【作者】 蔡永萍；

【导师】 周庚寅； P.C.W.Hogendoorn； A.M.Cleton-Janson；

【作者基本信息】 山东大学 ， 病理学， 2010， 博士

【摘要】 【研究背景】骨肉瘤是最常见的原发性恶性骨肿瘤,主要发生于青少年,常累及长骨干骺端,如股骨远端或肱骨、胫骨的近端。在青少年生长发育时,长骨的干骺端代谢活跃,而骨肉瘤常常发生于此,提示骨肉瘤的发生可能与快速骨生长有关。自本世纪70年代采取术前化疗以来,病人5年生存率得到明显提高。但近30年来,无论尝试新化疗药物或联合化疗,病人生存率没有得到提高。对于诊断时已有肺转移或术后复发的病人,愈后更差。现阶段对骨肉瘤的发生的分子学机制了解甚少,制约了新的靶向治疗的开展。近来研究发现Wnt信号途径在骨的发育中扮演重要角色。Wnt信号途径包括β-catenin依赖途径,即经典型Wnt信号通路和β-catenin非依赖途径,即所谓的非经典Wnt信号途径(包括Wnt／Ca2+和Wnt/JNK途径)。许多研究发现经典型Wnt信号途径是骨骼发生的重要调控因素,而非经典型Wnt通路的作用尚不清楚。因此,本研究主要涉及经典型Wnt信号途径。Wnt信号途径活化是间充质干细胞向骨细胞分化所必须的条件之一。后来,它通过调节骨原细胞的增值和分化来参与调控骨的发生。研究发现在成年小鼠的骨原细胞中,Wnt信号途径是活化的。骨肉瘤的诊断标准是高度异性的肿瘤细胞伴有肿瘤样骨样基质,肿瘤样骨样基质类似正常成骨中的骨基质,提示肿瘤细胞可能来自骨细胞,但失去终末分化的能力。Wnt信号途径活化是正常骨发育的重要因素,但它是否参与骨肉瘤的形成尚不清楚。大量研究证明Wnt信号途径成员的突变导致过度活化的Wnt信号通路促进内胚层上皮细胞来源的肿瘤发生,如肠道肿瘤。但是关于Wnt信号途径在中胚层间充质细胞起源的骨肉瘤发生中的作用尚不清楚。早先研究根据在骨肉瘤标本中检测到Wnt配体或细胞膜／浆β-catenin表达,推测活化的Wnt信号途径促进骨肉瘤的发生。但这种研究方法存在一定缺陷,因为复杂的Wnt信号通路由多个配体、受体、共同受体及细胞抑制因子组成,仅检测Wnt家族一员如Wnt配体不足以证明Wnt信号途径是激活的,而β-catenin进入细胞核是Wnt信号活化重要标志,因此检测细胞核β-catenin的表达是证明Wnt信号途径活化的可信方法。大量研究发现激活Wnt信号通路促进间充质干细胞向骨细胞分化,因而对Wnt信号通路的干预,成为治疗骨质疏松新的药物靶点或骨组织再生工程学的研究热点。以往的研究表明Wnt信号通路的过度激活与某些肿瘤的发生密切相关,因此这些研究最大的担忧是激活Wnt信号途径是否会诱发骨肿瘤。本文通过研究Wnt信号途径在骨肉瘤中发生的作用,对于解除这一担忧具有一定指导意义。本研究系统探讨Wnt信号途径在骨肉瘤发生中的作用,我们通过检测细胞核β-catenin在骨肉瘤、骨肉瘤细胞系和骨母细胞瘤(良性骨肿瘤)中的表达,同时检测调节Wnt信号途径对骨肉瘤细胞增殖和向骨细胞分化的影响。明确Wnt信号途径在骨肉瘤的作用不仅有助于深入认识骨肉瘤发生的机制,而且将为骨肉瘤的治疗提供新的思路和理论依据。【研究目的】1.明确Wnt信号途径在骨肉瘤中状态。2探索激活Wnt信号途径对骨肉瘤细胞增值和分化的影响,为今后临床治疗骨肉瘤提供新思路。【研究方法】1.免疫组化方法检测细胞核β-catenin在52例人骨肉瘤活检组织、15例人骨母细胞瘤(良性骨肿瘤)标本和骨肉瘤细胞系中的表达；2.运用Wnt荧光素酶报告基因检测骨肉瘤细胞MG-63、SJSA-1、HOS和U-2-OS中Wnt信号途径的活性,RT-PCR方法检测Wnt信号抑制剂DKK-1的表达；3.检测GIN (GSK3β抑制剂)和配体Wnt3a是否能激活Wnt信号途径：根据Wnt荧光素酶报告基因的活性、细胞核β-catenin的表达和Wnt信号通路的靶基因Axin2的表达的变化；4.MTS方法检测激活Wnt信号途径对骨肉瘤细胞增值的影响；5.通过检测碱性磷酸酶活性(alkaline phosphatase, ALP)和骨原结节的形成(Alizarin Red染色)探讨激活Wnt信号途径对骨肉瘤细胞向成骨细胞分化的影响。【实验结果】1.Wnt信号途径在骨肉瘤中失活首先运用免疫组织化学方法检测β-catenin在骨肉瘤活检标本中的表达。研究结果发现在骨肉瘤标本中,在90%的标本中细胞核β-catenin染色呈阴性,其余的10%标本中β-catenin弱核阳性,而细胞膜或胞浆β-catenin阳性见于90%(47／52)标本中。在15例骨母细胞瘤中,β-catenin染色呈核强阳性,同时细胞浆／膜也阳性,但其中的破骨细胞β-catenin染色为阴性。在骨肉瘤细胞系中,我们运用Wnt荧光素酶报告基因检测Wnt信号途径的活性。在阳性对照结肠癌细胞SW480,纠正后的Wnt荧光素酶值为10左右,而在所有骨肉瘤细胞中,其值只有0.002～0.01(比SW480低1000～5000倍),这个数值与阴性对照pGL-3强度相似。Wnt荧光素酶报告基因检测结果表明在骨肉瘤系中Wnt信号途径处于失活状态。免疫荧光检测β-catenin结果发现在所有的骨肉瘤细胞系中,细胞核β-catenin染色阴性,这一结果也表明Wnt信号途径是失活的。2.在骨肉瘤细胞中GIN激活Wnt信号途径前面的结果显示在骨肉瘤细胞中Wnt信号途径失活,而Wnt信号途径是正常骨细胞分化所必须的因素之一。因此,下一步检测GIN和Wnt3a是否能够激活Wnt信号途径。GIN通过抑制GSK3β阻止β-catenin的降解进而激活Wnt信号途径。在骨肉瘤细胞系MG-63、SJSA-1、HOS和U-2-OS中,GIN (0.2μM)处理24h后Wnt荧光素酶报告基因活性提高10-200倍(p<0.01)。同时,在DMSO对照组,免疫荧光显示细胞膜β-catenin阳性,而在GIN处理所有的骨肉瘤细胞系后,细胞核β-catenin染色强阳性。与DMSO处理组相比,GIN (0.2μM)处理后的细胞Wnt靶基因Axin2 mRNA表达增加10-60倍(p<0.01)。Wnt3a是常用的Wnt配体,与受体结合后激活Wnt信号途径。在阳性对照细胞C2C12中,50ng/ml;和100ng/mlWnt3a处理后Wnt荧光素酶报告基因活性呈浓度依赖性增加,而在所有骨肉瘤细胞系中,Wnt3a作用后,未发现Wnt荧光素酶报告基因活性增加,同时也未发现Wnt3a作用后促进细胞核表达β-catenin。