脂联素通过mTOR信号通路抑制骨肉瘤发生发展的机制研究

Osteosarcoma is a major kind of primary bone malignant tumor. Adiponectin is one of the significant adipocytokines secreted by adipocytes, which is ubiquitously involved in processes including metabolism. Recent studies have demonstrated that adiponectin played a negative role in regulating the occurrence and development of malignant tumors, yet the underlying mechanisms require further exploration. Our preliminary data showed that expression of serum adiponectin was significantly impaired in patients with osteosarcoma, while the adiponectin receopor (AdipoR1) was specifically expressed in human osteosarcoma tissue. We also found that AMPK，as one of the upregulation signaling of mTOR pathway, was enhanced with increased adiponectin concentration or stimulus duration, whereas that the expression of mTOR pathway was suppressed in a similar time- and concentration-dependent way. Meantime,we can not rule out the possible effect of PI3K/Akt/mTOR axis as it has been proved to be a classical upregulation signaling of mTOR pathway. Therefore, we hypothesize that adiponectin negatively regulates the occurrence and development of osteosarcoma through mTOR signaling pathways. We will use various research strategies, including osteosarcoma cell lines(SaOS-2, MG-63), adiponectin whole body knockout mice and Rheb1 osteoblast-specifically knockout mice, and human osteosarcoma tissue, to study the mechanism of regulation on osteosarcoma by adiponectin, and to study the effects of mTOR pathway both in vitro and in vivo. Our study will provide important information for the mechanisms of osteosarcoma proliferation and development, which could provide new thoughts and new drug targets for the clinical treatment of osteosarcoma.

骨肉瘤是起源于骨骼的高度恶性肿瘤，其发病率在原发性骨恶性肿瘤中居首位。脂联素是脂肪细胞分泌的一种脂肪因子，近年研究表明脂联素与恶性肿瘤的发生发展起着密切负相关作用，但其具体机制目前尚不明确。我们前期工作发现脂联素在骨肉瘤患者血清中表达水平显著下降，其受体在骨肉瘤组织亦有特异性表达，同时脂联素可随时间和浓度改变激活mTOR上游信号分子AMPK，从而抑制mTOR的表达，提示其可能通过调节骨肉瘤组织细胞中的mTOR信号途径发挥作用,据此提出假设脂联素通过mTOR信号通路抑制骨肉瘤的发生发展。我们将用骨肉瘤细胞系（SaOS-2，MG-63）以及脂联素基因敲除小鼠、成骨细胞特异性Rheb1基因敲除小鼠以及人体骨肉瘤组织活检，从离体、在体以及人体三个层面阐明脂联素对于骨肉瘤mTOR信号通路的作用及机制。该研究将为骨肉瘤的的发生和发展提供重要信息，为临床骨肉瘤的治疗提供新的思路和药物新靶点。

骨肉瘤是起源于骨骼的高度恶性肿瘤，其发病率在原发性骨恶性肿瘤中居首位。脂联素是脂肪细胞分泌的一种脂肪因子，近年研究表明脂联素与恶性肿瘤的发生发展起着密切负相关作用，但其具体机制目前尚不明确。本课题从骨肉瘤细胞系、临床患者标本等不同水平对脂联素通过调节骨肉瘤组织细胞中的mTOR信号途径发挥骨肉瘤发生发展调控作用的机制进行研究。我们发现脂联素在骨肉瘤患者血清中表达水平显著下降，而其受体在骨肉瘤患者组织及骨肉瘤细胞株中却高表达；同时证实脂联素具有抑制骨肉瘤细胞株增殖、迁移及侵袭和促进凋亡的作用，且其作用效果具有浓度依赖性及细胞株间差异性。此外，脂联素可随时间和浓度改变激活mTOR上游信号分子AMPK磷酸化水平和降低Akt（Ser473）磷酸化水平，从而抑制mTOR的表达，并可抑制骨肉瘤MG-63细胞中mTOR下游信号蛋白p70 S6K1（Thr389）、4EBP1（Thr47/46）的磷酸化水平，我们还通过慢病毒转染过表达骨肉瘤细胞株中mTOR蛋白双向证明此结论，这提示脂联素通过调节骨肉瘤组织细胞中的mTOR信号途径发挥作用。同时，我们还发现脂联素可协同增强PI3K/Akt特异性抑制剂LY294002对骨肉瘤细胞株的增殖、侵袭抑制或凋亡诱导作用。此外，已先后培养了骨肉瘤、滑膜肉瘤及尤文肉瘤等多种骨与软组织恶性肿瘤原代细胞。已完成37例临床不同性别和不同病程骨与软组织恶性肿瘤（包括17例骨肉瘤）患者的骨肉瘤人源性异种移植小鼠（PDX）模型操作（皮下移植和骨髓原位移植），解决了肿瘤样本运输处理和冻存、复苏关键技术难题，并经过PDX原代和传代模型与患者组织学、基因学检测一致性表明成功，目前骨与软组织恶性肿瘤PDX建模成功率接近50%。成功建立鼠源性K7M2骨肉瘤细胞荷瘤正常免疫Balb/c小鼠，成瘤率达80%。该研究将为骨肉瘤的的发生和发展提供重要信息，为临床骨肉瘤的治疗提供新的思路和药物新靶点。