趋化因子CCL15介导骨髓间充质干细胞靶向肝癌迁移的机制研究

In our previous study, human mesenchymal stem cells (hMSCs) were first reported to have the potential as an effective delivery vehicle for therapeutic genes in the treatment of Hepatocellular carcinoma (HCC). While the propensity of MSCs to migrate to tumors has been well documented,the molecular signals guiding this movement are not completely defined. To elucidate the mechanism of MSCs recruitment by HCC, a cytokine antibody array of conditioned media was performed. Among all the cytokines tested, CCL15 was found to be secreted by three HCC cell lines at the highest level, whereas there was least expression of this protein in normal liver cell line. It is assumed that CCL15 might contribute to the migration of hMSCs toward HCC. Results of prelminary expreriments showed that anti-CCL15 antibody inhibited chemotaxis of HCC cell-conditioned medium on hMSCs dramatically, and CCR1, a receptor of CCL15, was expressed by hMSCs. In this study, the role of CCL15 and its receptor, CCR1 on the migration of hMSCs toward HCC will be assesed in vitro through RNA interference (RNAi) or lentivirus-mediated gene transfection techniques. The downstream signaling during MSC migration in response to tumor cell-conditioned medium and recombinant CCL15 will also be explored in this study. Finally, the role of CCR1 on the migration of hMSCs and the safety of hMSCs as a vehicle of gene therapy will be evaluated in vivo using an orthotopic nude mice model of HCC. This study will not only help to elucidate the reciprocal intereaction between MSCs and HCC, but also help to develop novel vechiles with enhanced capability of migration, which may make it possible for hMSCs to trace and eliminate tumor cells more effectively.

申请者在前期试验中，首次报道MSCs具有靶向肝癌迁移的能力。通过蛋白芯片技术，本课题小组发现，趋化因子CCL15是肝癌细胞培养上清中表达水平最高的细胞因子。申请者推测，肝癌微环境分泌的CCL15可能是介导hMSCs发生迁移的主要因素。预实验结果显示，CCL15抗体能够显著抑制hMSCs的体外迁移，且hMSCs表达CCL15的受体CCR1。申请者拟在前期研究的基础上，利用RNA干扰和基因转染技术，体外研究 CCL15和 CCR1受体在hMSCs靶向肝癌迁移中的作用，并探讨CCR1激活后的下游信号转导机制；利用肝癌裸鼠原位模型，通过体内实验证实CCR1受体在hMSCs靶向肝癌迁移中的作用，并初步分析hMSCs作为基因治疗细胞载体的安全性。本研究的结果将有助于人们阐明肝癌与MSCs相互作用的机制及开发迁移能力更强的细载体，从而更有效地追踪、清除肿瘤细胞，特别是已发生转移的肿瘤细胞。

当前，基因治疗已成为医学领域的研究热点。然而，到目前为止，基因治疗在临床实验中，并没有取得令人满意的结果，其中一个重要的原因便是基因导入系统存在缺乏靶向性的问题。间充质干细胞 (mesenchymal stem cell，MSCs)具有高度自我更新能力、取材方便、体外易于培养和扩增、低免疫原性，并能稳定高效表达外源基因等优点，同时MSCs具有向肿瘤微环境趋向转移的特性，因此MSCs可以作为基因治疗的载体将目的基因带到肿瘤部位，从而增加肿瘤基因治疗的靶向性。申请者在前期试验中，首次报道MSCs具有靶向肝癌迁移的能力。通过蛋白芯片技术，本课题小组发现，趋化因子CCL15是肝癌细胞培养上清中表达水平最高的细胞因子。申请者在前期研究的基础上，利用RNA干扰和基因转染技术，体外研究 CCL15和 CCR1受体在hMSCs靶向肝癌迁移中的作用，并探讨CCR1激活后的下游信号转导机制；利用肝癌裸鼠原位模型，通过体内实验证实CCR1受体在hMSCs靶向肝癌迁移中的作用，并初步分析hMSCs作为基因治疗细胞载体的安全性。本研究结果显示，本研究的结果将有助于人们阐明肝癌与MSCs相互作用的机制及开发迁移能力更强的细载体，从而更有效地追踪、清除肿瘤细胞，特别是已发生转移的肿瘤细胞。