蛋白磷酸酶2A调控外源性化学物致血液毒性的机制研究

To date, a comprehensive description of key events or pathways involved in the environmental chemicals induced hematotoxicity is limited. The early biomarkers for prediction of hematotoxicity induced by low dose chemical exposure remain to be explored. Protein phosphatase 2A (PP2A) participates in regulating the phosphorylation process of key points in the signal pathways, and may also plays an important role in the process of exogenous chemical induced cytotoxicity. This research is designed to screen the key events or signaling pathways mediated by PP2A during the exogenous chemicals caused hematotoxicity by the gene knockout animal model and in vitro cell models. In our previous studies, we have established a specified bone marrow granulocyte knockout mouse model targeting PP2A A alpha subunit. With low dose benzene exposure, we found the homozygousgene knockout animal model showed an observably change in the differentiation and proliferation ability of hematopoietic stem cells. This result illustrates that the change of the PP2A activity may regulate the hematopoietic damage caused by benzene. Therefore, we intend to take this animal model combined with HL60 cell line and the primary cultured bone marrow cells isolated from gene knockout mouse, to reveal the key points or pathways in the process of chemicals induced hematotoxicity. In addition, we attempt to clarify the phosphorylation sites in order to provide new molecular targets for the prevention or therapy of chemicals caused hematological disorders and to identify early biomarkes for biological monitoring for chemicals exposure and hematological damage.

目前环境化学物致血液毒性的机制研究中缺乏对作用靶点和关键通路的全面描述，低剂量暴露致血液毒性的早期损伤标志物仍有待明确。蛋白磷酸酶2A可调控信号通路中关键分子的去磷酸化，并在外源化学物诱导细胞损伤过程中起重要作用。本课题旨在利用基因定位敲除动物及体外细胞模型筛查影响化学物致血液毒性的关键通路。应用已成功构建的骨髓粒细胞PP2A Aα基因敲除小鼠模型，我们在前期研究发现纯合子基因缺失小鼠低浓度苯暴露可引起造血干细胞增殖分化能力的改变，说明PP2A酶功能改变可调控苯导致的血液毒性。本项目拟选取六种代表性血液毒性化学物，利用骨髓粒细胞PP2A Aα敲除小鼠模型、基因敲除小鼠的原代培养骨髓细胞和Aα缺失的人HL-60细胞，结合免疫共沉淀和磷酸化蛋白芯片技术揭示化学物致血液毒性的关键通路和作用靶点，并明确蛋白磷酸化的位点，为化学物致血液系统疾病的防治提供新靶点，寻找血液毒性生物监测的早期生物标志物。

环境化学物致血液毒性的机制研究中缺乏对作用靶点和关键通路的全面描述，低剂量苯暴露致血液毒性的早期损伤标志物仍有待明确。蛋白磷酸酶2A可调控信号通路中关键分子的去磷酸化，并在外源化学物诱导细胞损伤过程中起重要作用。此外，外源化学物致血液毒性的早期通常伴随有氧化应激加重的现象，Nrf2是氧化应激过程的关键分子。因此，本研究通过利用蛋白磷酸酶2A敲除的小鼠及Nrf2基因敲除的小鼠，对苯致血液毒性的调控机制进行研究，并进一步通过收集职业苯暴露人群，对动物及细胞实验结果进行验证。结果发现低剂量苯暴露可诱导小鼠及职业苯暴露人群的红细胞计数升高，白细胞计数无明显变化，并激活CYP450代谢酶表达；但高剂量的苯暴露会导致动物体内的代谢酶表达、白细胞计数显著降低。通过基因敲除小鼠研究，我们发现PP2A-B56α，β-连环蛋白和Cyp2e1的级联调节可参与苯诱导的血液毒性；苯可诱导Nrf2-KO小鼠细胞毒性和氧化应激加剧，且Nrf2敲除还可干扰造血干细胞的增殖和分化。这对于我们制定苯的职业接触限值及探讨其致病机制和治疗方案时具有一定的参考意义。