3.DKK-1在骨肉瘤细胞中表达前面的研究结果发现GIN能够激活Wnt信号途径,而Wnt3a却不能,推测Wnt配体和受体的结合环节受到阻碍。为了进一步探索导致Wnt信号途径失活的原因,我们应用实时荧光定量PCR (qRT-PCR)检测Wnt抑制剂DKK-1 mRNA的表达。同Hela细胞相比,我们发现在MG-63、SJSA-1和HOS细胞中,DKK-1 mRNA的表达较高,但在U-2-OS细胞中未检测到DKK-1mRNA的表达。4.激活Wnt信号途径促进骨原细胞分化Wnt信号途径激活是间充质细胞向骨细胞分化必须的,而前面的研究结果发现Wnt信号途径在骨肉瘤中失活,因此下一步检测在骨肉瘤细胞中激活Wnt信号途径是否能促进其向骨原细胞分化。我们在诱导骨肉瘤细胞向骨原细胞分化的几个不同时间段激活Wnt信号途径,即在不同时间段加入GIN,如在分化开始前3天、分化开始前3天至分化开始后前3天、分化开始后的前3天和分化开始后的前7天。ALP活性(第7天)和骨原结节的形成(第21天)是骨原细胞分化早期和晚期的标志。在MG-63和U-2-OS中,未加GIN处理时ALP活性较低,同时没有骨原结节形成,在不同的时间段运用GIN处理激活Wnt信号途径对ALP活性和骨原结节没有影响。在SJSA-1和HOS细胞中,未加GIN处理时ALP活性较高,并有骨原结节形成。同对照组相比,结果发现激活Wnt信号途径组(分化开始后的前3天或前7天的GIN处理组)ALP活性增高(p<0.01)和骨原结节形成增多；而预先激活Wnt信号途径(分化开始前3天GIN处理组)对ALP活性和骨原结节形成没有影响。5.激活Wnt信号途径抑制骨肉瘤细胞增殖接下来,我们检测激活Wnt信号途径对骨肉瘤细胞增值的影响。与DMSO对照组相比,GIN (0.2μM)作用4天后对SJSA-1和HOS细胞增殖几乎没有影响,而在MG-63和U-2-OS,细胞增殖率只有对照组50%(p<0.01)。【结论】本研究首次报道Wnt途径在骨肉瘤中是失活的,这一结论与以往的研究观点不同。前人研究根据细胞膜和／或细胞浆β-catenin的表达得出骨肉瘤中Wnt信号途径激活的结论。本实验结果显示Wnt信号途径关键因子细胞核β-catenin在骨肉瘤标本和骨肉瘤细胞中不表达,而在骨母细胞瘤中细胞核β-catenin强阳性表达,并且在骨肉瘤细胞中激活Wnt信号途径抑制细胞增殖或促进向骨原细胞分化,因此我们推测骨正常发育所必须的Wnt信号途径的丢失是促进骨肉瘤发生因素。本研究对Wnt信号途径失活的机制进行初步探讨。GIN在骨肉瘤细胞中可以激活Wnt信号通路,而Wnt3a不能,同时检测到Wnt通路抑制剂DKK-1的高表达,表明Wnt抑制剂的过表达可能是导致Wnt通路失活的原因之一。大量研究表明,激活的Wnt信号途径在上皮性肿瘤中扮演促进肿瘤发生的角色,而我们的结果支持Wnt信号途径在间叶来源的骨肉瘤的发生中可能起抑癌的作用,我们这一发现提示Wnt信号途径的作用可能是器官或组织依赖性的。更多还原

【Abstract】 【Background】Osteosarcoma is the most common primary malignant nonhematological tumor of bone. Osteosaroma has a peak incidence in adolescence. Conventional osteosarcoma frequently affects young adolescents during their longitudinal growth spurt at the metaphysis of long bones, suggesting a correlation between rapid bone turnover and the pathogenesis of osteosarcoma. Prognosis remains less that 20% especially in patients with clinically detectable metastasis at diagnosis or relapsed disease. Limited improvement in survival has been achieved over the past 30 years, regardless of intensifying or modifying chemotherapy.. New target therapy has not been developed yet because of limited progress on the pathogenesis of osteosarocoma.Recently, accumulated evidence show Wnt/β-catenin pathway has emerged as an essential pathway in skeletal development. Wnt acts throughβ-catenin-independent pathways, the so-called non-canonical Wnt pathways (including the Wnt/Ca2+and Wnt/JNK pathways) and through aβ-catenin-dependent or canonical Wnt pathway. The role of the non-canonical Wnt pathway is poorly understood in bone formation, thus this study focus on canonical Wnt pathway.The Wnt pathway is crucial for physiological osteoblast development. Wnt pathway is required for promoting an osteogenic lineage in skeletal stem cells at an early stage. Later on, Wnts stimulate osteoblast proliferation and support osteoblast maturation. Wnt pathway has been shown