WWP2基因下调表达介导造血干细胞衰竭的分子调节机制研究

Aplastic anemia （AA）is a syndrome of bone marrow failure, and was considered to be relative to the intrinsic defects of hematopoietic stem / progenitor cells, functional defects of hematopoietic microenvironment and the abnormal immune response to injury hematopoietic stem / progenitor cells, but still no more comprehensive elucidated. Our previous studies suggest that AA may be a disease coeffected by the constitutive suppression of Akt signaling pathway,apoptosis and abnormal immunity activation in hematopoietic stem cells,which are mediated by the downgulation of gene expression induced by a variety of risk factors, and definitively leads to bone marrow failure. qRT-PCR and Western blot will be performed to detect the expression of WWP2 gene in bone marrow samples of AA .Epigenetics method will be used to analyze the molecular mechanisms of decreased expression of WWP2 gene. Knockout and over-expression of these genes mediated by lentiviral infection in hematopoietic stem cells from mice will be performed in vitro, and then analysis the biological properties of these transgenic cell lines, which will be transplanted to lethal doses of radiation BLAB/C mice. In vivo and vitro experiments and other methods will be applied to study the impact of these genes to hematopoietic reconstitution,and other molecular biology technology will be applied to study the molecular mechanisms and the relative signaling pathways of bone marrow failure. Our study will provide a theoretical foundation for new treatment targets and methods for AA.

再生障碍性贫血是骨髓造血衰竭性的疾病，一般认为与造血干/祖细胞内在缺陷、造血微环境功能缺陷和异常免疫反应损伤有关，但至今对其发病机制特别是分子机制仍无全面的阐明。我们的前期研究提示再障可能是由各种致病因素诱导的基因表达缺失所介导的骨髓造血干细胞中Akt信号通路抑制、细胞凋亡和免疫异常活化，从而造成骨髓造血细胞增生受限和免疫损伤，两者共同导致骨髓造血衰竭。本项目将通过QRT-PCR、Western blot检测再生障碍性贫血患者骨髓标本中WWP2与相关基因不同水平表达情况，应用表观遗传学和分子遗传学方法研究在再障骨髓细胞中WWP2基因表达缺失的分子机制，将siRNA和过表达慢病毒包装颗粒感染小鼠造血干细胞获得稳定表达细胞株，并移植至致死剂量BLAB/C小鼠，体内外实验研究WWP2基因缺失对小鼠的骨髓造血重建恢复影响以及分子机制和参与的信号通路，为再障的临床诊疗提供新的治疗靶点和理论基础。

获得性再生性障碍性贫血（AA）是一种骨髓衰竭性疾病，主要表现在骨髓造血干细胞(HSC)减少导致的外周全血细胞减少。免疫异常介导的细胞毒T细胞的活化，分泌相关细胞因子诱导造血干细胞凋亡是其主要病因之一。实验结果发现:(1)AA患者骨髓中WWP2和PTEN的表达水平明显低于正常组; (2)与WWP2-siRNA介导的PTEN和P73高表达组相比，WWP2过表达介导PTEN和P73表达下调，并诱导骨髓细胞G1期细胞比例增加，S期细胞比例下降，提示WWP2的表达可以诱导骨髓细胞G1/S细胞周期阻滞；(3) WWP2-siRNA介导的FasL的上调表达，可提高骨髓细胞的凋亡率，提示WWP2可能通过调控Fas/FasL途径，影响细胞凋亡进程；(4) WWP2过表达可上调CyclinD1的表达以及Akt磷酸化水平，可能是通过下调PTEN的表达所介导；(5)小鼠HSC移植后的骨髓造血重建监测结果提示，WWP2-siRNA组的小鼠外周血血象和骨髓象的增生情况，脾脏以及胸腺重量均低于WWP2过表达组，HE染色显示WWP2-siRNA组骨髓组织出现明显脂肪组织，提示WWP2表达异常可能介导骨髓衰竭。本研究初步确定WWP2的缺失表达将影响HSC移植小鼠骨髓的造血重建，并诱导骨髓衰竭。然而，WWP2是否通过对小鼠的骨髓免疫细胞的表型、活化和功能具有调控作用，从而影响骨髓造血重建，至今仍尚未阐明。下一步将分析移植小鼠的骨髓及外周造血组织中的免疫细胞表型和功能，探讨WWP2参与免疫调控及骨髓造血重建的作用机制，为临床的治疗提供理论依据。