青黄散联合健脾补肾方对MDS患者HDAC1基因去甲基化介导的HIF-1/EPO信号通路调节机制研究

Myelodysplastic syndromes are a group of malignant clonal tumors. The symptoms of many MDS patients are anemia. With the development of epigenetics,The recent studies have shown that pathogenesis and progress of MDS is associated with tumor suppressor genes hypermethylation.The project is based on the clinical and mechanisms research of Qinghuang Power combined with tonifying kidney and strengthening spleen prescription therapy on MDS. We think that the possible mechanism for increasing hemoglobin levels in the treatment of MDS is to make HDAC1 gene demethylation and Restore HDAC1 gene expression which can increase HIF-1α gene transcription activity and HIF-1α mRNA content.As the result, The HIF-1α protein levels rise, and then regulates the HIF-1/EPO pathway using HIF-1α as the core,which can activate its target gene——EPO.In this trial,we can explore mechanisms of Qinghuang Power combined with tonifying kidney and strengthening spleen prescription in treating MDS by detecting HDAC1 gene methylation,HIF-1α mRNA expression, HIF-1α protein levels in bone marrow and EPO in peripheral serum before and after carrying out this therapeutic regimen,using the techniques and methods such as methylation specific PCR(MSP),Real-Time PCR(RT-PCR),Flow Cytometry(FCM)and Elisa.The purpose of our project is to investigate how the regimen can increase hemoglobin levels,reveal its modern molecular biology mechanism of "phlegm stasis,detoxification of evil, replenishing qi and blood, nourishing spleen and kidney".and provide scientific molecular basis for clinical using of this therapeutic regimen,to further enrich its theoretical connotation.

骨髓增生异常综合征是一组恶性克隆性肿瘤，患者临床表现多有贫血症状。研究认为MDS发病与进展与抑癌基因高甲基化有关。本项目在前期应用青黄散联合健脾补肾方治疗MDS研究基础上，提出其改善血红蛋白可能机制是通过降低HDAC1基因甲基化水平，恢复HDAC1基因表达和功能，增加HIF-1α基因转录活性，提高HIF-1α mRNA含量，使HIF-1α蛋白水平升高，进而调节以HIF-1α为核心的HIF-1/EPO通路，升高EPO效应发挥作用。本研究拟通过MSP、RT-PCR、FCM、Elisa等科学方法，检测青黄散联合健脾补肾方治疗前后30例MDS患者骨髓细胞的HDAC1基因甲基化、HIF-1α mRNA表达、骨髓细胞核内HIF-1α蛋白含量以及血清EPO水平变化，探讨其提升MDS血红蛋白机理，揭示本方案“化痰瘀、解毒邪、益气血、补脾肾”分子生物学机制，为临床应用本方案提供科学依据，丰富其理论内涵。

骨髓增生异常综合征（Myelodysplastic Syndrome，MDS）是起源于造血干细胞的恶性克隆性疾病，包括一组以骨髓无效造血、病态造血改变、克隆性染色体异常和高风险向急性髓性白血病转化的临床表现高度异质性疾病。MDS总体发病率随年龄的上升而呈逐步增高的趋势，在未来的老龄化社会中MDS将可能成为血液病常见病种之一。青黄散联合健脾补肾方治疗MDS具有肯定的临床疗效。在治疗MDS有效患者中，其外周血象以红细胞、血红蛋白恢复最快。.本课题通过提取MDS患者治疗前后骨髓细胞DNA，应用亚硫酸氢盐测序法检测HDAC1基因甲基化、RT-PCR检测HIF-1α基因在mRNA水平上的表达、流式细胞仪检测治疗前患者骨髓细胞核内HIF-1α蛋白表达量、Elisa方法检测MDS患者应用本方案治疗前后血清EPO表达含量，验证青黄散联合健脾补肾方通过降低HDAC1基因甲基化而调节以HIF-1α为核心的HIF-1/EPO通路发挥升血红蛋白效应的机制。.我们的研究发现：1、经青黄散联合健脾补肾方治疗前30例MDS骨髓标本中，HDAC1基因启动子区CpG_1、CpG_5、CpG_12、CpG_23位点呈高甲基化，分别为58.67%、85.67%、63.89%、71.63%，治疗后，上述HDAC1基因启动子区4个甲基化位点呈低甲基化状态，分别为0.67%、0%、1.33%、1.33%（P＜0.01）；2、HIF-1α mRNA相对表达量治疗后较治疗前增加了7.5倍（P＜0.01）。所有患者治疗后淋巴细胞、单核细胞、有核红细胞的HIF-1α平均荧光强度较治疗前增强（P＜0.05），表明HIF-1α蛋白较治疗前上升。3、应用Elisa方法检测治疗前后外周血EPO的表达，结果表明治疗治疗前后EPO的表达量未见明显变化，差异无统计学意义（P>0.05）。.实验结果表明青黄散联合健脾补肾方通过降低MDS患者HDAC1基因高甲基化状态从而增加下游HIF-1αmRNA表达发挥提高外周血红蛋白作用，而HIF-1下游其中一个靶基因EPO的表达含量未受青黄散的影响。.所以，未来的研究可对HIF-1下游靶基因进行更加深入分析，以探索青黄散影响HIF-1的其他信号通路。