促红细胞生成素（EPO）和EPO受体（EPOR）是调节红细胞生成所必需的细胞因子。越来越多证据表明，EPO/EPOR在非造血器官包括心脏、大脑和骨骼的发育和再生中发挥重要作用。但目前人们对EPO/EPOR在骨骼发育和再生中的机制知之甚少。最近一些研究表明，系统性或局部注射EPO可促进骨形成。但是EPO/EPOR在骨骼发育和再生中的作用机制尚不明确。 / 实验发现EPO/EPOR在生长板的前肥大软骨和肥大软骨区富集，且可促进软骨细胞增殖。但利用siRNA技术将软骨细胞EPOR沉默后，软骨细胞增殖会受抑制。随后，我们用阿利新蓝对软骨细胞外基质中的的蛋白聚糖进行染色，发现EPO可促进软骨细胞分化。此外软骨细胞标志基因包括SOX9、SOX5、SOX6、2型胶原蛋白和蛋白聚糖表达上调。实验还发现EPO可促进MSCs增殖。同时EPO可促进上述软骨细胞标志基因的表达增加。当基因沉默EPOR或使用EPO封闭肽后，软骨细胞标志基因的表达降低。以上数据表明， EPO促进软骨细胞增殖和分化的功能至少部分通过其同源受体EPOR介导。 / 我们还探讨了在低氧环境下，EPO对软骨细胞的作用。在低氧环境中， EPO/EPOR和HIF-1α mRNA和蛋白质表达水平均上调，且EPO促进软骨祖细胞集落形成。此结果提示EPO在低氧条件下介导软骨细胞增殖。同时在软骨细胞中标，EPO可激活JAK2和STAT3磷酸化，该结果表明JAK/STAT信号介导软骨细胞的生物学功能。 / 体外内皮细胞出芽实验发现EPO明显促进跖骨表面内皮细胞出芽。在小鼠骨折处局部注射EPO 14天后μCT血管成像发现，EPO可促进骨折处血管生成。体外与体内试验结果同时证实EPO可促进血管生成。 / 在骨折术后第7天和14天，藏红O染色发现EPO促进软骨骨痂形成。在术后第28天，X光和μCT扫描发现，三维重建和定量分析显示EPO促进骨形成，且伴随着骨量和骨面积的增加，以及骨生物力学特性的改善。以上结果表明， EPO可有效促进骨折修复。 / 综上所述， EPO/ EPOR信号调控软骨细胞和MSCs增殖及其软骨细胞分化。EPO还调控软骨细胞在低氧环境下的生物学特性。EPO/ EPOR信号分子有助于骨愈合过程中血管生成和骨形成。因此，EPO/EPOR可作为一个新的治疗靶点促进骨骼修复与再生。 / Erythropoietin (EPO) and EPO receptor (EPOR) are essential cytokine signals regulating erythropoiesis. Growing evidences suggest that EPO/EPOR signaling involves in the development and regeneration of non-hematopoietic organs including heart, brain and bone, et al. Several recent studies indicate that administration of EPO locally or systemically promotes bone formation. However, the underlying mechanisms of EPO/EPOR in skeletal development and regeneration remain unknown. / Our results show that EPO and EPOR are abundantly expressed in the pre-hypertrophic and hypertrophic zone of the growth plates. The proliferation rate of chondro-progenitors is increased following EPO treatment Alcian blue staining for extracellular matrix proteoglycan indicates that EPO promotes the differentiation of chondrocytes. This is accompanied by up-regulated chondrogenic marker genes including SOX9, SOX5, SOX6, type 2 collagen and aggrecan. In a parallel study, the proliferation rate of MSCs is increased following EPO treatment. The mRNA expression of above chondrogenic marker genes is also up-regulated. These effects are eliminated following knockdown of EPOR in chondrocytes by siRNA or treatment with EPO block peptide. These findings indicate that EPO promotes the proliferation and differentiation of primary chondrocytes at least partially mediated by its cognate receptor EPOR. / We next examined the role of EPO in chondrocytes under hypoxia. The mRNA and protein levels of EPO/EPOR and HIF-1α are up-regulated under hypoxia. EPO also enhances the colony forming efficiency of chondro-progenitors under hypoxia. This result suggests that EPO may serve as a mediator to regulate proliferation of chondro-progenitors under hypoxic condition. In addition, we show that EPO up-regulates the phosphorylation states of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in chondrocytes, suggesting that the function of EPO in chondrocytes is mediated through JAK/STAT signaling. / To address the function of EPO in angiogenesis, we performed metatarsal endothelial sprouting assay. The endothelial sprouting is significantly enhanced in metatarsals treated with EPO. This coincide with our in vivo data that local delivery of EPO increases vascularity of the healing bone at day 14 post-fracture in mice as indicated by micro-CT angiography analysis. / Interestingly, in the mouse fracture model, EPO promotes cartilaginous callus formation at days 7 and 14 post-surgery. This results in accelerated osteogenesis at day 28 post-surgery indexed by the radiographical scoring and micro-CT analysis characterized by increased bone volume and bone surface. This is accompanied by improved biomechanical properties of the healing bone. These results indicate that administration of EPO may serve as an efficient therapy to facilitate bone regenerartion. / In conclusion, EPO/EPOR signal regulates of the proliferation and differentiation of chondrocytes and MSCs to promote chondrogenesis. EPO may also function as a positive mediator in chondrocytes in response to low oxygen tension during chondrogenesis. EPO/EPOR signaling also contributes to angiogenesis and osteogenesis during bone healing. Therefore, EPO/EPOR may serve as a novel therapeutic target to promote skeletal regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wan, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-152). / Abstracts also in Chinese. / Wan, Lin.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1077663 |
| Date | January 2014 |
| Contributors | Wan, Lin (author.), Wan, Chao (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences, (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource], electronic resource, remote, 1 online resource (18 unnumbered leaves, 152 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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