Netrin-1通过上调β3整合素表达激活下游FAK信号通路促进T-ALL细胞中枢神经系统浸润的研究

T acute lymphoblastic leukemia (T-ALL) was a malignant with poor prognosis. Central nervous system (CNS) infiltration is nervous symptom common in T-ALL due to the brain invasion of leukemic cells and CNS infiltration was demonstrated as a risk factor to poor prognosis of T-ALL patients. Netrin-1-integrinβ3-FAK pathway has been demonstrated an important mechanism for invasion and metastasis in many malignant. Previous experiments showed serum netrin-1 was high-regulated in the T-ALL patients with CNS infiltration which suggests a potential role of netrin-1 in brain invasion of T-ALL leukemic cells. Also, the results showed that netrin-1 could up-regulate the expression of integrin β3 and the phosphorilation of FAK. This study aims to investigate the mechanism of netrin-1-integrinβ3-FAK in promoting T-ALL CNS infiltration. We will analyze the adhesion and invasion promoting effect of netrin-1 in T-ALL leukemic cell line Jurkat and MOLT4. Further we will detect the expression of integrin β3, MMPs and also the phosphorilation of FAK pathways to explore the pathway through which netrin-1 regulate the leukemic cell infiltration. This mechanism will be further identified by the applying of integrin β3 interferred cells and FAK inhibitor treatments. In vitro Blood Brain Barrier (BBB) model will be constructed to identify this mechanism through which T-ALL cells infiltration through BBB. Then T-ALL mice model constructed in immune-deficiency mice will be applied to demonstrate the promotion effect of netrin-1-integrinβ3-FAK pathway on CNS infiltration of T-ALL cells. This study will provide new mechanism for understanding of T-ALL CNS infiltration and suggest new targets for the therapy to manage this pathological process.

T急淋是预后不良的恶性肿瘤疾病，中枢神经系统浸润是T急淋的不良预后因素。轴突导向因子-1（Netrin-1）-整合素-黏着斑激酶（FAK）是参与多种恶性肿瘤的侵袭和转移的重要通路。预实验结果提示netrin-1在T急淋中枢浸润患儿外周血高表达，并可以上调β3整合素表达和FAK活性。本课题拟阐明netrin-1上调β3整合素表达并活化FAK信号促进T急淋中枢浸润的机制。通过重组netrin-1的处理，分析其对T-ALL细胞侵袭能力的影响，进而分析下游β3整合素，MMP2/9的表达和FAK通路磷酸化水平，探索netrin-1调控机制。应用β3整合素干扰细胞和FAK抑制剂验证该调控机制。利用体外血脑屏障模型和免疫缺陷小鼠白血病模型探索并验证该机制对中枢浸润的影响。从而为理解T急淋中枢浸润提供新的机制，为进一步治疗T急淋中枢浸润提供新的靶点和靶通路。

Netrin-1是神经诱导因子家族的重要成员，其参与多种肿瘤的发展和转移过程，研究者前期发现netrin-1可以调控integrinβ3表达进而影响T-ALL细胞迁移。基于此，我们对netrin-1通过integrinβ3调控T-ALL细胞迁移能力的机制进行了探索和验证。我们分析了netrin-1对integrinβ3及其下游FAK-MAPK信号途径的影响，但我们发现integrinβ3-FAK-MAPK信号途径并不直接参与netrin-1对于T-ALL细胞迁移能力的影响，于是我们通过IP检测了netrin-1在T-ALL细胞上的受体并检测了其他下游信号途径的磷酸化水平，发现neogenin1及PI3K-AKT信号途径参与该过程，并在细胞水平和体外血脑屏障模型中验证了这一调控机制。该研究结果提示在白血病细胞增殖、迁移的调控过程中，netrin-1具有很强的浓度依赖性，通过在不同浓度差异结合受体启动差异下游信号途径从而实现增殖和迁移的分别控制，为T-ALL细胞浸润中枢神经系统提供了潜在的机制参考。

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