从炎性信号通路失衡研究肺癌微环境中MSCs恶性转化的分子机制及黄芪调控作用研究

Bone marrow mesenchymalstem cells (MSCs) is one kind of ideal stem cells in tumor cytological therapy, safety is concerned increasingly in the tumor microenvironment. Subject group preliminarily confirmed co-culture of MSCs and lung cancer A549 cells can produce stable genetic change. Based on the theory that the inflammatory signaling pathway imbalance can induce carcinogenesis and theory that Astragalus can culture normal Qi to expel evilness, co-culture system MSCs and different lung cancer cell lines is to be established with Transwell, technology such as biochip is to be utilized to screen for key inflammatory factors that induce malignant transformation of MSCs and for inflammatory signaling pathway, and lung cancer-associated inflammatory factor culture system is to be simulated. Protective effect of effective Astragalus parts on malignant transformation of MSCs induced by inflammatory cytokines and its regulation of expression imbalance in promoting / inhibiting malignant factors in NF-κ B and STAT3 inflammatory pathway will be analyzed by means of protein chip, confocal laser cell analysis, cell-to- tumor experiments. The molecular mechanism of Astragalus in prevention and treatment of tumor inflammatory microenvironment to induce stable genetic change of MSCs through Astragalus function in culturing normal Qi to expel evilness, regulating intracellular environment to get balance of yin and yang will be revealed. The theoretic and experimental basis of combining Astragalus and MSCs in treating tumor and regulating inflammatory signaling pathway imbalance will be provided to prevent malignant change of MSCs.

骨髓间充质干细胞（MSCs）是肿瘤细胞生物学治疗的理想干细胞之一，但其在肿瘤微环境中的安全性问题日渐受关注，课题组初步已证实MSCs与肺癌A549共培养可发生恶性变化，但其机制尚待探明。本项目基于炎性信号通路失衡可诱发癌变及黄芪的扶正祛邪等理论，以Transwell小室建立MSCs与不同类型肺癌细胞株共培养体系，利用生物芯片等技术筛选诱发MSCs恶变的关键性炎性因子（或组合）及其信号通路，模拟建立诱发MSCs恶变的肺癌相关炎性因子培养体系；结合蛋白芯片、激光共聚焦技术、细胞成瘤实验等分析黄芪有效部位对炎性因子诱发MSCs恶性转化的抑制作用及其对NF-κB等炎性信号通路中促/抑恶变因子表达失衡的调控作用；揭示黄芪通过扶正祛邪、调控细胞内环境阴平阳秘而抑制炎性微环境诱发MSCs恶性变化的分子机制，并为黄芪联合MSCs防治肿瘤以及以调控细胞炎性信号通路失衡为靶点防治MSCs恶变提供理论与实验依据

项目背景：. BMSCs的优质特性为其在临床广泛应用创造良好条件，但其所处微环境发生变化时（如炎症、肿瘤等）是否仍然能保持遗传稳定与定向分化争议较多。如何在确保安全的前提下高效地开展BMSCs的临床应用是亟待解决的问题。课题组初步已证实MSCs与肺癌A549共培养可发生恶性变化，但其机制尚待探明。.主要研究内容：. 建立诱发BMSCs恶变的肺癌共培养微环境并检测相关炎性因子，模拟建立诱发培养恶性转化的炎性微环境体系，研究炎性微环境下BMSCs形态、增殖、周期、免疫表性、TAFs相关蛋白、遗传稳定性等变化，分析mRNA表达谱的变化，观察黄芪多糖对BMSCs 恶性转化的干预作用，并从IL-6、TNF-α通路揭示黄芪多糖发挥防护作用的可能调控机制。.重要结果和关键数据 ：. 结合课题组前期研究基础，利用生物信息学分析方法，运用炎性因子IL-6和TNF-α模拟建立诱发BMSCs稳定性下降的炎性微环境体系。研究结果显示，IL-6和TNF-α可诱导BMSCs出现形态、细胞周期、TAFs蛋白表达水平等多种生物学特性变化；CCK-8法、流式细胞术、激光共聚焦显微技术等方法检测显示免疫表性维持能力降低、缝隙连接通讯能力下降和异常增殖等；定向诱导分化实验检测显示成骨、成脂定向分化能力下降；HE染色观察细胞表现异型性；Western blot 技术检测肿瘤相关基因p53、PTEN、Ras、C-myc表达水平明显变化；RT-PCR及激光共聚焦显微技术检测干性基因表达水平出现明显变化；动物实验未见移植细胞成瘤；基因芯片技术检测分析mRNA表达谱发生显著改变；Western blot 技术检测IL-6/JAK、TNF-α/NF-KB、Wnt等信号传导通路中关键基因JAK2、IKKβ、P-GSK-3β、β-Catenin、GSK-3β等蛋白的表达出现明显变化。黄芪多糖对BMSCs出现生物学稳定性下降的显著变化发挥防护作用。其保护机制可能与其调控IL-6/JAK、TNF-α/NF-KB、Wnt等信号通路有密切关联。.科学意义：. 本课题通过研发以信号通路为调控手段维持BMSCs干性稳定及预防其恶性转化的生物制剂，以保障和促进BMSCs的临床应用更加安全、有效，为推动中医药与BMSCs 联合应用提供了理论依据和实践参考。