to be active in osteoblasts in adult mice. The exact role of Wnt coordination osteoblast proliferation and differenation is unclear. Osteosarcoma is characterized by tumor osteoid, which suggest tumor cells retain part of capability as osteoblast but is unable to go terminal differentation. Wnt pathway plays an essential role in bone development, howerer, its role in osteogenic tumor osteosarcoma remains largely unknown.Since mutation of key components APC of Wnt pathway has been found to cause colorectal tumor, increasing evidence suggest that activation of Wnt pathway contributes to a variety of epithelial tumor types development. However, its role in the mesenchymal cells derived tumor osteosarcoma remains unclear. Previous studies have suggested that the Wnt pathway is active in osteosarcoma, based on the detection of Wnt ligands or cytoplasmicβ-catenin staining. However, those approaches are inadequate to demonstrate whether this pathway is active or not given the large family of Wnt pathway, which includes a number of ligands, receptors, co-receptors, and inhibitors. Nuclearβ-catenin expression is the node of Wnt pathway, thus detection nuclearβ-catenin expression is a relatively reliable method.Accumulating evidences suggest that activation of Wnt pathway promotes MSC toward osteoblast development, therefore, manipulating MSC is a promising direction for treatment of osteoporosis and bone regeneration. However, concerns remain that activation of Wnt pathway contributes to tumor development given that numerous studies related active Wnt pathway with tumor development.Thus, in this study, we aim to explore the role of Wnt pathway in osteosarcom by evaluating nuclear P-catenin expression in osteosarcoma biopsies and osteosarcoma cell lines as well as osteoblastomas. Moreover, we assessed the effect of modulating this pathway on cell growth and osteogenic differentiation. Our study will advance the understanding of mechanism of osteosarcoma development and provide a new therapy target for osteosarcoma treatment.【Objectives】1. To determine Wnt pathway status in osteosarcoma.2. To explore the role of Wnt pathway in osteosarcoma cells proliferation and differentiation. [Methods】1. Nuclear P-catenin expression was examined in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours) and human osteosarcoma cell lines by immunohistochemistry.2. Wnt pathway activity was measured in osteosarcoma cell lines MG-633. SJSA-11. HOS and U-2-OS by Wnt luciferase reporter assay. Wnt pathway inhibitor DKK-1 expression was examined by qRT-PCR.3. Wnt/β-catenin pathway activity was modulated using a drug GIN (GSK3βinhibitor) and Wnt 3 a and the effect was determined by Wnt luciferase reporter assay, nuclear P-catenin expression and change of Wnt target gene Axin2 mRNA expression.4. The effect of activation Wnt pathway on osteosarcoma cells growth was evaluated by the MTS assay.5. ALP activity and mineralization formation was measured to determine the effect of activation Wnt pathway on osteosarcoma cells differentiation toward osteoblasts.