骨髓间充质干细胞外泌体选择性转运miR-15a激活Notch信号通路介导慢粒细胞对TKIs耐药的研究

Chronic myeloid leukemia (CML) could acquire resistance to the tyrosine kinase inhibitors(TKIs) through a certain therapy tolerance mechanism that remain poorly understood. This kind of TKIs resistance was believed to be closely linked with mesenchymal stem cells(MSCs) in the bone marrow microenvironment. However, the pathway remained unclear. Applicants found that compared with exosomes secreted by normal MSCs, exosmes secreted by CML-MSCs contain a low expression of miR-15a. CML cells englobe the exosomes and down-regulate the apoptosis induced by TKIs and up-regulate the expression of Notch1 and NF-κB. These results strongly suggested that exosmes secreted by CML-MSCs could activate notch signal pathway, which mediates the survival of CML cells and contributes to drug resistance of TKIs. Therefore, the current proposal aims to further optimize the effect of exosomes secreted by CML-MSCs on CML cells and probe the roles of Notch1 pathway in drug resistance of CML cells to TKIs. To fulfill the goals, Spinning disk confocal, RNA interference or gene recombination techniques will be used to interfere with miRNA-15a or Notch, and the consequences of which will be investigated. We will also examine the downstream elements in Notch signaling system. Through the proliferation and apoptosis capacities of CML cells in vitro and in vivo in the immune-deficient mouse model, we observe the sensitivity variation of CML-MSCs after treated TKIs. We believe this study will help elucidate the mechanism of exosmes secreted by CML-MSCs promoting CML cells resistant to TKIs and explore new intervention strategy of directing CML cells drug resistance. The expected results might also provide a new thought for the further investigation about targeted CML cells to restore the sensitivity of CML cells to TKIs.

慢粒白血病（CML）对酪氨酸激酶抑制剂（TKIs）耐药的发生与骨髓微环境中间充质干细胞（MSCs）联系密切，但具体机制未明。申请者前期发现：CML-MSCs外泌体较正常MSCs外泌体含有低表达的肿瘤抑制因子miR-15a，且其被CML细胞吞噬后可下调TKIs对后者诱导的凋亡，并伴Notch1-NF-κB表达上调。故推测：CML-MSCs外泌体可通过激活CML细胞内Notch信号通路，保护CML细胞存活，促进其对TKIs耐药。本项目拟利用共聚焦显微成像技术、qPCR、western blot、基因转染或干扰技术，分别从细胞、基因和蛋白水平，观察与MSCs外泌体共培养后CML细胞在体内外对TKIs敏感性及Notch信号分子的变化，阐明MSCs外泌体通过激活Notch信号通路促CML细胞对TKIs耐药的机制，初步探索CML对TKI治疗耐药的新靶点，为研究逆转其对TKIs耐药提供理论依据。

目前酪氨酸激酶抑制剂（tyrosine kinase inhibitor，TKI）在治疗慢性粒细胞性白血病（chronic myeloid leukemia，CML）上已取得了显著的效果，但有少部分患者对TKI原发耐药、还有部分患者在治疗过程中对TKI产生耐药导致疾病复发，这是治愈CML所面临需克服的重大难题。前期研究发现：CML-MSCs外泌体较正常MSCs外泌体含有低表达的肿瘤抑制因子miR-15a，且其被CML细胞吞噬后可下调TKIs对后者诱导的凋亡，并伴Notch1-NF-κB表达上调。故推测：CML-MSCs外泌体可通过激活CML细胞内Notch信号通路，保护CML细胞存活，促进其对TKIs耐药。本项目利用共聚焦显微成像技术、qPCR、western blot、基因转染或干扰技术，分别从细胞、基因和蛋白水平，观察与MSCs外泌体共培养后CML细胞在体内外对TKIs敏感性及Notch信号分子的变化，结果表明BMMSC-Exo通过转运miR-15a抑制慢粒增殖、将细胞周期阻滞在G0/G1期，并且体内外实验证明可诱导慢粒耐药，阐明MSCs外泌体促CML细胞对TKIs耐药的机制，初步探索CML对TKI治疗耐药的新靶点，为研究逆转其对TKIs耐药提供理论依据。