Notch信号通路在ALDH1+/Musashi+胆囊癌干细胞中功能研究

Gallbladder cancer is common primary malignant tumor of the digestive system. The full understanding of incidence and metastasis mechanisms is largely lacking. Cancer stem cells (CSCs) are cancer cells that possess characteristics associated with normal stem cells, specifically the ability to generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Our previous studies found that ALDH-1, Musashi-1 may serve as markers of cancer stem cells and its exprssion levels higly correlated with gallbladder cancer staging and patient prognosis. Human primary gallbladder cancer cells were sorted by Flow cytometry and can be cultured, passaged in vitro. The ALDH1+ subpopulations were confirmed maintainance of stem cell properties both in vitro and in vivo. A preliminary study shown that Notch signaling mechanisms may play an important role in gallbladder adenocarcinoma tumor stem cell. In current project, we will use gallbladder adenocarcinoma cancer stem cells as model to in-depth study how Notch signaling pathway regulating CSCs self-renewal , cell proliferation, invasion and migration in vitro and in vivo. By carring out this project, we will finally figure out ALDH1+/Musashi+ can be used as markers of gallbladder cancer CSCs and their molecular mechanisms , suggesting the potential therapeutic targets and diagnostic biomarkers for clinical diagnosis and treatment of gallbladder cancer.

胆囊癌是较常见的恶性消化系统原发肿瘤，发病和转移机制有待研究。肿瘤干细胞为肿瘤组织中仅有极少数具有成瘤潜能的细胞，具有自我更新和增殖等干细胞特性。前期研究发现肿瘤干细胞分子标志物ALDH-1、Musashi-1表达水平与胆囊癌的病理分期和病人预后相关。流式细胞分选获得原代人胆囊癌细胞，并能体外稳定传代，初步体外体内试验证实分选获得的该细胞亚群具有肿瘤干细胞特性。初步机制研究发现Notch信号通路可能在胆囊腺癌肿瘤干细胞生物学功能中扮演重要角色。本项目应用胆囊腺癌肿瘤干细胞为模型，深入研究Notch信号通路对干性维持与自我更新，细胞增殖、侵袭、迁移能力及体外成瘤能力的功能影响。研究内容可望阐明ALDH-1和Musashi-1可作为胆囊癌中CSCs标记物以及其分子机制，发现潜在的治疗靶标和诊断的生物标志物，有望为临床诊断和治疗提供新策略和方法。

本研究通过CD133磁珠分选出CD133+胆囊癌干细胞，通过流式检测发现ALDH1、Musashi1在CD133+胆囊癌干细胞中高表达，因此我们以CD133+、ALDH-1+、Musashi-1+胆囊癌干细胞为模型，研究ALDH-1、Musashi-1和Notch信号通路对胆囊癌干细胞的影响。研究发现胆囊癌干细胞的增殖、迁移、侵袭、成球及体外成瘤能力均强于胆囊癌细胞，干性相关基因在胆囊癌干细胞中的表达高于胆囊癌细胞。通过RNA干扰，发现ALDH-1和Musashi-1可以促进囊癌干细胞的增殖、迁移、侵袭、成球及体内成瘤。通过Notch抑制剂LY-411575处理胆囊癌干细胞，发现LY-411575可以抑制胆囊癌干细胞的增殖、迁移、侵袭、成球及体内成瘤，促进细胞凋亡。通过RNA干扰分别敲减Jagged1、DLL4、Hes1和Hey1，发现胆囊癌干细胞的增殖、迁移、侵袭、成球及体外成瘤能力均受到抑制；细胞凋亡增加。在胆囊癌干细胞敲减ALDH-1或Musashi-1后，发现Hes1和Hey1的表达下调。为了验证ALDH-1和Musashi-1是否通过Hes1和Hey1影响胆囊癌干细胞的特性，在已敲减ALDH-1或Musashi-1的胆囊癌干细胞中，分别过表达Hey1或Hes1，比较胆囊癌干细胞的生物学特性变化。研究发现在已敲减ALDH-1或Musashi-1的胆囊癌干细胞中分别过表达Hey1或Hes1，胆囊癌干细胞的增殖、侵袭、迁移、成瘤能力增强，细胞凋亡减少，这提示ALDH-1和Musashi-1通过Notch信号通路影响胆囊癌干细胞的生物学特性。通过WB和qRT-PCR法检测Notch信号通路相关基因、ALDH-1和Musashi-1在胆囊癌癌和癌旁组织中的表达，结果显示这些基因在胆囊癌组织中的表达均高于癌旁组织。通过免疫组化法研究Jagged1、DLL4、Notch1、Notch3、Hes1和Hey1在胆囊鳞癌/腺鳞癌和胆囊腺癌中的表达及其临床病理意义，研究发现Jagged1、DLL4、Notch1、Notch3、Hes1和Hey1的阳性表达与胆囊癌的临床病理特征密切相关。本研究发现ALDH1和Musashi1可以作为胆囊癌干细胞的标志物，ALDH-1和Musashi-1通过Notch信号通路影响胆囊癌干细胞的生物学特性。

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