/L groups, especially in the 5 nmol/L group. Both 2 and 5 nmol/L lithium chloride upregulated the expression of phosphorylated glycogen synthase kinase 3β and β-catenin proteins, but showed no difference in the expression of glycogen synthase kinase 3β. In conclusion, lithium chloride can promote the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells, and the possible mechanism is through the regulation of Wnt/β-catenin signaling pathway-related protein expression.
引文
[1] Phinney DG, Kopen G, Isaacson RL, et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice:variations in yield, growth, and differentiation.J Cell Biochem. 1999;72(4):570-585.
[2] Cohen MA, Markoulaki S, Jaenisch R. Matched Developmental Timing of Donor Cells with the Host Is Crucial for Chimera Formation. Stem Cell Reports. 2018;10(5):1445-1452.
[3] Ali EHA, Ahmed-Farid OA, sman AAE.Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model.Neural Regen Res. 2017;12(12):1990-1999.
[4] Atashi F, Modarressi A, Pepper MS. The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation:a review. Stem Cells Dev. 2015;24(10):1150-1163.
[5]王皓沿,海鑫.白藜芦醇对小鼠骨髓间充质干细胞成骨/成脂分化的影响[J].哈尔滨医科大学学报,2018,52(2):99-104.
[6] Yamaguchi M, Zhu S, Weitzmann MN, et al. Curcumin analog UBS109 prevents bone marrow osteoblastogenesis and osteoclastogenesis disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro. Mol Cell Biochem. 2015;401(1-2):1-10.
[7] Azuma K, Zhou Q, Kubo KY. Morphological and molecular characterization of the senile osteoporosis in senescenceaccelerated mouse prone 6(SAMP6). Med Mol Morphol.2018;51(3):139-146.
[8] Abuna RP, Stringhetta-Garcia CT, Fiori LP, et al. Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis. J Appl Oral Sci. 2016;24(4):376-382.
[9] Ghali O, Broux O, Falgayrac G, et al. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation. BMC Cell Biol. 2015;16:9.
[10] Patel R, Rodriguez A, Yasmeen T, et al. Impact of Obesity on Osteoporosis:Limitations of the Current Modalities of Assessing Osteoporosis in Obese Subjects. Clinical Reviews in Bone and Mineral Metabolism. 2015;13(1):36-42.
[11] Li J, Liu X, Zuo B, et al. The Role of Bone Marrow Microenvironment in Governing the Balance between Osteoblastogenesis and Adipogenesis. Aging Dis. 2015;7(4):514-525.
[12]谢小伟,裴福兴,康鹏德,等.锶盐联合淫羊藿苷对大鼠骨髓基质干细胞成骨及成脂分化影响的研究[J].中国矫形外科杂志, 2015,23(5):450-457.
[13] 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):755-765.
[14]杨斌,向萌娟.氯化锂诱导毛囊干细胞定向分化中Wnt信号通路介导基因的调控[J].中国组织工程研究与临床康复, 2011,15(49):9202-9206.
[15] Wils J, Favre J, Bellien J. Modulating putative endothelial progenitor cells for the treatment of endothelial dysfunction and cardiovascular complications in diabetes. Pharmacol Ther. 2017;170:98-115.
[16] Esfandiari F, Fathi A, Gourabi H, et al. Glycogen synthase kinase-3 inhibition promotes proliferation and neuronal differentiation of human-induced pluripotent stem cell-derived neural progenitors. Stem Cells Dev. 2012;21(17):3233-3243.
[17] Huang J, Guo X, Li W, et al. Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes. Sci Rep. 2017;7:40716.
[18] Zhang L, Chan C. Isolation and enrichment of rat mesenchymal stem cells(MSCs)and separation of single-colony derived MSCs. J Vis Exp. 2010;(37):1852.
[19]聂义珍,闫朝岐,付红梅,等.冷刺激对骨髓间充质干细胞增殖及成骨分化的影响[J].中华骨质疏松和骨矿盐疾病杂志, 2018,11(6):577-583.
