去卵巢大鼠血清来源外泌体促进原代成骨细胞的增殖
|
摘要
背景:骨代谢过程中,成骨细胞的数量和功能的变化影响骨的生物学特性,外泌体能够通过细胞间的传递进行信号传递,具有促进细胞增殖、分化的潜能。目的:探讨骨质疏松大鼠血清中外泌体的性质及其对成骨细胞的作用。方法:32只成年健康雌性SD大鼠,24 h新生SD大鼠若干只用于原代成骨细胞分离培养,实验动物由广东省医学动物实验中心提供。(1)建立去卵巢骨质疏松大鼠模型,以假手术组大鼠为对照组。通过ExoQuick提取对照组和骨质疏松大鼠血清中外泌体,用电镜、nanosight、Western blotting进行鉴定;(2)取对数期的原代成骨细胞接种于96孔细胞培养板中,接种浓度为2×10~4/孔。实验分为空白对照、外泌体对照组、骨质疏松外泌体组,分别加入DPBS、正常血清外泌体和骨质疏松血清外泌体,100μL/孔,CO_2培养箱培育1d,采用四甲基偶氮唑盐比色法检测成骨细胞的增殖;酶联免疫吸附试验法检测外泌体中血管内皮生长因子的浓度。结果与结论:(1)成功提取大小在100nm以下外泌体颗粒;(2)蛋白定量检测12周组外泌体蛋白质量浓度最高;不同时间点外泌体颗粒数12周组略高,但未见明显差异;(3)骨质疏松大鼠血清来源外泌体与空白对照组及正常外泌体组相比较,明显促进成骨细胞的增殖,且外泌体内血管内皮生长因子升高;(4)结果说明,骨质疏松大鼠血清中外泌体能够促进成骨细胞的增殖。
BACKGROUND: During bone metabolism, changes in the number and function of osteoblasts influence the biological characteristics of bone tissues. Exosomes can mediate cell-to-cell transmission, and have the potential to promote cell proliferation and differentiation. OBJECTIVE: To study the properties of exosomes in serum of rats with osteoporosis and to investigate their effects on osteoblasts. METHODS: Several newborn Sprague-Dawley rats at 24 hours of birth were used for primary osteoblast isolation and culture. A rat model of ovariectomized osteoporosis was established in 32 healthy female adult Sprague-Dawley rats. Rats undergoing sham operation were used as control group. All the experimental animals were provided by Guangdong Medical Animal Experimental Center, China. Exosomes were extracted from the serum of control rats and ovariectomized rats using ExoQuick and identified by electron microscopy, nanosight and western blot. Primary osteoblasts in logarithmic phase were seeded into 96-well culture plates with an inoculation concentration of 2×10~4/well. Then, the cells were divided into blank control group, exosome group, and osteoporosis+exosome group, and DPBS, normal serum-derived exosomes, and osteoporosis serum-derived exosomes, 100 μL/well, were added, respectively. The cells in each group were cultured in a CO_2 incubator for 1 day. The proliferation of osteoblasts was detected by MTT colorimetric assay. Enzyme-linked immunosorbent assay was used to detect the concentration of vascular endothelial growth factor in exosomes. RESULTS AND CONCLUSION: Successfully extracted exosomes particles were below 100 nm in size. The serum exosome protein concentration in osteoporotic rats was highest at 12 weeks after ovariectomized surgery. The number of exosome particles was also highest at 12 weeks after ovariectomized surgery, but there was no significant difference in the osteoporotic rats at different time points. Compared with the blank control and exosome group, exosomes from the serum of osteoporotic rats significantly promoted the proliferation of osteoblasts, and the level of vascular endothelial growth factor in exosomes increased. These findings indicate that exosomes from the serum of osteoporotic rats truly promote the proliferation of osteoblast.
