PDGF/PDGFR于原发性血小板增多症发病机制中作用及机理的研究

Platelet-derived growth factor (PDGF) and its receptor (PDGFR) has special affinity with megakaryocytes (MKs) and platelets. Our previous data showed that PDGF potently enhanced the recovery of MK and platlets in the radiation-induced myelosuppression mouse model, and this effect may be induced by the activation of PDGFR and the subsequent PI3K/AKT signal pathway, which lead to a reduction of MK apoptosis. However, the exact role of PDGF/PDGFR in MK/platelets related disease, for example, essential thrombocythemia (ET) was still unknown. .Recently, researches demonstrated that the expression level of PDGF increased remarkably in ET patient-derived serum, suggesting a potential relation between the PDGF over expression and ET development. In this study, we'd like to set up the PDGF-overexpression mouse model, from which the proliferative and differentiation function of MK and the amount of peripheral platelets will be determined. We also plan to establish the PDGF-overexpression mesenchymal stromal cell model which will be co-cultured with the CHRF-288-11 MK cells. CHRF cells will then be collected and the phosphorylation level of PI3K/AKT and JAK/STAT on these cells will be detected by flow cytometry and western blot. The tyrosine kinase inhibitor, imatinib mesylate will be given to the mouse model. The MK/platelet function will then be tested. Although there were some data regarding the application of imatinib on BCR/ABL positive ET patients, none of them was targeting on PDGFR. Results from this study will be the first to point out the role of PDGF/PDGFR in ET development and the potential molecular mechanism. More importantly, PDGFR, but not BCR/ABL will be recognized as the molecular drug inhibition target for imatinib in ET patient, and the PDGF plasma level will be considered as a new ET diagnostic index, making much more benefit for the patients.

血小板衍生生长因子（PDGF）及其受体PDGFR被证实与巨核细胞（MK）/血小板关系密切，我们的前期工作表明PDGF显著地促进骨髓抑制小鼠中MK及血小板计数恢复，但此通路在MK及血小板相关疾病，如原发性血小板增多症（ET）中的作用尚不明确。研究表明PDGF在ET患者血清中表达显著增高，提示PDGF的高表达与ET发病有潜在的联系。本项目拟在PDGF超表达的小鼠及体外模型上，观察MK及血小板功能及下游的分子信号通路；并予酪氨酸激酶抑制剂伊马替尼灌饲小鼠，观察MK及血小板功能变化。虽有报道使用伊马替尼治疗BCR/ABL阳性的ET患者，但无一针对PDGFR。通过此研究，我们将首次证实PDGF/PDGFR在ET发病中的作用及机制；确定PDGFR，而非BCR/ABL为伊马替尼治疗ET的分子靶点，并将PDGF血清表达水平作为诊断及评价ET疗效的新指标，使更多的ET患者得到有效的诊疗。

血小板衍生生长因子（PDGF）及其受体PDGFR被证实与巨核细胞（MK）/血小板关系密切，我们的前期工作表明PDGF显著地促进骨髓抑制小鼠中MK及血小板计数恢复，但此通路在MK及血小板相关疾病，如原发性血小板增多症（ET）中的作用尚不明确。本研究旨在研究PDGF/PDGFR于原发性血小板增多症发病中的作用，并探索针对该机制的治疗方法。用ELISA方法检测ET患者及健康人的骨髓血清中PDGF-BB的表达水平；用流式细胞技术检测PDGFR-β(CD140)在ET患者及健康人骨髓细胞的表达；建立PDGF-BB高表达的动物模型，观察其对外周血血小板的影响，并对比加入PDGFR抑制剂伊马替尼后，巨核细胞/血小板计数的变化；用流式细胞及western blotting技术分别检测PDGF-BB对JAK2/STAT3及PI3K/AKT信号通路的活化作用，并观察伊马替尼对该作用的影响。本研究结果提示,PDGF在ET患者骨髓血清中表达水平显著高于健康患者；且ET患者骨髓中PDGFRβ的表达显著高于健康患者；高浓度的PDGF明显促进小鼠体内血小板的生成，而伊马替尼可以阻断该作用；在体外实验中，PDGF可刺激巨核细胞JAK/STAT及PI3K/AKT信号通路的活化，且该促进作用可被伊马替尼阻断。结论：PDGF-BB表达水平的异常增高可能是JAK2/V617F突变阴性的原发性血小板增多症发生的机制之一；其作用机理可能是通过结合PDGFR，顺而激活JAK/STAT及PI3K/AKT信号通路，刺激巨核细胞的增殖和抑制其凋亡；而酪氨酸激酶抑制剂可通过阻断PDGFR，发挥治疗原发性血小板增多症的作用。通过此研究，我们将首次证实PDGF/PDGFR在ET发病中的作用及机制；确定PDGFR，而非BCR/ABL为伊马替尼治疗ET的分子靶点，并将PDGF血清表达水平作为诊断及评价ET疗效的新指标，使更多的ET患者得到有效的诊疗。