骨髓基质干细胞EMT/MET表型转化在Hp慢性感染致胃癌发生中的作用

Although the novel concept on contribution of the bone marrow-derived mesenchymal stem cells(MSCs) in Helicobacter pylori-associated gastric cancer has been discussed quite promptly in recent years, the mechanism underlying this process remains controversial. Houghton et al have shown that MSCs could repopulate the gastric epithelium and progress to cancer. However, recent data has demonstrated that carcinoma associated fibroblasts (CAFs) in stomach, which promote gastric cancer progression, originated from MSCs. These clear evidences raised a model that MSCs contribute to gastric cancer as both epithelial and mesenchymal phenotype. Our previous data have investigated that MSCs present as two difference phenotype in vitro: epithelial-like MSCs(eMSCs, FSP-1(fibroblast specific protein-1)negtive expression) and fibroblast-like MSCs(fMSCs, FSP+), which might give an explanation for the phenotypic plasticity of MSCs shown in gastric cancer. Therefore, we address the hypothesis that the EMT/MET of MSCs effect on the inflammation-induced gastric tumorgenesis. The subsets of MSCs(eMSCs and fMSCs) were obtained through FACS-sorting with FSP1 and/or the induction of EMT/MET with TGF-β and BMP-7. The effect of MSCs-EMT/MET were evaluated by the ratio of eMSCs and fMSCs, the subset MSCs characters of proliferation, differentiation, immune-regulation, homing, interaction with epithelial cell and tumor formation. Wnt signaling pathway was investigated as the central regulation factor of EMT/MET, tumogenesis and MSCs self-balance. .To understand the real pathophysiology meaning of MSCs EMT/MET in gastric cancer holds the insightful therapeutic potential for gastric cancer patients.

骨髓基质干细胞(MSCs)参与慢性感染后胃癌发生是近年关于肿瘤形成的新观点，但具体机制尚不明确。现有研究表明MSCs可能以上皮细胞（肿瘤细胞）、间质细胞（构成肿瘤基质）两种不同的细胞类型参与肿瘤发生。本课题基于我们的前期研究结果"MSCs存在两种不同形态的细胞亚群- - 上皮样细胞及纤维样细胞"，借用在跨胚层分化以及肿瘤转移过程中均发挥重要作用的上皮细胞/间质细胞表型转化（EMT/MET）模型，首次提出MSCs表型转化参与胃癌发生的观点。通过体外分选或诱导混合培养模型，及尾静脉或胃肠道在体移植模型，以同时对EMT/MET转化、肿瘤发生及MSCs自调控具有通用性的Wnt信号通路为切入点，对不同亚群MSCs相互间的调控作用，两者独自或协作参与胃癌发生的分子机制及慢性感染微环境的作用进一步探索。MSCs作为一种良好的基因工程细胞，研究其表型转化在胃癌发生中的作用机制，可为胃癌的治疗提供新的思路。

骨髓间充质干细胞体外培养过程中可以发现两种不同形态的细胞亚群—上皮样细胞（Epithelia-like MSCs, eMSCs）及纤维样细胞（Fibroblast-like MSCs, fMSCs）。研究发现骨髓间充质干细胞与幽门螺旋杆菌感染下胃癌的形成相关，但eMSCs/fMSCs干细胞亚群细胞具体的生物学特以及各自在胃癌发生中的作用尚不明确。第一部分，本实验利用BMP-7体外诱导建立经典间质上皮表型转化（MET）模型，建立诱导模型后，以 PCR、Western blot、免疫荧光技术检测诱导前后骨髓间充质干细胞上皮/基质细胞表面标志物确认诱导转化效率。利用流式细胞术、Transwell、PCR、Western blot、免疫荧光等手段检测eMSCs/fMSCs优势转化亚群的细胞生物学特性，包括增殖能力、迁移能力、干细胞特性、Wnt通路和致瘤能力。第二部分，进一步研究了在幽门螺杆菌感染下eMSCs/fMSCs的生物学特性的变化，包括增殖能力、迁移能力和Wnt信号通路。并且在裸鼠体内建立前胃癌细胞和eMSCs/fMSCs移植模型以及慢性幽门螺旋杆菌感染balb/c小鼠移植eMSCs/fMSCs模型，探讨eMSCs/fMSCs两种不同亚群对胃癌发生发展的作用。结果显示MET的经典诱导剂BMP-7可以成功诱导骨髓间充质干细胞发生MET表型转化（上皮细胞的标志物E-cadherin表达升高，而间质细胞的标志物FSP-1和Vimentin的表达降低）。此外，eMSCs/fMSCs干细胞亚群表现出不同的生物学特性：相比较于fMSCs, 通过MET表型诱导的 eMSCs增殖能力增强、迁移能力减弱、Wnt通路激活、干细胞特性下降；在幽门螺杆菌感染下eMSCs/fMSCs的生物学特性不同：与fMSCs相比，eMSCs与幽门螺杆菌上清液共培养后增殖能力增强、迁移能力增强、Wnt通路激活更显著；动物模型证实eMSCs/fMSCs均能促进胃癌发生发展。由上述实验结果证实eMSCs /fMSCs干细胞亚群具有不同的生物学特性，均参与了胃癌的发生发展，并且部分通过Wnt信号通路实现。明确MSCs参与胃癌发生的统一机制，有望为寻找胃癌治疗靶点提供依据。