BMSCs转染原癌基因ras向肺癌干细胞分化及黄芪的干预作用研究

Bone marrow mesenchymal stem cells (BMSCs) is one of the important cell such as lung cancer microenvironment, it is a great significance to prove its genetic stability and its relevance to tumor stem cells for the prevention and treatment of cancer. Radix Astragali has the function of supplementing qi and nourishing essence, supporting healthy energy, BMSCs is the embodiment of the bone marrow nutrients at the cellular level. In previous studies, the genetic stability of BMSCs in lung cancer microenvironment was significantly increased by Radix Astragali. It is based on carcinogenesis induced by ras gene mutation in C. elegans and will set up a co-culture model of typeⅱalveolar epithelial cells ( AEC) and BMSCs transfected by ras gene. Using FCM、CCK-8、and confocal microscopy to detect cytoskeleton morphology and proliferation of BMSCs; Using RT-PCR、immunohistochemistry and western blot to test Occludin and CK-18 on AEC、CD133 and CD44 on lung cancer stem cells、ras and myc gene in lung cancer cells; The formation of tumor cells in nude mice was observed; The effects of cytoskeleton morphology and proliferation of Radix Astragali on BMSCs were also tested. That will provide a scientific basis for the prevention and treatment of tumors in Radix Astragali, the ras mutation of BMSCs and the association of lung cancer and lung cancer stem cells.

骨髓间充质干细胞（BMSCs）是肺癌等组织微环境中重要细胞之一，探明其遗传稳定性及其与肿瘤干细胞关联性对肿瘤防治研究具有重要意义。黄芪具有益气生精、扶正固本作用，BMSCs是骨髓精气在细胞层面的体现，课题前期研究证实黄芪可显著提高肺癌细胞微环境中BMSCs遗传稳定性。本项目在前期ras基因突变诱发线虫癌变的基础上，以ras基因转染BMSCs，并与肺上皮细胞共培养，利用FCM术、CCK8、激光共聚焦法等检测BMSCs细胞骨架形态、增殖性；采用RT-PCR、免疫组化及Western blot等技术检测Occludin和CK-18等肺上皮细胞标记物、CD133和CD44等肿瘤干细胞标记物、及ras和myc等基因及蛋白的表达；观察裸鼠实验分化细胞的成瘤性；并进一步利用黄芪干预观察对上述指标的影响；以期为BMSCs的ras突变与肺癌及其肺癌干细胞关联性，以及黄芪通过扶正固本防治肿瘤提供科学依据。

背景：肺癌中的肿瘤微环境在肿瘤的进展和转移中起著重要的作用。前期研究表明与 A549 肺癌细胞共培养的骨髓源间充质干细胞（BMSCs）显示出形态变化，细胞增殖增加和细胞迁移。本课题旨在探讨BMSCs在肺癌微环境中的变化以及中药黄芪多糖（APS）对骨髓间充质干细胞变化的调控作用。一方面：中药黄芪多糖（APS）对 A549肺癌细胞体外诱导的骨髓间充质干细胞变化的影响；另一方面：原癌基因转染对BMSCS的影响。.材料/方法：BMSCs与A549细胞（Co-BMSC）共培养。将共培养的BMSCs和经50μg/ml APS（Co-BMSCs + APS）处理的A549细胞与未处理的Co-BMSCs行比较。使用细胞计数试剂盒8（CCK-8）测定法测量细胞增殖。流式细胞仪评估了细胞周期。使用用于mRNA表达的微阵列测定和用于蛋白表达的蛋白质印迹。将携带原癌基因Kras、CMYC、Bmi-1质粒载体进行慢病毒包装后，转染BMSC。.结果：与未处理的Co-BMSCs相比，APS处理Co-BMSCs可改善细胞形态，减少细胞增殖，并抑制细胞周期停滞。有丝分裂原活化蛋白激酶（MAPK）/核因子-κB（NF-kB）途径，TP53，caspase-3，乙酰化H4K5，乙酰化H4K8和乙酰化H3K9 参与了调控过程。转染后的实验组H9772，H9773，H9775的细胞增殖能力有所提高，此研究有待进一步深入探讨，后续研究成果将不断发布。.结论：APS可以逆转Co-BMSCs中异常的形态变化，增加细胞增殖和细胞周期停滞。这些作用受有丝分裂原激活的蛋白激酶（MAPK）/核因子-κB（NFkB）途径，TP53，caspase-3，乙酰化的 H4K5，乙酰化的H4K8 和乙酰化的 H3K9 调节。APS处理对A549肺癌细胞体外诱导的BMSCs 的细胞增殖和形态变化具有保护作用。