从组蛋白乙酰化调控细胞凋亡角度探讨骨髓增生异常综合征中医“正虚”、“瘀毒”证型特点的表观遗传学机制

Abnormal epigenetics regulation is the important pathogenesis of Myelodysplastic Syndrome(MDS).It is essential to find effective and safe interventions of epigenetics regulation by the current clinical research.In recent years,some clinical studies have confirmed the effect of the Chinese Medicine(TCM) in MDS.In order to further investigate the targeting effect in epigenetics regulation of TCM in MDS,we will clarify the correlation between abnormal epigenetics regulation and TCM syndromes features of Vital Qi Deficiency and Stasis Toxin,which will prove the scientific connotation of TCM syndromes transformation and therapeutic principles.Our previous studies have indicated the Chinese Medicine syndrome of MDS,Vital Qi Deficiency and Stasis Toxin,which is correlated with western medicine risk degrees and dysregulated apoptosis.This time,we will examine acetylation sites and enrichment of genome-wide histone H3 and H4 of the normal and the MDS of different risk degree by chip-seq,which is to analysis the dynamic changes of acetylation,risk degree,and malignancy trend of MDS.Then we will observe the different therapeutic mechanisms with the dynamic changes of the Chinese medicine recipe with different composition of the 'vital-Qi' and the 'eliminating evil' medicine, all that is the TCM syndromes transformation theory on MDS,Vital Qi Deficiency and Stasis Toxin,explained by the epigenetics mechanism.

表观遗传学异常是MDS发病的重要病理机制，寻找有效、安全的改变患者表观遗传表达的干预措施是当前临床研究的重要方向。临床已证实"健脾补肾、活血解毒"中药治疗MDS具有良好疗效，为深入探讨其在改善患者表观遗传学方面的靶向作用，阐释异常表观遗传学环节与中医"正虚"、"瘀毒"证型特点的内在联系，证明MDS证型演变的科学内涵和相应的中医治则治法机理，本研究在既往MDS中医"正虚"、"瘀毒"证型演变与西医危度分层、凋亡机制相关性研究的基础上，以"组蛋白乙酰化介导的细胞凋亡通路"为切入点，应用chip-seq技术分析正常人、不同IPSS危度分组MDS全基因组组蛋白H3和H4发生乙酰化修饰的位点及富集程度，分析其与MDS危度分层、恶性程度及转恶趋势相关的动态性变化，观察不同"扶正"、"祛邪"药物构比对此特征性动态变化的差异作用，用以方测证法阐释MDS中医"正虚瘀毒"证型演变理论的表观遗传学机制。

表观遗传学异常是MDS重要发病机制。本研究采用免疫磁珠分选骨髓CD34+细胞,运用荧光比色法、组蛋白试剂盒、RT-PCR、流式细胞仪检测等方法从TLR2mRNA/βarrestin 1/组蛋白乙酰化通路机制分析不同危度、不同中医分型MDS组蛋白乙酰化、基因转录、细胞凋亡水平的差异性。研究分析结果显示：1.低中危组凋亡率、TLR2mRNA均明显高于中高危组及正常组（P<0.01）；低中危组TLR2mRNA与凋亡率表达呈正相关；低中危组β-arrestin1mRNA表达及组蛋白H4乙酰化水平均高于中高危组（P<0.01），H4乙酰化水平与凋亡率、TLR2表达呈正相关。研究证实TLR2mRNA高表达水平可能促进MDS骨髓 CD34 +细胞凋亡，TLR2/βarr1/组蛋白乙酰化主要介导低中危MDS细胞凋亡。2.体外研究表明TLR2激动剂（MALP2）处理CD34+细胞组蛋白乙酰转移酶p300mRNA的表达明显增加（P＜0.01）；βarr1shRNA组p300mRNA的表达下降程度明显高于MALP2+βarr1shRNA组（P＜0.01）；各组CBPmRNA表达无统计学差异（P>0.05）。结果证明MALP2激活TLR2上调βarr1mRNA表达，促使其向细胞内转移, 使p300mRNA的表达水平增高，增加H4乙酰化及相关靶基因转录进而影响细胞凋亡。3.正虚型MDS TLR2 mRNA表达与细胞凋亡水平呈正相关，说明TLR2mRNA高表达促进正虚型MDS骨髓 CD34 +细胞凋亡；正虚型MDS的HAT活性水平较高，其上调介导了MDS凋亡增加，随着疾病进展后期“瘀毒型”MDS TLR2、β-arrestin1、H4乙酰化水平相对减少。本研究证实低中危组乙酰化水平高于中高危组，组蛋白去乙酰化酶抑制剂可用于MDS的治疗。βarr1 mRNA表达与TLR2mRNA其表达水平在MDS不同危险度分组之间表达存在差异，且有相同的进展趋势，可能与MDS危险程度分级以及疾病进展密切相关；证明TLR2mRNA/βarrestin 1及组蛋白乙酰化相关指标所体现的疾病内在病理机制与中医证型的关系，进一步充实MDS中医证治规律研究。