[20]李忠海.不同浓度锶对MC3T3-E1细胞增殖、ALP活性及成骨分化的影响[J].中国骨与关节杂志, 2016, 5(3):221-225.
[21]董冰子,孙晓方.骨质疏松症治疗新进展:从分子机制到药物靶点[J].中华骨质疏松和骨矿盐疾病杂志,2018,11(6):620-627.
[22] Varela A, Chouinard L, Lesage E, et al. One Year of Abaloparatide, a Selective Activator of the PTH1 Receptor,Increased Bone Formation and Bone Mass in Osteopenic Ovariectomized Rats Without Increasing Bone Resorption. J Bone Miner Res. 2017;32(1):24-33.
[23] Song L, Liu M, Ono N, et al. Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes. J Bone Miner Res. 2012;27(11):2344-2358.
[24] Neth P, Ciccarella M, Egea V, et al. Wnt signaling regulates the invasion capacity of human mesenchymal stem cells.Stem Cells. 2006;24(8):1892-903.
[25]李理.新型氯化锂复合磷酸钙骨水泥对成骨细胞增殖及分化的影响[D].南宁:广西医科大学,2016.
[26] Sieber MH, Thomsen MB, Spradling AC. Electron Transport Chain Remodeling by GSK3 during Oogenesis Connects Nutrient State to Reproduction. Cell. 2016;164(3):420-432.
[27] Weng J, Wang YH, Li M, et al.GSK3βinhibitor promotes myelination and mitigates muscle atrophy after peripheral nerve injury.Neural Regen Res. 2018;13(2):324-330.
[28] Desiderio V, Tirino V, Papaccio G, et al. Bone defects:molecular and cellular therapeutic targets. Int J Biochem Cell Biol. 2014;51:75-78.
[29] Kim do Y, Park EY, Chang E, et al. A novel miR-34a target,protein kinase D1, stimulates cancer stemness and drug resistance through GSK3/β-catenin signaling in breast cancer.Oncotarget. 2016;7(12):14791-14802.
[30] Li VS, Ng SS, Boersema PJ, et al. Wnt signaling through inhibition ofβ-catenin degradation in an intact Axin1 complex.Cell. 2012;149(6):1245-1256.
[2] Cohen MA, Markoulaki S, Jaenisch R. Matched Developmental Timing of Donor Cells with the Host Is Crucial for Chimera Formation. Stem Cell Reports. 2018;10(5):1445-1452.
[3] Ali EHA, Ahmed-Farid OA, sman AAE.Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model.Neural Regen Res. 2017;12(12):1990-1999.
[4] Atashi F, Modarressi A, Pepper MS. The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation:a review. Stem Cells Dev. 2015;24(10):1150-1163.
[5]王皓沿,海鑫.白藜芦醇对小鼠骨髓间充质干细胞成骨/成脂分化的影响[J].哈尔滨医科大学学报,2018,52(2):99-104.
[6] Yamaguchi M, Zhu S, Weitzmann MN, et al. Curcumin analog UBS109 prevents bone marrow osteoblastogenesis and osteoclastogenesis disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro. Mol Cell Biochem. 2015;401(1-2):1-10.
[7] Azuma K, Zhou Q, Kubo KY. Morphological and molecular characterization of the senile osteoporosis in senescenceaccelerated mouse prone 6(SAMP6). Med Mol Morphol.2018;51(3):139-146.
[8] Abuna RP, Stringhetta-Garcia CT, Fiori LP, et al. Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis. J Appl Oral Sci. 2016;24(4):376-382.
[9] Ghali O, Broux O, Falgayrac G, et al. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation. BMC Cell Biol. 2015;16:9.
[10] Patel R, Rodriguez A, Yasmeen T, et al. Impact of Obesity on Osteoporosis:Limitations of the Current Modalities of Assessing Osteoporosis in Obese Subjects. Clinical Reviews in Bone and Mineral Metabolism. 2015;13(1):36-42.