BACKGROUND: During bone metabolism, changes in the number and function of osteoblasts influence the biological characteristics of bone tissues. Exosomes can mediate cell-to-cell transmission, and have the potential to promote cell proliferation and differentiation. OBJECTIVE: To study the properties of exosomes in serum of rats with osteoporosis and to investigate their effects on osteoblasts. METHODS: Several newborn Sprague-Dawley rats at 24 hours of birth were used for primary osteoblast isolation and culture. A rat model of ovariectomized osteoporosis was established in 32 healthy female adult Sprague-Dawley rats. Rats undergoing sham operation were used as control group. All the experimental animals were provided by Guangdong Medical Animal Experimental Center, China. Exosomes were extracted from the serum of control rats and ovariectomized rats using ExoQuick and identified by electron microscopy, nanosight and western blot. Primary osteoblasts in logarithmic phase were seeded into 96-well culture plates with an inoculation concentration of 2×10~4/well. Then, the cells were divided into blank control group, exosome group, and osteoporosis+exosome group, and DPBS, normal serum-derived exosomes, and osteoporosis serum-derived exosomes, 100 μL/well, were added, respectively. The cells in each group were cultured in a CO_2 incubator for 1 day. The proliferation of osteoblasts was detected by MTT colorimetric assay. Enzyme-linked immunosorbent assay was used to detect the concentration of vascular endothelial growth factor in exosomes. RESULTS AND CONCLUSION: Successfully extracted exosomes particles were below 100 nm in size. The serum exosome protein concentration in osteoporotic rats was highest at 12 weeks after ovariectomized surgery. The number of exosome particles was also highest at 12 weeks after ovariectomized surgery, but there was no significant difference in the osteoporotic rats at different time points. Compared with the blank control and exosome group, exosomes from the serum of osteoporotic rats significantly promoted the proliferation of osteoblasts, and the level of vascular endothelial growth factor in exosomes increased. These findings indicate that exosomes from the serum of osteoporotic rats truly promote the proliferation of osteoblast.
引文
[1]Capulli M,Paone R,Rucci N.Osteoblast and osteocyte:games without frontiers.Arch BiochemBiophys.2014
[2]Kolhe R,Hunter M,Liu S,et al.Gender-specific differential expression of exosomalmiRNA in synovial fluid of patients with osteoarthritis.Sci Rep.2017;7(1):2029.
[3]Xie Y,Gao Y,Zhang L,et al.Involvement of serum-derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone-related proteins.Aging Cell.2018;17(3):e12758.
[4]Li D,Liu J,Guo B,et al.Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation.Nat Commun.2016;7:10872.
[5]Gy?rgy B,Pálóczi K,Kovács A,et al.Improved circulating microparticle analysis in acid-citrate dextrose(ACD)anticoagulant tube.Thromb Res.2014;133(2):285-292.
[6]Rager TM,Olson JK,Zhou Y,et al.Exosomes secreted from bone marrow-derived mesenchymal stem cells protect the intestines from experimental necrotizing enterocolitis.JPediatr Surg.2016;51(6):942-947.
[7]Van der Meel R,Krawczyk-Durka M,van Solinge WW,et al.Toward routine detection of extracellular vesicles in clinical samples.Int J Lab Hematol.2014;36(3):244-253.
[8]Deng L,Wang Y,Peng Y,et al.Osteoblast-derived microvesicles:a novel mechanism for communication between osteoblasts and osteoclasts.Bone.2015;79:37-42.
[9]Weilner S,Keider V,Winter M,et al.Vesicular Galectin-3levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.Aging(Albany NY).2016;8(1):16-33
[10]Cui Y,Luan J,Li H,et al.Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.FEBS Lett.2016;590(1):185-192.
[11]Ge M,Wu Y,Ke R,et al.Value of osteoblast derived exosomes in bone diseases.J Craniofac Surg.2017;28(4):866-870.
[12]Peth?A,Chen Y,George A.Exosomes in Extracellular Matrix Bone Biology.CurrOsteoporos Rep.2018;16(1):58-64.
[13]Weilner S,Keider V,Winter M,et al.Vesicular Galectin-3levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.Aging(Albany NY).2016;8(1):16-33
[14]Birmingham E,Niebur GL,McHugh PE,et al.Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche.Eur Cell Mater.2012;23:13-27.
