【作者】 毛英杰；

【导师】 谷志远；

【作者基本信息】 浙江大学 ， 口腔医学， 2011， 博士

【摘要】 破骨细胞的骨吸收活动与成骨细胞的骨形成活动相互作用调节,形成一种特殊的耦联机制,维持骨平衡。该耦联机制调节发生异常,会破坏骨重建和骨稳态,引发骨硬化症、骨质疏松症等骨量平衡失调所致的疾病以及肿瘤、炎症等所致的骨质异常表型。因此,该耦联的分子机制一直是骨生理和骨疾病研究、治疗及药物研发的重点对象。近几十年,关于该耦联的分子机制研究最多的就是早期提出的RANKL/RANK信号系统,即成骨细胞及骨髓基质细胞释放的RANKL,与破骨前体细胞膜表面的受体RANK结合,促进破骨前体细胞分化融合为成熟的破骨细胞。而近几年,Eph/ephrin信号转导通路异军突起,凭借对成骨细胞、破骨细胞耦联活动中关键问题的成功诠释,在骨组织领域受到越来越多的关注。骨组织中,EphB4/ephrinB2双向信号转导同时促进成骨细胞分化,而抑制破骨细胞分化。这是膜蛋白Eph受体与其配体ephrin相互作用的一个重要特征,即ephrin配体兼具受体样的功能,同时产生双向信号转导。其中,向Eph表达细胞胞内转导信号,称之为正向信号；反向ephrin自身表达细胞胞内转导信号,称之为逆向信号。EphB4/ephrinB2逆向信号对破骨细胞诱导分化的抑制作用依赖于ephrinB配体胞内区的C末端YKV基序,YKV基序含PDZ结构域蛋白的结合位点,提示有潜在的PDZ结构域蛋白参与到EphB4/ephrinB2逆向信号下游转导途径。这个或这些未知的PDZ结构域蛋白是调节骨重建的重要潜在靶点本课题研究拟在体外破骨细胞诱导分化过程中,筛选出参与到EphB4/ephrinB2逆向信号下游信号转导途径的潜在PDZ结构域蛋白种类,并初步探索其表达变化是否受EphB4/ephrinB2逆向信号调控。第一部分建立EphB4/ephrinB2逆向信号抑制RANKL诱导的Raw264.7破骨细胞分化模型目的：考虑到原代细胞培养间存在个体差异性及来源选择的局限性等不利因素,细胞株因其标准的实验试剂培养步骤及结果的可重复性、可复制性等优点越来越多的用于生命科学基础研究中。本课题拟以单核巨噬细胞系细胞Raw264.7细胞株作为潜在的破骨前体细胞,研究EphB4/ephrinB2逆向信号对RANKL诱导的Raw264.7破骨细胞分化行为及关键转录因子(c-Fos, c-Jun, NFATcl)基因表达的影响,分析其是否适合本课题研究需要。方法：在RANKL刺激下,诱导Raw264.7细胞分化为破骨样细胞。用免疫荧光细胞化学、荧光实时定量RT-PCR技术及Western blot技术检测分析细胞分化前后是否表达ephrinB2。以加入Fc蛋白为对照,加入EphB4-Fc蛋白到上述培养液中为实验组,诱导培养4天后,用TRAP染色试剂盒分析各组细胞的耐酒石酸盐酸性磷酸酶活性变化,用荧光实时定量RT-PCR技术分析各组细胞破骨细胞分化相关标志酶基因的表达变化,及细胞中RANKL/RANK信号系统下游关键转录因子(c-Fos, c-Jun, NFATcl)基因的表达变化。结果：免疫荧光细胞化学、荧光实时定量RT-PCR技术及Western blot检测结果示：经RANKL诱导分化的Raw264.7破骨样细胞有ephrinB2蛋白表达,未经诱导分化的Raw264.7细胞未见ephrinB2蛋白表达。Raw264.7诱导培养液中加入EphB4-Fc蛋白后,TRAP阳性多核破骨细胞数量明显减少(P<0.01),破骨细胞分化相关标志酶基因的表达也明显减少(P<0.01),关键转录因子(c-Fos, c-Jun, NFATc1)基因的表达亦明显下降(P<0.01)。结论：EphB4/ephrinB2逆向信号抑制RANKL诱导的Raw264.7破骨细胞分化。Raw264.7细胞可用作研究EphB4/ephrinB2逆向信号的细胞模型,适合本课题研究的需要。第二部分筛选参与EphB4/cphrinB2逆向信号下游信号转导途径的潜在PDZ结构域蛋白目的：膜蛋白ephrinB2有一个跨膜结构域和很短的胞内区,胞内区域是高度保守的,含有多重酪氨酸,而C末端YKV基序含PDZ结构域蛋白的结合位点。本课题研究拟筛选RANKL诱导的Raw264.7破骨细胞分化过程中参与EphB4/ephrinB2逆向信号下游信号转导途径的潜在PDZ结构域蛋白。方法：免疫荧光细胞化学及Western blot检测分析在RANKL诱导的Raw264.7破骨细胞分化前后PDZ结构域蛋白(PDZ-RGS3, Dv12, Pick1, Syntenin, GRIP1, GRIP2, FAP-1 and Par3)的表达。免疫共沉淀技术分析诱导分化后有表达的PDZ结构域蛋白是否能与ephrinB2共沉淀,从生物化学角度,初步筛选出可能参与EphB4/ephrinB2逆向信号下游信号转导的潜在PDZ结构域蛋白。EphB4-Fc蛋白加入到含RANKL的Raw264.7细胞培养液中,诱导培养4天后,荧光实时定量RT-PCR及Western blot技术比较分析潜在PDZ结构域蛋白的表达变化。结果：免疫荧光细胞化学及Western blot检测结果示：Raw264.7经RANKL诱导分化4天后的破骨细胞内8种PDZ结构域蛋白都有明显表达。免疫共沉淀分析结果示：仅有Dvl2蛋白能与ephrinB2共沉淀,其他蛋白未见共沉淀结果。Western blot及荧光实时定量RT-PCR技术分析结果示：在EphB4实验组中Dvl2的蛋白表达明显低于Fc对照组(P＜0.01), mRNA表达亦低于Fc对照组,但不具有统计学差异(P>0.05)。另外,虽然免疫共沉淀实验未显示Pickl和syntenin与ephrinB2结合,但Western blot及荧光实时定量RT-PCR技术分析结果示：在EphB4实验组中Pick1的蛋白表达明显强于Fc对照组(P＜O.01), mRNA表达量亦明显高于Fc对照组(P<0.01);而在EphB4实验组及Fc对照组间syntenin蛋白及mRNA表达量无明显差异(P>0.05)。结论：RANKL诱导的Raw264.7破骨细胞分化过程中,Dvl2蛋白是参与EphB4/ephrinB2逆向信号下游信号转导途径的潜在PDZ结构域蛋白分子。更多还原

【Abstract】 Osteoclastic bone resorption and osteoblastic bone formation are coordinated as a coupled mechanism to maintain bone homeostasis. This coupling is of great importance because a large majority of the diseases of bone that result in changes in bone mass, such as osteoporosis and osteopetrosis, are due to disruption of the coupling. The molecular mechanisms that are responsible for osteoclast-osteoblast communication remain one of the central issues in bone cell biology. Over the past decades, the pivotal roles of the receptor activator of NF-κB (RANK), its ligand RANKL. and the natural decoy receptor for RANKL, osteoprotegerin (OPG) have been well-documented. The binding of RANKL, which is released by osteoblasts and bone marrow cells, to its receptor RANK has an essential role in the differentiation