细胞粘附介导的耐药(CAM-DR)是导致非霍奇金淋巴瘤不可治愈的重要原因。其中β1整合素的表达水平及在细胞中的内化循环与CAM-DR形成密切相关。本课题组前期研究发现，Numblike在粘附模型中表达有差异，并且除了PTB区域外，羧基端也能够与β1整合素相互作用，并参与调控细胞周期及FAK、促存活信号通路PI3K/AKT激活。本研究拟通过纤粘蛋白及骨髓基质细胞包被构建淋巴瘤细胞CAM-DR模型，验证并探讨Numblike与β1整合素的相互作用及调节机制，分析Numblike是否通过羧基端对β1整合素的调节影响细胞周期进程及PI3K/AKT信号通路的激活，从而促进淋巴瘤细胞CAM-DR形成。课题的研究结果将为我们进一步揭示CAM-DR发生发展的具体分子机制，并且确认Numblike在淋巴瘤细胞CAM-DR形成过程中的重要作用。这一发现也将为临床治疗淋巴瘤提供新的靶点。

Cell adhesion mediated drug resistance (CAM-DR) is an important reason why non-Hodgkin lymphoma is not curable. And the expression level of β1 integrin and its internalization circulation in cells are closely related to the CAM-DR formation. Our preliminary studys have found that, Numblike is differentially expressed in adhesion model, and in addition to PTB domain, the carboxyl end can interact with β1 integrin as well，and can be involved in regulating the cell cyclin and activating the PI3K/AKT pro-survival signal pathway. This study intends to verify and discuss the interaction and the regulation mechanism between Numblike and β1 integrin through the establishment of the CAM-DR model of lymphoma cells coated with fiber mucin and bone marrow stromal cells, and analysis whether Numblike influences the cell cycle process and the activation of PI3K/AKT signal pathway through the adjustment of its carboxyl end to the β1 integrin so as to promote the formation of CAM-DR of lymphoma cells. The results of our subject will further reveal the specific molecular mechanisms of the occurrence and development of CAM-DR for us, and confirm that Numblike plays an important role in the process of the formation of the CAM-DR of lymphoma cells.

细胞粘附介导的耐药（CAM-DR）是非霍奇金淋巴瘤（NHL）患者不可治愈的重要原因。β1整合素（Integrinβ1）的表达水平及其在细胞中的内化循环与CAM-DR形成密切相关。根据实验室前期工作基础及相关领域的研究进展，课题组首先利用纤粘蛋白（FN）及骨髓基质细胞（HS-5）包被构建了NHL细胞粘附模型，免疫印记检测并分析Numblike (Numbl)、Integrinβ1在粘附及悬浮培养过程中的表达差异。免疫共沉淀检测NHL细胞中Numbl与Integrinβ1的相互作用；通过分别构建Numbl与Integrinβ1截短突变质粒，工具细胞中分析两者相互作用的结构域，发现Numbl通过其羧基端结构域（C-terminal domain, CTD）与Integrinβ1的455-802aa结构域结合。免疫荧光双标进一步验证上述两分子在NHL细胞中存在显著共定位。接下来通过构建Numbl过表达、小干扰及结构域缺失质粒，分析干预Numbl表达后对Integrinβ1蛋白水平的影响，结果显示Numbl高表达可以促进Integrinβ1蛋白表达。进一步的钙黄绿素实验显示干预两者相互作用能够抑制NHL细胞与骨髓基质细胞HS-5或FN的粘附率。利用化疗药物处理NHL细胞，细胞活力分析实验显示干预Numbl与Integrinβ1的相互作用可以显著改善由于细胞粘附导致的NHL对化疗药物的耐受情况。流式细胞检测结果显示Numbl和Integrinβ1相互作用可导致NHL细胞周期阻滞在G0/G1期，引起细胞周期蛋白p27Kip1表达增加，活化FAK及PI3K信号通路。阻断PI3K信号通路可抑制Numbl过表达导致的NHL细胞对化疗药物耐受。课题的研究结果将为我们进一步揭示CAM-DR发生发展的具体分子机制，并确认Numbl与Integrinβ1的相互作用在NHL细胞CAM-DR形成过程中的作用具有重要意义。按照合同约定，课题研究成果以论文形式提交。在该基金的资助下，目前已发表论文SCI收录论文15篇，共培养硕士研究生3名。