趋化因子CCL19/受体CCR7轴介导活化成纤维细胞促进云锡矿粉诱发肺癌演进的作用及机制

In the process of tumor development，the interaction between tumor cells and stromal cells is concerned. Stromal fibroblast has become a new target for cancer research and anti-cancer therapy. Our previous study found that activated fibroblast is an important factor that affects progression of Yunnan tin mine dust-induced lung cancer and this is related closely with chemokine CCL19 and receptor CCR7. In this research, we will establish the models of Yunnan tin mine dust-induced activated lung fibroblasts, transformated lung epithelia and their co-culture. Through antibody blocking, the role of CCL19/CCR7 axis in progression of Yunnan tin mine dust-induced lung cancer promoted by activated fibroblasts will be studied. Phosphorylation levels of associated kinase and transcription factors will be detected by protein microarray hybridization and Western blot. After blocking the signal pathways with kinase-specific antagonist, the active state of the signal transduction pathways initiated by CCL19/CCR7 axis will be analyzed. Immunohistochemistry will be used to detect the changes of expression and/or phosphorylation of CCL19, CCR7 and other associated factors in tree shrew animal experiments specimens and clinical specimens. Through the above study, we will interpret the molecular mechanisms of carcinogenesis induced by Yunnan tin mine dust from the perspective of the tumor stroma, reveal the intrinsic relationship between the pulmonary fibrosis and lung cancer，explore a new way to block and/or reverse carcinoma progression through antagonism and intervention of chemokine/receptor signal pathways.

肿瘤演进过程中，肿瘤细胞与基质细胞的相互作用备受关注。实体瘤中含量丰富的肿瘤基质成纤维细胞已成为研究肿瘤发生发展和治疗的新靶标。我们前期研究发现活化成纤维细胞是影响云锡矿粉诱发肺癌演进的重要因素，并与趋化因子CCLl9及其受体CCR7密切相关。本研究拟通过建立矿粉诱导肺成纤维细胞活化、肺上皮细胞转化及共培养模型，以抗体阻断等技术探索CCLl9/CCR7轴介导活化成纤维细胞促进矿粉诱发肺癌演进的作用；蛋白芯片杂交等技术检测相关激酶及转录因子磷酸化水平，分析相关信号转导途径的活性状态及关键节点，激酶特异拮抗剂阻断相关信号通路功能，探索肺上皮转化过程中CCLl9/CCR7信号转导机制；并在矿粉诱发树鼩肺癌动物模型及云锡矿工肺癌临床标本层面进行验证。从肿瘤基质角度阐明矿粉诱癌机理并诠释肺纤维化与肺癌之间的内在关系及其分子事件，探索通过干预趋化因子与其受体的相互作用以阻断和/或逆转肿瘤演进的方法。

肿瘤演进过程中，肿瘤细胞与基质细胞相互影响，相互作用。实体瘤中含量丰富的肿瘤基质成纤维细胞已成为研究肿瘤发生发展和治疗的新靶标，其确切的分子机制尚不明了。本研究通过体外细胞培养模型, 建立矿粉诱导肺上皮细胞恶性转化、肺成纤维细胞活化及上皮细胞和成纤维细胞共培养模型，模拟体内矿粉诱癌过程中上皮细胞－基质细胞的相互作用。采用抗体阻断技术、芯片杂交、免疫印迹技术等探索CCLl9/CCR7轴介导活化成纤维细胞促进云锡矿粉诱癌演进的作用；同时，我们以近灵长类攀鼩目小型哺乳动物——树鼩为对象，建立矿粉诱癌模型，模拟肺癌的动态发生发展过程。并应用云锡矿工肺癌临床标本进行验证。结果发现：云锡矿粉可以诱导肺成纤维细胞表达α-SMA及分泌CCL19；云锡矿粉可以诱导肺泡Ⅱ型上皮细胞转化及表达CCR7，CCR7中和抗体可延缓矿粉诱导上皮细胞转化时间及减弱上皮细胞增殖活性；CCL19可上调云锡矿粉打击上皮细胞中Akt的磷酸化水平及Bcl-2蛋白表达水平，下调Bax蛋白表达水平； CCR7中和抗体处理后，可抑制CCL19提高上皮细胞的Akt磷酸化及Bcl-2；趋化因子受体CCR7在多种肺癌细胞株及肺癌组织中存在高表达； CCL19促进肺癌细胞的迁移及侵袭能力， 低表达CCR7减弱CCL19的促进作用；CCL19不影响A549细胞的增殖；高表达 CCL19抑制了A549失巢性凋亡；CCL19下调A549细胞上皮标志物E-cad的表达，同时上调间质标记物 snail、N-cad及Akt磷酸化的表达，MK2206可抑制CCL19提高A549细胞Akt的磷酸化水平及snail、N-cad的表达；CCL19及CCR7在淋巴结转移组肺癌中的表达较正常组及非淋巴结转移肺癌组中的表达高；树鼩支气管灌注云锡矿粉悬液后肺组织经历：肺部炎症反应→肺间质纤维增生→肺支气管黏膜上皮和肺泡上皮增生→非典型增生→癌变的过程。以上结果说明趋化因子CCL19及其受体CCR7与肺癌细胞的迁移侵袭密切相关，其可能通过配体受体结合，激活肿瘤细胞内Akt信号通路，诱导上皮-间质转化，介导肿瘤细胞发生侵袭和转移。 研究提示趋化因子CCL19及受体CCR7是调控肺癌细胞侵袭转移的一个关键因素，其机制可能与PI3K/Akt通路活化有关。从趋化因子角度诠释了肿瘤细胞侵袭转移的分子机制，为临床肺癌转移防治提供了新的思路。

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