SCR-3在雌激素促巨核细胞分化中的作用及机制研究

It has been studied that estrogen can promote megakaryocytes differentiation and platelets production, but its mechanism is still unclear. SRC-3 is an important co-activator of estrogen receptor (ER), and SRC-3-/- mice displayed significantly reduced peripheral blood platelets. Bioinformatics analysis shows that there exists binding sites with higher scores between ER and the promoter regions of GATA-1 which participates in the regulation of differentiation and maturation of megakaryocytes. Meanwhile, our previous study also found that estrogen could significantly increase the expression of GATA-1 in megakaryocytes, while this effect was blocked when SRC-3 was knocked out. These suggest that ER may promote differentiation and maturation of megakaryocytes through regulating the transcription of GATA-1, and SRC-3 may exert an important role in this progress. Therefore, in this study, by application of MACS, flow cytometry, IP and ChIP, we will firstly illustrate the role of SRC-3 in the promotion of megakaryocytes differentiation and maturation by estrogen. Then we plan to explore whether ER can combine with the promoter of GATA-1 to regulate its transcription, and the role of SRC-3 in the transcription regulation of GATA-1 by ER. The aim of this study is to clarify the mechanism of the regulation of megakaryocytes differentiation by estrogen, and to provide theoretical basis for its clinical application as well.

已有研究表明雌激素能够促进巨核细胞分化、血小板生成的作用，但其机制不明。SRC-3是雌激素受体（ER）的重要辅活化因子，SRC-3-/-小鼠其外周血血小板水平显著降低，多倍体受到抑制。生物信息学预测显示ER与巨核细胞成熟分化调控因子GATA-1的启动子区存在得分较高的结合位点，我们前期研究也发现雌激素能够显著上调GATA-1的表达，而SRC-3缺失会阻断雌激素对GATA-1的表达调控作用，提示ER可能通过调控GATA-1的转录而促进巨核细胞分化与成熟，SRC-3在此过程中可能发挥着重要的调节作用。为此，本项目拟采用免疫磁珠分选、IP、ChIP等技术，首先明确SRC-3在雌激素促巨核细胞分化成熟中的作用，然后分析ER是否能够与GATA-1启动子结合并调控其转录，并探讨SRC-3在ER调控GATA-1转录生成的作用，从而深入阐明雌激素调控巨核细胞分化的作用机制，为其临床应用提供理论依据。

已有研究表明雌激素能够促进巨核细胞分化、血小板生成的作用，但其机制不明。SRC-3是雌激素受体（ER）的重要辅活化因子，SRC-3-/-小鼠其外周血血小板水平显著降低，多倍体受到抑制。生物信息学预测显示ER与巨核细胞成熟分化调控因子GATA-1的启动子区存在得分较高的结合位点，我们前期研究也发现雌激素能够显著上调GATA-1的表达，而SRC-3缺失会阻断雌激素对GATA-1的表达调控作用，提示ER可能通过调控GATA-1的转录而促进巨核细胞分化与成熟，SRC-3在此过程中可能发挥着重要的调节作用。为此，本项目采用免疫磁珠分选、荧光素酶表达系统、IP、ChIP、SiRNA等技术，利用人脐血原代巨核细胞、小鼠原代巨核细胞以及不同时期巨核细胞株为模型，首先证实了雌激素受体β（ERβ）是雌激素促进巨核细胞分化的主要受体，运用siRNA技术，反向证实了雌激素通过ERβ促进巨核细胞的多倍体形成；明确了GATA1是雌激素作用后表达最明显的基因，采用生物信息测序分析技术和ChIP技术证实ERβ可以直接与GATA1的启动子区域结合，同时干扰GATA1后，能完全抑制雌激素促巨核细胞多倍体形成能力；进一步证实SRC-3在雌激素促巨核细胞分化成熟中的作用，然后分析ER能够与GATA-1启动子结合并调控其转录，进一步调节STAT1促进巨核细胞血小板前体形成和多倍体的形成，并探讨SRC-3在ER调控GATA-1转录生成的作用，深入阐明雌激素调控巨核细胞分化的作用机制，为其临床应用提供理论依据。。