【Results】1. Wnt pathway is inactive in osteosarcoma The expression ofβ-catenin in osteosarcoma was examined by immunohistochemistry. Negative staining of nuclear P-catenin was detected in 90% (47/52) of cases and the remaining five cases showed weak nuclear staining. Most of the cases (47/52) showed membranous and/or cytoplasmic staining. Strong membranous and nuclear P-catenin staining was seen in all 15 osteoblastomas and absence ofβ-catenin staining was found in osteoclasts.Wnt luciferase assay was used to determine the functional activity of Wnt pathway. The corrected Wnt-luciferase activity was around 10 in the positive control colorectal carcinoma cell line SW480; however, it was around 0.002-0.01 (1000-5000 times lower than the positive control) in osteosarcoma cell lines, comparable with the negative control. Consistently, none of the cell lines showed nuclear P-catenin staining.2. Restoration of Wnt signalling by GIN and Wnt3aInactive Wnt pathway was observed in osteosarcoma and active Wnt pathway is required for osteoblast development, thus we decided to investigate whether Wnt pathway can be actived in osteosarcoma cell lines.GIN stimulates Wnt pathway by inhibiting GSK3, thereby preventingβ-catenin degradation in the cytoplasm. Around a 10-to 200 fold increase in Wnt pathway activity occurred in all osteosarcoma cell lines upon GIN (0.2μM) treatment, as determined by the luciferase assay (p< 0.01). Accordingly, strong nuclear p-catenin staining was observed by immunofluorescence in all cell lines after GIN (0.2μM) treatment. Moreover, a 10-to 60 fold increase in Axin2 mRNA expression was found compared with DMSO-treated cells. Wnt3a is a ligand commonly used to activate Wnt signalling upon binding to the receptors. In the positive control C2C12 cell line, BAT-luc activity increased in a dose-dependent manner upon treatment with Wnt3a (50ng/ml,100ng/ml). However, neither a change of BAT-luc activity nor a change of nuclearβ-catenin staining was observed upon rWnt3a treatment in osteosarcoma cell lines.3. DKK1 expression in osteosarcoma cell linesGIN can stimulate Wnt pathway, however, Wnt3a failed, implying the link between Wnt ligands and receptor is impaired.To determine the possible cause of Wnt pathway inactivity, we used qRT-PCR to assess expression of the Wnt inhibitor DKK-1 at the mRNA level. Compared with HeLa cells, relatively high or similar levels