α-SMA（+）肌成纤维细胞在胃癌组织肿瘤干细胞向循环肿瘤干细胞转化中的作用及机制的研究

The existence of cancer stem cell and circulating cancer stem cell which play an important role in the carcinogenesis and progress of tumor have been proved in the previous researches. The circulating cancer stem cell could originate from tissue cancer stem cell through epithelial-mesenchymal transition (EMT). We have captured the gastric tissue cancer stem cell and circulating cancer stem cell previously and also demonstrated that the lung metastatic ability of circulating cancer stem cell was higher than that of tissue cancer stem cell, with the higher expression of AKT in circulating cancer stem cell. Researches have found myofibroblasts involving EMT closely could contribute to carcinogenesis and expansion of gastric cancer through SDF-1/CXCR4 signal pathway, in which AKT is an important member of downstream molecules. On account of these, the effect of myofibroblasts in the transfomation of gastric tissue cancer stem cell to circulating cancer stem cell and whether myofibroblasts promote the transformation process through SDF-1/CXCR4 signal pathway need to be resolved in this research. The research aims to investigate the role and mechanism of myofibroblasts in the transfomation of gastric tissue cancer stem cell to circulating cancer stem cell by RT-PCR, Western blot, immunofluorescence and RNA interference, which might show the theoretic basis of anti-metastasis comprehensive therapies targeting transfomation process of gastric tissue cancer stem cell to circulating cancer stem cell.

研究已证实肿瘤干细胞及循环肿瘤干细胞的存在，其在肿瘤发生、发展过程中起着重要作用。循环肿瘤干细胞可通过EMT过程来源于组织肿瘤干细胞。我们前期已同时分离出胃癌组织肿瘤干细胞及循环肿瘤干细胞；同时发现组织肿瘤干细胞的肺转移能力明显低于循环肿瘤干细胞，AKT在循环肿瘤干细胞中高表达。研究表明与EMT关系密切的肌成纤维细胞可主要通过活化SDF-1/CXCR4轴导致胃癌的发生发展，而AKT为该轴重要下游分子。基于此，肌成纤维细胞是否能促进胃癌组织肿瘤干细胞向循环肿瘤干细胞转化？是否可能经SDF-1/CXCR4通路促转化？均为本课题拟解决的关键问题。本研究拟通过RT-PCR、免疫印迹、免疫荧光和RNA 干扰等研究肌成纤维细胞在胃癌组织肿瘤干细胞向循环肿瘤干细胞转化中所起作用及其机制。本项目或能为进一步探索组织肿瘤干细胞向循环肿瘤干细胞转化的机制，通过靶向该过程进而抑制胃癌转移提供理论基础。

肿瘤干细胞被认为是肿瘤发生发展的根源，微环境对肿瘤干细胞的生物学作用十分复杂。肿瘤相关成纤维细胞（CAF）微环境中的重要组分，其对胃癌肿瘤干细胞的影响还不清楚。本课题旨在研究CAF对胃癌肿瘤干细胞侵袭迁移的影响及其分子调控机制。通过原代培养分离并鉴定了CAF，发现CAF高表达FN而胃癌肿瘤干细胞不表达FN；与CAF共培养后，胃癌肿瘤干细胞侵袭迁移能力显著提高，但EMT表型无明显改变；通过基因芯片检测和生物信息学分析发现与CAF共培养后，胃癌肿瘤干细胞中Laminin-332表达增高，且Laminin-332可促进胃癌肿瘤干细胞侵袭迁移，同时上调p-AKT的表达，而PI3K抑制剂可抑制胃癌肿瘤干细胞的侵袭迁移。Laminin-332中的亚基LAMA3过表达后，胃癌肿瘤干细胞的侵袭迁移能力提高，且LAMA3高表达与胃癌患者疾病进展和不良预后显著相关。而FN可促进胃癌肿瘤干细胞侵袭迁移及上调LAMA3的表达。本课题初步发现CAF可能通过分泌FN，促进胃癌肿瘤干细胞LAMA3的表达，并通过PI3K介导促进胃癌肿瘤干细胞的侵袭迁移，为进一步了解胃癌肿瘤干细胞的侵袭迁移分子机制及其与微环境的关系提供了实验依据。