血浆EFEMP1用于评估预测骨肉瘤发生与发展的临床和实验研究

At present, there is no effective index on the prediction and evaluation of prognosis and progression in osteosarcoma. Our previous study showed that EFEMP1 is specifically upregulated in osteosarcoma that may contribute to the migrating and invasive ability of osteosarcoma cells. The association of tumour up-regulation with EFEMP1 was significantly stronger in osteosarcoma patients with hematogenous metastasis than in those without. Multivariate survival analysis indicated that EFEMP1 expression level was an independent prognostic factor of patient survival. The log-rank test showed that survival time between the low and high EFEMP1 expression groups was signifcantly different. So we think that EFEMP1 is not only a poor prognostic indicator of osteosarcoma, but also a candidate for a molecular targeted therapy. EFEMP1 is a secreted extracellular glycoprotein, so we speculated that plasma EFEMP1 would be an available molecular markers to forecast and evaluate prognosis and progression of osteosarcoma. In this study, we intends to measure plasma EFEMP1 levels in osteosarcoma patients by ELISA, comparing with the normal people, and the patients of benign bone tumors, osteoarthritis, inflammatory disease or fractures. The sensitivity, specificity and efficiency of plasma EFEMP1 on osteosarcoma would be confirmed. At the same time, we would analysis the relation between plasma EFEMP1 and bone destruction and new bone formation, combining with pathological and imaging changes. In addition, we would make the plasmid concluding GFP and EFEMP1.Then the secretion of EFEMP1 protein by osteosarcoma cells would been analysed in vitro throught confocal laser scanning microscope. At the same time, we would analyse the variety of extracellular EFEMP1 through the change of intracellular EFEMP1. Aim to find the influence which intracellular EFEMP1 of osteosarcoma cells effected intracellular EFEMP1, and the relation between EFEMP1 and the epidermal growth factor (EGF). Moreover, through osteosarcoma cell transplantation and lung metastasis in animal models, we will further discuss the dynamic change of plasma EFEMP1 in the process of osteosarcoma occurrence, development and metastasis. In conclusion, our objective is to find an effective clinical molecular marker for the prediction and evaluation of prognosis and tumor progression of patients osteosarcoma. Meanwhile, its possible mechanism would be clarified. It has the important theoretical and application value.

临床缺乏评价骨肉瘤进展的有效指标。我们前期发现骨肉瘤EFEMP1过表达，可促进侵袭转移，与不良预后相关。由于EFEMP1是一种分泌性细胞外糖蛋白，故我们推测血浆EFEMP1测定可预测骨肉瘤进展。本课题拟通过ELISA测定骨肉瘤患者血浆EFEMP1，并与正常人群和其他骨疾病患者比较，评价血浆EFEMP1测定对骨肉瘤诊断的敏感性、特异性及诊断效率。同时分析骨质破坏及骨质形成对血浆EFEMP1的影响。此外，利用基因融合技术构建EFEMP1-GFP融合质粒转染骨肉瘤细胞，激光共聚焦显微镜体外观察活细胞分泌EFEMP1过程；并改变细胞内EFEMP1表达，观察细胞外EFEMP1变化，明确骨肉瘤EFEMP1过表达对EFEMP1分泌的影响，并分析与表皮生长因子受体的关系。再进一步通过骨肉瘤荷鼠及肺转移模型，观察骨肉瘤发生、发展及转移不同阶段血浆EFEMP1动态变化。希望寻找到评价骨肉瘤进展的临床分子指标。

骨肉瘤是最常见的原发性间叶源性恶性骨肿瘤，好发于儿童和青少年。虽然已有众多研究一直努力在探索骨肉瘤发生、发展的分子调节机制，也已发现多种基因可通过不同的分子机制参与了其中，但至今尚未能完全明确影响骨肉瘤发生、发展的决定性因素。寻找预测及评价骨肉瘤患者的预后和肿瘤进展的分子标记和影响因素，是当前骨肉瘤分子研究领域的重要任务之一。在本研究中我们首先再次明确了骨肉瘤患者中无论是肿瘤组织，还是患者血浆的EFEMP1都呈高表达，均高于正常人对照组。同时，在收集的其他骨肿瘤中发现骨巨细胞瘤、软骨肉瘤的血浆EFEMP1表达也较正常人群明显升高，这提示EFEMP1蛋白不是骨肉瘤的特异性指标。此外，还发现骨肉瘤患者血浆EFEMP1水平在手术后较手术前明显升高，考虑可能血浆EFEMP1水平可能受手术损伤引起机体组织修复增生的影响。由于EFEMP1是一种细胞外分泌糖蛋白，这提示EFEMP1作为一种细胞外分泌糖蛋白对骨肉瘤微环境起到了一定的影响作用。其次，我们在体外细胞学研究中也证实了EFEMP1作为一种细胞外分泌糖蛋白，可通过改变肿瘤微环境，影响骨肉瘤细胞的迁移和侵袭能力。再次，我们通过动物模型模拟了肿瘤发生发展过程，发现随着荷鼠模型的肿瘤体积增加和进展，血浆EFEMP1表达有逐渐升高的趋势。因此，本项目如期通过从临床患者、细胞水平和动物模型的研究，均明确了骨肉瘤的血浆 EFEMP1的水平是明显升高的，与患者预后相关，可用于评价肿瘤的进展。同时，在肿瘤发生发展过程中，EFEMP1的作用也是逐渐增加的，有动态变化的过程。随后，我们在部分研究内容上进行了扩展与延伸，发现NF-kB信号通路可能是调节EFEMP1基因的主要途径之一。另外，还发现TEM8高表达也是骨肉瘤患者不良预后因素之一，在骨肉瘤细胞株中下调TEM8表达后，可显著抑制p-ERK1/2、cyclinD1和MMP2的表达，EFEMP1调节骨肉瘤的侵袭部分可能通过TEM8/MMP-2的途径。为今后骨肉瘤的研究打下扎实的基础，具有重要的理论意义和应用价值。