目的：本论文深入探究黎药海南地不容抗乳腺癌活性化合物氧化克班宁对微管网络的破坏能力，研究氧化克班宁在分子层面和细胞层面对微管网络稳态的影响。方法：首先采用EBI位点竞争法和分子对接来确定氧化克班宁对微管位点的占据力；其次，采用Western Blot法检测STAT3、PKA1、CAMK4和PKA 检测氧化克班宁对微管蛋白的影响。结果：EBI位点竞争法结果显示，EBI与β⁃微管蛋白加合物比β⁃微管蛋白出现在分子量更小的区域（ctrl2）。分子对接结果显示，氧化克班宁可以占据微管蛋白的秋水仙碱位点。Western Blot结果显示，低、中、高3个浓度的氧化克班宁或阳性药Taxol作用MCF⁃7细胞48 h后，显著降低STAT3蛋白表达水平（P<0.01），显著降低PKA1和CAMK4蛋白表达水平（P<0.05，P<0.01），并且可降低PKA蛋白表达水平，与对照组差异均有统计学意义（P<0.01）。结论：氧化克班宁结合在微管蛋白秋水仙碱位点扰乱微管蛋白聚合，造成MCF⁃7细胞有丝分裂，从而导致MCF⁃7细胞死亡。氧化克班宁可通过抑制MCF⁃7细胞中STAT3、PKA1、CAMK4和PKA蛋白的表达水平，干扰纺锤体形成，最终造成MCF⁃7细胞有丝分裂灾难。

Objective： To determine the destructive ability of oxocrebanine， an anti⁃breast cancer active compound obtained from Stephania hainanensis H. S. Lo et Y. Tsoong， on microtubule network， and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels. Methods： the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine. Western Blot was used to detect the effect of oxocrebanine on microtubule⁃associated proteins including STAT3， PKA1， CAMK4， and PKA. Results： The results of EBI site competition assay showed that the binding of EBI to β⁃Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight than β⁃tubulin （ctrl 2）. Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins. As revealed by Western Blot， the expression of STAT3 protein was decreased after MCF⁃7 cells have been treated with low， medium， and high concentration of oxocrebanine or the positive drug taxol for 48 h （P <0.01）. The expression levels of PKA1 and CAMK4 proteins aslo showed significant reductions （P<0.05， or P<0.01）. Oxocrebanine also decreased the PKA protein in MCF⁃7 cells compared to the control group （P<0.01）. Conclusion： Oxocrebanine， a ligand that binds at the colchicine site of tubulin， perturbs tubulin polymerization and causes mitosis in MCF⁃7 cells， thus leading to MCF⁃7 cell death. Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3， PKA1， CAMK4， and PKA proteins in MCF⁃7 cells. Oxocrebanine interfers with spindle formation， and ultimately causes mitotic catastrophe in MCF⁃7 cells.

海南省自然科学基金项目（820RC776）