基于microRNA差异表达的桂枝茯苓丸加味治疗子宫肌瘤的分子机制

Ovarian steroid hormone and abnormal cell proliferation and apoptosis played a key role in evolving process of uterine fibroids in which microRNA was the bridge between the two previously mentioned. The literature and our preclinical study indicated that Guizhi Fuling pill was effective in the treatment of uterine fibroids, so we hypothesizes that Guizhi Fuling pill exerts it's therapeutic effect by intervening estrogen and progesterone levels, changing expression of microRNA, affecting proliferation and apoptosisin of uterine leiomyoma cells in hysteromyoma patients. This study uses techniques of gene chip, realtime quantitative PCR, western-blotting and immunohistochemistry, and study the intervention of herbal medicine on microRNA by clinical study and animal experiments. It validates the objectivity of this hypothesis from the whole - cell - gene - protein levels to expound the molecular mechanism of this classical prescription in the treatment of uterine fibroids, provide the scientific basis of it's clinical application and promotion, and reveal in part the scientific connotation of blood-activating and mass-eliminating method treating uterine fibroids. Similar reports have not seen inland.

卵巢甾体激素和细胞增殖、凋亡异常在子宫肌瘤的发生、发展中起了关键作用，microRNA 是子宫肌瘤发病过程中连接卵巢甾体激素与细胞增殖、凋亡异常的"桥梁"。文献及我们的前期临床研究发现，桂枝茯苓丸（加味）能有效治疗子宫肌瘤，因此提出"桂枝茯苓丸加味可能通过干预子宫肌瘤雌、孕激素水平，改变microRNA的表达，影响肌瘤细胞的增生和凋亡而发挥治疗作用"假说。本课题将通过临床基础及动物实验研究，采用基因芯片、实时定量PCR、Western-blotting、免疫组化等技术，通过中药对microRNA途径的干预研究，从整体－细胞-基因-蛋白等层次验证该假说的客观性，以阐释经方治疗子宫肌瘤的分子机制，为经方的临床应用和推广提供科学的依据，从一个侧面揭示"活血消癥"治法治疗子宫肌瘤的科学内涵。国内未见同类报道。

卵巢甾体激素和细胞增殖、凋亡异常在子宫肌瘤的发生、发展中起了关键作用，microRNA 是子宫肌瘤发病过程中连接卵巢甾体激素与细胞增殖、凋亡异常的“桥梁”，桂枝茯苓丸是治疗子宫肌瘤的著名经方，对处方进行优化可缩小子宫肌瘤体积。本课题通过临床基础及动物实验研究，采用基因芯片、实时定量PCR、Western-blotting、免疫组化等技术，通过中药对microRNA途径的干预研究，从整体－细胞-基因-蛋白等层次验证该假说的客观性，以阐释经方治疗子宫肌瘤的分子机制。.临床研究表明桂枝茯苓丸加味可显著降低子宫肌瘤患者的的肌瘤月增长体积（p＜0.05）。血清中有28个 microRNA表达量发生显著变化，经生物信息学分析得到的信息通路中有4条与子宫肌瘤密切相关，分别是MAPK信号通路、Wnt信号通路、mTOR信号通路、PI3K-Akt信号通路。动物实验研究表明，与模型组相比，中药大、小剂量组、预防给药组、西药组子宫湿重、子宫分脚下最大直径显著减小（p＜0.01），中药小剂量组、预防性给药组分脚横径显著减小（p＜0.05）。中药大、小剂量组、预防性给药组子宫形态明显接近正常组。与模型组相比，中药小剂量组、预防给药组血清E2R水平明显下降（p＜0.05）；模型组子宫局部miR-187-3p、miR-330-5p表达上调。对生物信息学分析MAPK信号通路的靶基因进行检测，与模型组相比，中药大、小剂量组Ras、PKC、Bax基因表达显著升高，中药大剂量组Raf1、EGF、EGFR、SRF、TNF-α基因表达均显著降低；预防给药组Bax基因表达显著升高，中药大、小剂量组、预防性给药组GCK、BCL-2基因表达显著降低。中药大剂量组、预防给药组Raf蛋白表达明显下降（p＜0.05），中药大、小剂量组、预防给药组、西药组E2R蛋白表达均显著下降，而Bax蛋白表达明显升高（p＜0.01或p＜0.01），中药小剂量组、预防性给药组、西药组PR、BCL-2蛋白表达明显降低（p＜0.01或p＜0.01）。.结论：桂枝茯苓丸加味具有阻断子宫肌瘤增长的作用，其机理与该药影响相关的micRNA及相关信号通路的靶基因的表达、促进细胞凋亡有关。