Exosomes in serum of ovariectomized rats promote primary osteoblast proliferation

doi:10.3969/j.issn.2095-4344.1071

Abstract

Abstract:

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 2×104/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 CO2 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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteoporosis, Exosomes, Osteoblasts, Vascular Endothelial Growth Factors, Tissue Engineering

CLC Number:

R446，R318

Gao Kun1, Zhu Wenxiu2, Li Heng1, Liu Weidong1, Li Quan1, Yu Weiji1, Wang Lixin1, Cao Yafei1. Exosomes in serum of ovariectomized rats promote primary osteoblast proliferation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(7): 958-989.

Figures/Tables 2

2.1 实验动物数量分析实验选用SD大鼠32只，分为2组，实验过程无脱失，全部进入结果分析。 2.2 骨质疏松大鼠血清内外泌体的提取及鉴定在电镜下检测外泌体颗粒，可见呈椭圆形，大小不一，颗粒大小均在200 nm以下(图1A)。通过nanosight检测外泌体颗粒的布朗运动，从而得出其颗粒大小的分布图，可见直径为83 nm的颗粒最多，大部分颗粒在100 nm以下(图1B，C)。通过Westernblot检测外泌体表面的标志蛋白，可见CD9、CD63在提取的外泌体表面高表达，而在提取过程中的上清中则未检测到明显的标志蛋白表达(图1D)。 2.3 骨质疏松大鼠不同时期血清内外泌体的含量变化将骨质疏松大鼠在不同的时间点处死，分别为4，8，12，16周，取其血清中的外泌体，进行蛋白定量和颗粒数统计。Bradford法蛋白定量可检测到在相同体积血清内12周组蛋白质量浓度最高，与对照组及4周组相比差异有显著性意义(P < 0.05)，随着骨质疏松程度进展，蛋白质量浓度升高，随着骨质疏松进展，16周蛋白质量浓度下降(图2A)。Nanosight检测不同时间点颗粒数，发现相同体积血清内，12周颗粒数略高，但未见明显差异(图2B)。 2.4 骨质疏松大鼠血清来源外泌体促进成骨细胞的增殖将骨质疏松大鼠血清中外泌体提取出来，在体外水平刺激原代成骨细胞，通过Transwell检测成骨细胞的增殖能力，可见与空白对照及外泌体对照组相比，骨质疏松外泌体组对原代成骨细胞有促进增殖功能，差异有显著性意义(P < 0.05，图3A)。为了检测该增殖作用，是否与细胞因子相关，通过Elisa检测外泌体内的血管内皮生长因子水平，可发现骨质疏松外泌体组明显高于外泌体对照组，差异有显著性意义(P < 0.05，图3B)。"

References

[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] György 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. J Pediatr 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-3 levels 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-3 levels 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 MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res (Amst) 2010;4:214-222.

[18] Nakamura Y, Miyaki S, Ishitobi H et al. Mesenchymal- stem-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.