[11] Li J, Liu X, Zuo B, et al. The Role of Bone Marrow Microenvironment in Governing the Balance between Osteoblastogenesis and Adipogenesis. Aging Dis. 2015;7(4):514-525.
[12]谢小伟,裴福兴,康鹏德,等.锶盐联合淫羊藿苷对大鼠骨髓基质干细胞成骨及成脂分化影响的研究[J].中国矫形外科杂志, 2015,23(5):450-457.
[13] 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):755-765.
[14]杨斌,向萌娟.氯化锂诱导毛囊干细胞定向分化中Wnt信号通路介导基因的调控[J].中国组织工程研究与临床康复, 2011,15(49):9202-9206.
[15] Wils J, Favre J, Bellien J. Modulating putative endothelial progenitor cells for the treatment of endothelial dysfunction and cardiovascular complications in diabetes. Pharmacol Ther. 2017;170:98-115.
[16] Esfandiari F, Fathi A, Gourabi H, et al. Glycogen synthase kinase-3 inhibition promotes proliferation and neuronal differentiation of human-induced pluripotent stem cell-derived neural progenitors. Stem Cells Dev. 2012;21(17):3233-3243.
[17] Huang J, Guo X, Li W, et al. Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes. Sci Rep. 2017;7:40716.
[18] Zhang L, Chan C. Isolation and enrichment of rat mesenchymal stem cells(MSCs)and separation of single-colony derived MSCs. J Vis Exp. 2010;(37):1852.
[19]聂义珍,闫朝岐,付红梅,等.冷刺激对骨髓间充质干细胞增殖及成骨分化的影响[J].中华骨质疏松和骨矿盐疾病杂志, 2018,11(6):577-583.
[20]李忠海.不同浓度锶对MC3T3-E1细胞增殖、ALP活性及成骨分化的影响[J].中国骨与关节杂志, 2016, 5(3):221-225.
[21]董冰子,孙晓方.骨质疏松症治疗新进展:从分子机制到药物靶点[J].中华骨质疏松和骨矿盐疾病杂志,2018,11(6):620-627.
[22] Varela A, Chouinard L, Lesage E, et al. One Year of Abaloparatide, a Selective Activator of the PTH1 Receptor,Increased Bone Formation and Bone Mass in Osteopenic Ovariectomized Rats Without Increasing Bone Resorption. J Bone Miner Res. 2017;32(1):24-33.
[23] Song L, Liu M, Ono N, et al. Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes. J Bone Miner Res. 2012;27(11):2344-2358.
[24] Neth P, Ciccarella M, Egea V, et al. Wnt signaling regulates the invasion capacity of human mesenchymal stem cells.Stem Cells. 2006;24(8):1892-903.
[25]李理.新型氯化锂复合磷酸钙骨水泥对成骨细胞增殖及分化的影响[D].南宁:广西医科大学,2016.
[26] Sieber MH, Thomsen MB, Spradling AC. Electron Transport Chain Remodeling by GSK3 during Oogenesis Connects Nutrient State to Reproduction. Cell. 2016;164(3):420-432.
[27] Weng J, Wang YH, Li M, et al.GSK3βinhibitor promotes myelination and mitigates muscle atrophy after peripheral nerve injury.Neural Regen Res. 2018;13(2):324-330.
[28] Desiderio V, Tirino V, Papaccio G, et al. Bone defects:molecular and cellular therapeutic targets. Int J Biochem Cell Biol. 2014;51:75-78.
[29] Kim do Y, Park EY, Chang E, et al. A novel miR-34a target,protein kinase D1, stimulates cancer stemness and drug resistance through GSK3/β-catenin signaling in breast cancer.Oncotarget. 2016;7(12):14791-14802.
[30] Li VS, Ng SS, Boersema PJ, et al. Wnt signaling through inhibition ofβ-catenin degradation in an intact Axin1 complex.Cell. 2012;149(6):1245-1256.