and fusion of precursors into mature osteoclasts. There is recent evidence to suggest a role for the Eph receptors and their ephrin ligands in bone biology, a role that may affect the coupling between osteoblasts and osteoclasts.An important characteristic of interactions between Eph receptors and ephrin ligands is the bidirectional signaling, due to activation of signaling pathways in both the receptor-expressing and the ligand-expressing cells. Activation of the EphB receptors by the ephrinB ligands is designated as "forward signaling". Conversely, activation of the ephrinB ligands by the EphB receptors is referred to "reverse signaling". The reverse inhibitory effect in osteoclasts is dependent on the cytoplasmic domain of ephrinB2, while the C-terminal YKV motif is critical for signal transduction, indicating that downstream PDZ-domain proteins are involved.This pilot_study was designed to preliminarily identify the potential PDZ-domain proteins involved downstream of ephrinB2 during the osteoclast differentiation of Raw264.7 cells in vitro, and to find out whether the expression levels of potential PDZ-domain proteins are regulated by EphB4/ephrinB2 reverse signaling.Part I Establishing the osteoclastogenesis model that EphB4/ephrinB2 reverse signaling inhibits RANKL-induced osteoclast differentiation of Raw264.7 cellsPurpose:The first step of our study was to select the type of osteoclastic precursors to be used for further research. However, inter-individual variability and insufficient material are disadvantages of working with primary cell cultures, and cell lines are being increasingly recognized as standard experimental reagents and as essential components to ensure reproducible and reliable results in life science research. Our study used Raw264.7 cells, a macrophage/monocyte lineage cell line, as osteoclast precursors, and to examine the effect of EphB4/ephrinB2 reverse signaling on the RANKL-induced osteoclastogenesis and the gene expression of critical transcriptional factors such as c-Fos. c-Jun and NFATc 1.Methods:Raw264.7 was used in this study and could be differentiated into osteoclast-like cells on activation by RANKL. The detection of ephrinB2 protein was examined by immunofluorescence techniques and the western blot. The EphB4 protein was added to nutrient solution to find out the effect of EphB4/ephrinB2 reverse signaling on the osteoclast differentiation of Raw264.7 cells. After 4 days, the cultured cells were stained by a leukocyte acid phosphatase kit to analyze the cytochemical tartrate-resistant acid phosphatase (TRAP). The Expression of the marker enzyme gene of osteoclast differentiation of Raw264.7 cells, and transcriptional factors such as c-Fos, c-Jun and NFATc1, were assessed by quantitative real-time RT-PCR.Results:By immunofluorescence techniques and the western blot, the expression of ephrinB2 protein was detected in the RANKL induced osteoclast differentiation of Raw264.7 cells, but was not detected in the absence of RANKL. After four days in the conditioning culture, TRAP staining was performed, and TRAP-positive MNCs in each well were counted as osteoclasts. The number of TRAP-positive MNCs was significantly lower in the EphB4-Fc treated group than that in the Fc treated group (P<0.01). Total RNA was collected and analyzed, and the results showed that the EphB4-Fc treatment reduced the mRNA expression levels of osteoclast specific genes such as TRAP, cathepsin K and calcitonin receptor