[15]Deng L,Wang Y,Peng Y,et al.Osteoblast-derived microvesicles:a novel mechanism for communication between osteoblasts and osteoclasts.Bone.2015;79:37-42.
[16]Herrera MB,Fonsato V,Gatti S,et al.Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatec-tomized rats.J Cell Mol Med.2010;14(6b):1605-1618.
[17]Lai RC,Arslan F,Lee MM et al.Exosome secreted by MSCreduces myocardial ischemia/reperfusion injury.Stem Cell Res(Amst)2010;4:214-222.
[18]Nakamura Y,Miyaki S,Ishitobi H et al.Mesenchymalstem-cell-derived exosomes accelerate skeletal muscle regeneration.FEBSLett 2015;589:1257-1265.
[19]Maes C,Coenegrachts L,Stockmans I,et al.Placental growth factor mediates mesenchymal cell development,cartilage turnover,and bone remodeling during fracture repair.J Clin Invest.2006;116:1230-1242.
[2]Kolhe R,Hunter M,Liu S,et al.Gender-specific differential expression of exosomalmiRNA in synovial fluid of patients with osteoarthritis.Sci Rep.2017;7(1):2029.
[3]Xie Y,Gao Y,Zhang L,et al.Involvement of serum-derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone-related proteins.Aging Cell.2018;17(3):e12758.
[4]Li D,Liu J,Guo B,et al.Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation.Nat Commun.2016;7:10872.
[5]Gy?rgy B,Pálóczi K,Kovács A,et al.Improved circulating microparticle analysis in acid-citrate dextrose(ACD)anticoagulant tube.Thromb Res.2014;133(2):285-292.
[6]Rager TM,Olson JK,Zhou Y,et al.Exosomes secreted from bone marrow-derived mesenchymal stem cells protect the intestines from experimental necrotizing enterocolitis.JPediatr Surg.2016;51(6):942-947.
[7]Van der Meel R,Krawczyk-Durka M,van Solinge WW,et al.Toward routine detection of extracellular vesicles in clinical samples.Int J Lab Hematol.2014;36(3):244-253.
[8]Deng L,Wang Y,Peng Y,et al.Osteoblast-derived microvesicles:a novel mechanism for communication between osteoblasts and osteoclasts.Bone.2015;79:37-42.
[9]Weilner S,Keider V,Winter M,et al.Vesicular Galectin-3levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.Aging(Albany NY).2016;8(1):16-33
[10]Cui Y,Luan J,Li H,et al.Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.FEBS Lett.2016;590(1):185-192.
[11]Ge M,Wu Y,Ke R,et al.Value of osteoblast derived exosomes in bone diseases.J Craniofac Surg.2017;28(4):866-870.
[12]Peth?A,Chen Y,George A.Exosomes in Extracellular Matrix Bone Biology.CurrOsteoporos Rep.2018;16(1):58-64.
[13]Weilner S,Keider V,Winter M,et al.Vesicular Galectin-3levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.Aging(Albany NY).2016;8(1):16-33
[14]Birmingham E,Niebur GL,McHugh PE,et al.Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche.Eur Cell Mater.2012;23:13-27.
[15]Deng L,Wang Y,Peng Y,et al.Osteoblast-derived microvesicles:a novel mechanism for communication between osteoblasts and osteoclasts.Bone.2015;79:37-42.
[16]Herrera MB,Fonsato V,Gatti S,et al.Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatec-tomized rats.J Cell Mol Med.2010;14(6b):1605-1618.
[17]Lai RC,Arslan F,Lee MM et al.Exosome secreted by MSCreduces myocardial ischemia/reperfusion injury.Stem Cell Res(Amst)2010;4:214-222.
[18]Nakamura Y,Miyaki S,Ishitobi H et al.Mesenchymalstem-cell-derived exosomes accelerate skeletal muscle regeneration.FEBSLett 2015;589:1257-1265.
[19]Maes C,Coenegrachts L,Stockmans I,et al.Placental growth factor mediates mesenchymal cell development,cartilage turnover,and bone remodeling during fracture repair.J Clin Invest.2006;116:1230-1242.