益髓颗粒调控DNA甲基化治疗骨髓增生异常综合征分子机制研究

Aberrant DNA methylation is currently known as an important pathogenetic mechanism of myelodysplastic syndrome (MDS) worldwide.Wnt signaling plays an essential role during the onset of MDS, and the aberrant DNA methylations of its key genes are prognostic factors of MDS. Our previous studies confirmed that "qi and yin deficiency, combined with blood stasis" was the major clinical syndrome of MDS patients. Yi Sui Granules (YSG), manufactured by our hospital, have the function of supplementing qi, nourishing yin and promoting blood circulation by removing blood stasis according to traditional Chinese medicine. Clinically, YSG was proved safe and effective in treating patients with MDS, delaying progresses into AML, improving quality of life and prolonging overall survival. Basic research also showed YSG had the effects of improving dysplasia by inhibiting proliferation and promoting differentiation, and regulating Wnt signaling. Our preliminary studies indicated that the DNMTs mRNA level was reduced in patients benefiting from YSG treatment, which indicated demethylation might be the mechanism of YSG’s efficacy in treating MDS. Therefore, in this project the rat model of MDS will be established, YSG will be administered, peripheral and bone marrow smears will be observed, and the expression and DNA methylation level of the key genes(including Wnt3a, FZD, β-catenin, TCF, SFRP) in canonical Wnt signaling will be detected in order to explore, from a DNA methylation point of view, the mechanism of YSG in treating MDS.

目前国际上公认，DNA甲基化异常是骨髓增生异常综合征（MDS）的重要发病机制。Wnt通路与MDS发病密切相关，其关键基因甲基化异常直接影响MDS预后。我们既往临床观察发现，“气阴两虚、血瘀内阻”是MDS临床主要病机，具有“益气养阴活血”功效的院内制剂“益髓颗粒”能够延缓MDS转化为白血病时间、提高患者生活质量与生存期，并有改善骨髓病态造血（抑制增殖、促进分化）、调节Wnt通路等作用。同时，益髓颗粒治疗有效的MDS患者DNA甲基转移酶（DNMTs）水平下调，表明益髓颗粒治疗MDS获效机制可能与去甲基化有关。因此，本课题将构建MDS大鼠模型，以益髓颗粒为干预药物，通过观察血象与骨髓缓解情况，检测DNMTs表达水平、DNA甲基化水平、Wnt通路关键基因Wnt3a、FZD、β-catenin、TCF、SFRP表达及甲基化水平，从DNA甲基化角度探讨益髓颗粒治疗MDS疗效机制。

去甲基化药物（HMA）明显提高了骨髓增生异常综合征（MDS）临床疗效，但不能显著延长患者生存期。具有“益气养阴活血”功效的“益髓颗粒”临床治疗MDS安全有效，为阐明其作用机理，我们构建并验证SKM-1裸鼠移植瘤模型，应用不同剂量益髓颗粒及HMA干预，观察裸鼠一般状态、体重变化、生存期及移植瘤生长情况，并采用BSAS、Real-time PCR、Western blot、免疫荧光等方法检测DNA甲基转移酶（DNMT）表达水平、Wnt 信号通路相关基因或蛋白表达及甲基化水平。研究首次发现：（1）益髓颗粒高剂量可抑制SKM-1移植瘤生长，其机理可能与单纯去甲基化作用不同；（2）与HMA相比，益髓颗粒具有更好的安全性；（3）与HMA相似，益髓颗粒可下调DNMT表达，发挥抑制MDS作用，且其作用具有剂量依赖性，去甲基化可能是益髓颗粒抑制MDS作用机理之一；（4）SFRP5基因DNA异常甲基化与MDS不良预后密切相关，益髓颗粒剂可能通过抑制DNMT1表达，降低SFRP5基因甲基化程度，上调SFRP5 mRNA及蛋白的表达，通过竞争性阻碍Wnt3a与受体结合，从而降低了 Wnt 信号通路异常激活；另一方面益髓颗粒剂可直接下调Wnt配体的表达，使得Wnt配体与其受体结合减少，进而阻止Wnt信号通路的激活；最终导致β-catenin被降解蛋白复合体磷酸化而降解，使得进入细胞核中的β-catenin蛋白减少，导致C-myc、CyclinD1等靶基因表达降低，而发挥抑制MDS移植瘤生长作用的。（5）与HMA不同，益髓颗粒可延长荷瘤裸鼠生存期，其机理与抑制MDS移植瘤生长机制不同，值得深入探索。