软坚散结中药抗肿瘤的生物力学机制研究

According our previous study, the softening and resolving hard mass drugs were demonstrated could soften tumor and reduce the tumor' density by inhibiting tumor angiogenesis, lymphangiogenesis and interstitial fibrosis. The increasing rigidity of tumor matrix improves the contractility and proliferation activity of tumor cells and promotes the proliferation and metastasis of tumor by impacting the integrin-induced force-chemical signaling conduction and cell signaling transmission. Therefore, we proposed the softening and resolving hard mass drugs could inhibit tumor growth and metastasis by reducing the rigidity of tumor matrix and changing the biomechanics property and force-chemical signaling conduction of tumor cells. This study measures the content of fibers, changing of the integrin-induced Rac1/Cdc42, RhoA and Ras pathways as well as the biomechanics properties like the enduring mechanical force, cellular adherence, deformation, motion, cytoskeleton, migration and invasion of tumor cells by co-culturing the tumor cells and cancer-associated fibroblasts. The nude mice transplanted models were established to measure the rigidity of tumor tissue, tumor inhibition rate, number of metastatic sites, the content of proteins in the integrin-induced signaling pathways and the content of fibers in the matrix. This study links the softening biological activity with resolving biological activity of the softening and resolving hard mass drugs organically and reveals the scientific connotation of traditional Chinese medicine theory via advanced technology of biomechanics.

前期研究我们发现软坚散结中药能抑制肿瘤血管、淋巴管生成与间质纤维化，导致肿瘤密度降低。肿瘤基质硬度的增加通过整合素介导的力-化学信号转换与细胞信号传递，引起细胞骨架重排，细胞收缩和增殖活性增强，促进肿瘤细胞增殖与迁移。据此我们提出软坚散结药物通过降低肿瘤组织的硬度，改变其生物力学性质与力-化学信号传导，抑制肿瘤生长与转移。本研究通过肿瘤细胞与肿瘤相关成纤维细胞共培养，检测纤维含量及整合素介导的RhoA/ROCK、Rac1/Cdc42与Ras通路改变，同时检测肿瘤细胞所受的机械力与细胞粘附、变形、运动、细胞骨架等生物力学性质，以及细胞增殖、迁移与侵袭能力。建立裸鼠移植瘤模型，测量肿瘤硬度，计算抑瘤率与转移灶数目，检测整合素介导的信号蛋白表达和基质纤维含量。本研究将软坚散结药物的"软坚"与"散结"功能有机联系，利用生物力学的前沿手段揭示传统中医理论的科学内涵。

前期研究我们发现软坚散结中药能抑制肿瘤血管、淋巴管生成与间质纤维化，导致肿瘤密度降低。软坚散结中药守宫中的守宫硫酸糖肽（GSPP）可以降低乳腺癌肿瘤微环境中bFGF的水平并增加TGF-β的水平。本研究中我们发现，利用细胞因子TGF-β1和bFGF受体阻滞剂，活化肿瘤细胞TGF-β通路的同时抑制bFGF通路可以诱导肝癌细胞向良性成纤维细胞方向分化，并且能抑制肿瘤细胞的生长和迁移。此外，研究还发现GSPP不仅能够抑制肿瘤细胞的增殖和侵袭，还能通过抑制肝癌细胞整合素通路相关蛋白表达，发挥对整合素通路上生物力-化学信号转导的影响，进而影响细胞生物力学特性和细胞骨架蛋白的重组和分布，使得细胞形变、迁移以及侵袭能力下降，从而发挥抑制肿瘤转移的作用。体内和体外实验证明，软坚散结中药瓜蒌中的有效成分葫芦素B能有效地抑制乳腺癌细胞的迁移和侵袭。进一步研究表明葫芦素B下调了乳腺癌细胞中F-actin/vimentin/FAK的表达，改变了骨架蛋白在细胞内的分布。此外，葫芦素B能够抑制整合素下游Rho GTPases家族和RAC1/Cdc42/RhoA的信号转导。综上所述，软坚散结中药对于肿瘤细胞生物力学特性的改变，为 “软坚”的肿瘤治疗思路提供了新的实验证据，为软坚散结中药的实验和临床应用提供了新的思路。