温肾壮骨方干预BMSCs促肿瘤EMT化抗乳腺癌骨转移的机制研究

Bone is the most common site of breast cancer metastasis (its incidence rate reach 70%). Bone metastasis is associated with reduced survival and impaired quality of life due to skeletal-related events. Bone marrow-derived mesenchymal stem cells (BMSCs) in tumor microenvironment can promote breast cancer cell invasion and metastatic potential by inducing epithelial-to-mesenchymal transition (EMT). BMSC-tumor cell interaction is considered a target for drug intervention. Our study group proposes that “kidney-deficiency and bone marrow-empty” is the key pathogenesis of breast cancer bone metastasis. Clinical practice and experimental research demonstrated that Wenshen-Zhuanggu Formula could inhibit BMSC-tumor cell interaction, reduce osteolytic bone destruction, and prevent the development of bone metastases in patients with breast cancer. Based on previous studies, the project will establish co-culture system of BMSCs with breast cancer cells and the nude mice model of breast cancer bone metastasis, perform the techniques of cytobiology, molecular biology, LC-MS/MS analysis, investigate inhibitory effect and mechanism of Wenshen-Zhuanggu Formula on the process of BMSCs inducing EMT in breast cancer cells, and reveal its main material basis for therapeutic potentials and pharmacokinetic properties which will suggest some new ideas and evidences to explain the biological characteristics of “kidney-deficiency and bone marrow-empty” theory and illustrate the scientific connotation of “warming kidney and activating yang, resolving masses and relieving pain” method.

骨是乳腺癌细胞优先转移的部位（70%发生率），骨转移导致的骨相关事件严重影响乳腺癌患者的生活质量。失巢的骨髓间充质干细胞（BMSCs）诱导肿瘤细胞上皮间充质转化（EMT），促进其侵袭和转移。BMSCs与肿瘤细胞的相互作用成为药物干预肿瘤转移的潜在靶点。“肾虚髓空”是乳腺癌骨转移的关键病机，前期研究显示，临床有效方—温肾壮骨方能显著抑制BMSCs与乳腺癌细胞间相互作用，阻止骨转移发展。本项目以BMSCs和乳腺癌细胞共培养体系及乳腺癌骨转移裸鼠模型为研究对象，应用细胞生物学、分子生物学、LC-MS/MS等技术，以TGF-β/Smads信号途径为切入点，研究BMSCs诱导乳腺癌细胞EMT的机理，探索温肾壮骨方干预BMSCs促肿瘤EMT化的作用靶点及分子机制，揭示温肾壮骨方药效物质基础及体内药代动力学规律，探讨乳腺癌骨转移“肾虚髓空”理论的现代生物学特征，阐释“温肾通阳、散结止痛”治法的科学内涵。

乳腺癌居中国女性肿瘤发病率首位，骨转移严重危害乳腺癌患者的生活质量。“肾虚髓空”是乳腺癌骨转移的关键病机，失巢的骨髓间充质干细胞（BMSCs）能诱导肿瘤细胞EMT的发生进程。本项目以TGF-β/Smads信号途径为切入点，研究BMSCs诱导乳腺癌细胞上皮间充质转化（EMT）的机理，探索临床有效方—温肾壮骨方干预BMSCs促肿瘤EMT化的作用靶点及分子机制，揭示复方的药效物质基础及体内药代动力学规律，探讨乳腺癌骨转移“肾虚髓空”理论的现代生物学特征，阐释“温肾通阳、散结止痛”治法的科学内涵。研究结果表明：（1）BMSCs与不同乳腺癌细胞直接共培养能显著促进其生长、侵袭及迁移能力；（2）BMSCs能通过分泌TGF-β降低乳腺癌细胞上皮表型蛋白、升高间质表型蛋白及转录因子Twist、Slug、Snail、Zeb2、Zeb1 mRNA的表达，促进肿瘤EMT的发生；BMSCs能够上调MDA-MB-231BO乳腺癌细胞中TGF-βRІ、Smad4、pSmad2、pSmad3蛋白表达水平；（3）BMSCs/MDA-MB-231BO细胞共注射构建乳腺癌裸鼠骨转移模型；温肾壮骨方能减少骨转移灶数目，减轻骨中肿瘤负荷，保护骨结构及降低溶骨性损伤，起到显著的治疗作用；温肾壮骨方能够逆转骨转移灶中上皮-间质表型蛋白的表达及干预TGF-β/smad信号途径，阻止BMSCs介导的乳腺癌细胞EMT进程；（4）与正常动物比较，温肾壮骨方中补骨脂素、异补骨脂素、蛇床子素、花椒毒素、欧前胡素、佛手柑内酯6种香豆素成分在病理模型动物体内吸收速度加快但吸收量较少，同时体内清除加快。推测可能与乳腺癌骨转移导致的胃肠道功能紊乱及胃肠道菌群失调有关。本研究证实了BMSCs在乳腺癌细胞EMT启动程序中具有重要作用；温肾壮骨方发挥抗骨转移效应的分子机制与逆转BMSCs诱导乳腺癌细胞EMT进程有关；温肾壮骨方中活性成分的药代动力学结果为深入探讨温肾壮骨方抗骨转移的效应物质基础及指导中药复方临床治疗剂量提供有价值的信息。