间充质干细胞通过IL-6/STAT3和Notch通路协同作用促进骨肉瘤化疗耐药的机制研究

It is not clear how environment affects the chemoresistance of tumors. We previously demonstrated that MSCs promoted the proliferation and chemoresistance of Saos-2 cells in vitro.Moreover, our un-published data showed that MSCs enhanced miR-21 exprssion in osteosarcoma cells, and over-activated STAT3 as well as over-expressed Notch1 were observed in chemotherapy-resistant osteosarcoma samples. Based on these evidences, we propose that MSCs activate STAT3 pathway in osteosarcoma via secreting IL-6. On the other hand, by contacting with the tumor cells directly, MSCs activate the Notch pathway, which then promotes the persistent activation of STAT3. Finally, the activated STAT3 up-regulates the expression of miR-21 and enhances the chemoresistance of osteosarcoma. In order to verify this hypothesis,we use an in vitro co-culture model and an in vivo nude mice osteosarcoma model to investigate the effects of MSCs on osteosarcoma. By using in vivo imaging, FACS, immunohistochemistry, Lenti-virus transfection,RNA interference,western blot et al, we try to understand the mechnisams involved in the acquired chemoresistance process and hope to provide new methods for the treatment of osteosarcoma.

环境因素对肿瘤细胞产生化疗耐药的作用仍知之甚少，我们之前研究发现MSCs能通过IL-6促进骨肉瘤的生长和体外化疗抵抗，预实验中发现MSCs可以促进miR-21的表达,且临床耐药的骨肉瘤样本中也有STAT3和Notch1的过度激活/表达。我们因而提出假说：MSCs一方面通过分泌IL-6激活骨肉瘤中STAT3通路，而另一方面MSCs通过和骨肉瘤细胞的直接作用激活Notch通路，从而使骨肉瘤中STAT3通路持续激活，最终持续激活的STAT3上调miR-21的表达，促进骨肉瘤的化疗抵抗。为验证该假说，我们通过两种细胞共培养以及荧光素酶标记的骨肉瘤裸鼠模型，采用活体成像、流式细胞、免疫组化、慢病毒转染、RNA干扰、Western blot等手段，从分子细胞、动物以及临床样本水平探讨MSCs对骨肉化疗耐药的影响。本研究试图通过了解微环境中MSCs促进骨肉瘤化疗耐药的分子机制，为骨肉瘤的治疗提供新思路。

目前对骨肉瘤耐药机理的研究主要集中在基因突变等内源性致病因素的分析上，而对肿瘤所处微环境在骨肉瘤发生获得新耐药过程中的作用知之甚少。本研究分析了肿瘤微环境中骨髓间充质干细胞（MSCs）与骨肉瘤细胞相互作用产生化疗耐药的分子机制，主要研究发现有: （1）骨肉瘤细胞条件培养基可以抑制MSCs细胞的成骨分化，并通过分泌TGF-β促进干细胞分泌更多的促肿瘤生长因子，包括IL-6和VEGF，进而促进骨肉瘤生长及存活。（2）MSCs可以激活骨肉瘤细胞内IL-6/STAT3信号通路，促进耐药相关基因表达使肿瘤细胞获得化疗抵抗能力。（3）借助临床样本分析，进一步明确IL-6/STAT3通路在骨肉瘤临床耐药中的重要作用。这些结果提示，环境因素和肿瘤细胞的相互作用可以诱导骨肉瘤细胞发生化疗耐药，而其中STAT3通路是一个关键因素，有效抑制该通路激活可能为骨肉瘤治疗提供治疗靶点。