compared with the Fc treatment (P0.01). The mRNA level of these transcription factors was also reduced in the EphB4-Fc treated group compared with the Fc treated group (P<0.01).Conclusions:These results show that EphB4/ephrinB2 reverse signaling inhibits RANKL-induced osteoclast differentiation of Raw264.7 cells.PartⅡA preliminary identification of potential PDZ-domain proteins downstream of ephrinB2 during osteoclast differentiation of Raw264.7 cellsPurpose:The intracellular domain of ephrinB ligands, particularly the last 33 C-terminal amino acids, is highly conserved and contains multiple tyrosine residues, and the C-terminal YKV motif is a binding site for PDZ (postsynaptic density protein, disks large, zona occludens) domain-containing proteins. This pilot study was designed to preliminarily identify the potential PDZ-domain proteins involved downstream of ephrinB2 during the osteoclast differentiation of Raw264.7 cells in vitro.Methods:The immunofluorescence staining and western blot analysis were used to identify potential PDZ-domain proteins (PDZ-RGS3. Dv12, Pickl/PHIP, Syntenin, GRIP1, GRIP2, FAP-1 and Par3) expressed during the RANKL-induced osteoclast differentiation of Raw264.7 cells. To investigate whether ephrinB2 ligands associate with any of these potential PDZ-domain proteins, we immunoprecipitated ephrinB2 proteins from the cell lysis buffer with a goat polyclonal antibody, and the precipitate was assayed for coimmunoprecipitated PDZ-domain proteins by immunoblotting. Finally, to find out whether the EphB4-ephrinB2 reverse signaling affects the expression level of potential PDZ-domain proteins, preclustered soluble EphB4-Fc or Fc fragments were added to the RANKL-induced osteoclastogenic cultures. After four days in the conditioning culture, the expressions of the potential PDZ-domain proteins were assessed by the western blot and quantitative real-time RT-PCR.Results:The immunofluorescence staining and western blot analysis revealed that the eight PDZ-domain proteins were all expressed in the RANKL+ group after four days in the conditioning culture. However, among the eight proteins, only Dv12 showed the expected size coprecipitated with ephrinB2, and the reverse experiment (immunoprecipitation by the Dv12 antibody and immunoblotting by the ephrinB2 antibody) confirmed the relationship. The other PDZ-domain proteins were not detected at the expected sizes in the precipitate after immunoprecipitation with the ephrinB2 antibody in three independent experiments. After four days in the conditioning culture, the western blot showed that the band for Dv12 in the EphB4-Fc treated group was weaker than that in the Fc treated group (P<0.01), and the mRNA level of Dvl2 in the EphB4-Fc treated group was a little lower than in the Fc treated group, albeit with no statistical significance (P>0.05). What is more, we studied the effects of reverse signaling on the expression levels of Pickl and syntenin, PDZ-domain proteins which could not coprecipitate with ephrinB2. The western blot and quantitative real-time RT-PCR results showed that, in contrast to the similar expression level of syntenin between EphB4-Fc treated group and Fc treated group (P>0.05), the protein and mRNA expression levels of Pickl were obviously enhanced in EphB4-Fc treated group compared with Fc treated group (P<0.01).Conclusions:Dv12 may be the potential downstream PDZ-domain protein that is the binding partner for the C-terminal YKV motif of ephrinB2.更多还原