胃癌微环境MSC调节中性粒细胞极化作用及分子机制

We have demonstrated that human bone marrow-derived mesenchymal stem cells (BM-MSC) and their derived conditioned medium and microvesicles can favor the growth of gastric cancer in vivo. We have recently isolated MSC-like cells from H. pylori-positive gastric cancer tissues and found that the gastric cancer tissue derived MSC are more activated than those from adjacent normal tissues and bone marrow, which possess the typical characteristics of carcinoma-associated fibroblasts (CAF). Thus, it deserves further investigation to elucidate the correlation between cancer derived MSC and cancer development and progression. This project is designed to study the roles of gastric cancer MSC in modulating tumor microenvironment, with special focus on tumor-associated neutrophils. Cytokine array will be performed to screen the differentially expressed cytokines and chemokines between gastric cancer and adjacent normal tissue derived MSC. The candidate cytokines and chemokines will be validated in in vitro cell model and in vivo animal model. The key molecules and signaling pathways by which tumor MSC-derived cytokines and chemokines mediate the polarization of neutrophils to acquire a protumor phenotype will be further investigated. The proposed study will help understand the roles and mechanisms of action of MSC in gastric cancer and will provide novel targets for anti-gastric cancer therapy.

我们发现骨髓间质干细胞(BM-MSC)及其培养上清(CM)、分泌的微囊泡(MVs)可以在体内促进胃癌生长。我们从H. pylori阳性的胃癌组织中分离获得MSC, 并发现胃癌MSC较骨髓和癌旁组织来源的MSC更为活化，具有肿瘤相关成纤维细胞的性质。胃癌来源的MSC与胃癌发生和恶性转化的关系值得研究。本课题以胃癌来源MSC为研究对象，以中性粒细胞为切入点，研究在慢性炎症中MSC参与调节肿瘤微环境的作用。我们采用Luminex液相芯片筛选，在体外细胞模型和体内肿瘤模型中应用分子干预技术加以验证，确定胃癌MSC调节中性粒细胞促瘤表型极化的关键分子及其作用靶点。本研究将为阐明MSC在胃癌发生中的作用和分子机理提供新的证据，同时也为寻找新的胃癌治疗靶点提供实验依据，具有重要的科学意义。

项目研究背景：.本项目主要探讨胃癌微环境中的间质干细胞（GC-MSCs）对中性粒细胞极化的调节作用、机制及生物学意义，为揭示MSCs通过调控中性粒细胞促进胃癌的作用提供理论基础和实验依据。.项目研究内容：.采用GC-MSCs条件培养基处理中性粒细胞，MTT和流式细胞术检测中性粒细胞存活和凋亡情况；实时荧光定量PCR检测中性粒细胞IL-8、OSM、CCL2和TNF-α基因表达情况；检测中性粒细胞对胃癌细胞迁移能力和内皮细胞管形成能力的影响。Western blot检测中性粒细胞STAT3和ERK信号通路活化情况。采用小分子抑制剂阻断STAT3或ERK信号通路活化，检测对中性粒细胞凋亡、活化和功能的影响。采用Luminex液相芯片筛选GC-MSCs调节中性粒细胞的关键因子，并用实时荧光定量PCR和ELISA进行验证。采用特异性抗体进行阻断，检测对GC-MSCs活化中性粒细胞的影响。采用活化的中性粒细胞上清作用于正常MSCs，western blot和免疫荧光检测其CAFs相关标志物表达，并评价作用后的MSCs对胃癌细胞迁移能力的影响。.项目关键数据：.GC-MSCs条件培养基促进中性粒细胞存活，并抑制其自发性凋亡，上调其IL-8、CCL2和TNFα等炎症因子基因表达。GC-MSCs条件培养基活化的中性粒细胞显著促进胃癌细胞迁移和内皮细胞管形成。GC-MSCs条件培养基主要通过激活中性粒细胞STAT3-ERK1/2信号通路而活化中性粒细胞。阻断STAT3-ERK1/2信号通路显著抑制GC-MSCs条件培养基对中性粒细胞凋亡保护、炎症因子基因表达诱导和促胃癌细胞迁移和内皮细胞管形成作用。Luminex分析揭示GC-MSCs分泌高水平的IL-6。拮抗IL-6/IL-6R作用明显抑制GC-MSCs对中性粒细胞的功能活化。GC-MSCs活化的中性粒细胞上清能诱导正常MSCs向CAFs分化并促进胃癌细胞迁移。.项目科学意义：.GC-MSCs通过IL-6-STAT3信号通路调节中性粒细胞表型改变和功能活化，且活化的中性粒细胞诱导正常MSCs向CAFs分化。GC-MSCs与中性粒细胞的双向调节作用共同促进胃癌进展。本研究揭示了MSCs在胃癌微环境中的新作用，为全面认识MSCs与胃癌发生发展提供了新证据，同时也为胃癌的干预治疗提供了潜在靶点。