of DKK-1 mRNA were found in MG-63, SJSA-1, and HOS cells, although DKK-1 mRNA expression was hardly detectable in U-2-OS cells.4. Restoration of Wnt signalling promotes osteoblast differentiation in SJSA-1 and HOS cells Since Wnt signalling is essential for osteogenic differentiation, we examined whether restoration of Wnt signalling could promote differentiation of osteosarcoma cells. ALP activity (on day 7)and mineralization formation (on day 21) are early and late stage markers of osteoblast differentiation, respectively. We planned to activate Wnt pathway at different time period by adding GIN at different time point, i.e. prior 3 days, prior 3 days till first 3 days, first 3 days and first 7days. Low levels of ALP activity and absence of mineralization were observed in U-2-OS and MG-63, and stimulation of the Wnt pathway did not rescue this phenotype. High levels of ALP activity and mineralization were seen in SJSA-1 and HOS cells. Stimulation of the Wnt pathway by GIN for 3 or 7 days (when differentiation starts) induced a further increase in ALP activity and mineralization. Three day prior treatment with GIN did not induce ALP activity or mineralization.5. Activation of the Wnt pathway inhibits cell proliferation in MG-63 and U-2-OS cellsThe effect of stimulation of the Wnt pathway on cell proliferation was examined. A slight decrease of cell numbers was found for SJSA-1 and HOS cells upon GIN treatment compared with DMSO control group. In contrast, around 50%inhibition of proliferation was observed in MG-63 and U-2-OS upon GIN treatment.【Conclusions】To the best of our knowledge, this is the first time we report that Wnt pathway is inactive in osteosarcoma. Our view is different from previous conclusion that Wnt pathway is active in osteosarcoma, which based on membranous and/or cytoplasm staining ofβ-catenin.We observed negative nuclearβ-catenin expression in most osteosarcoma specimen and osteosarcoma cell lines and strong nuclearβ-catenin staining in osteoblastoma. Moreover, activation of the Wnt pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Thus, we propose that loss of Wnt/β-catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development.We also explored the reason for inactivation of Wnt pathway. GIN stimulated Wnt pathway, but Wnt3a failed. High expression of DKK-1 was detected in osteosarcoma cells. Our data suggest high expression of Wnt inhibitor may be the cause of inactive Wnt pathway.The oncogenic role of the Wnt/β-catenin pathway in epithelial tumors has been well studied; however our data support a tumor suppressor role of Wnt pathway in osteosarcoma, which originate from mesenchymal cells, implicating the role of Wnt pathway acts in a context dependent manner.更多还原