神经递质5-HT在巨核细胞/前血小板生成反馈调控中的分子机制

Thrombocytopenia (low platelet count) is a major treatment-related problem in cancer patients undergoing radiotherapy, chemotherapy or bone marrow transplantation.And there is an urgent need for the development of effective and specific new treatment. Recently, we have shown that serotonin (5-HT) promotes haematopoiesis and has anti-apoptotic effect on megakaryocytes. Our earlier studies also demonstrated that serotonin has mitogenic effect on megakaryocytopoiesis. However, the mechanism of action remains unknown. Here, we hypothesize that serotonin exerts its effect primarily through the binding of 5-HT2 receptors, activating ERK/AKT pathway, which block the initiation of apoptosis and induce cell proliferation in megakaryocytic progenitors, and activate F-actin reorganization in mature megakaryocytes which lead to platelet formation...In this study, we will test these hypothesis through three objectives: (1) To determine the role of 5-HT2 receptors and ERK1/2 pathway play in the proliferation of megakaryocytic progenitors by serotonin; (2) To determine the role of 5-HT2 receptors, PI3K/AKT play in the anti-apoptotic effect of serotonin on megakaryocytic cells; and (3) To determine if serotonin activates 5-HT2 receptors, ERK1/2 pathway, F-actin reorganization on mature megakaryocytes, which lead to platelet formation. ..More specifically, we will use the following experimental techniques: (1) CFU-MK and proplatelet assays will be used to test cell proliferation or platelet formation; (2) the expression of 5-HT2 receptors of megakaryocytic cells will be determined by RT-PCR, immunofluoresence staining, flow cytometry and Western blot analysis; (3) the apoptosis of primary megakaryocytic cells will be determined by Annexin V, propidium iodide, Caspase-3, and JC-1 assays with flow cytometry; (4) F-actin reorganization in these cells will be studied by imunofluoresence staining with rhodamine-conjugated phalloidin; (5) Phosphorate-ERK1/2 and AKT will be examined by immuno-staining with flow cytometry and Western blotting; and (6) Inhibitors will be used to block the corresponding pathways. Specifically, 5-HT2R antagonists, PD98059 and LY294002 will be used to block the activation of 5-HT2 receptors, ERK1/2 and PI3K/AKT respectively. Results from this study will lay the foundation for future development of a treatment option for thrombocytopenia and haematopoietic stem/progenitor cell expansion.

血小板减低是肿瘤患者接受放疗，化疗或者骨髓移植之后主要的副作用和死因。至今，多种促进血小板生成的药物仍处于临床试验阶段。我们在国际上首次证实神经递质5-HT能促进巨核细胞和造血干细胞增殖及对巨核细胞抗凋亡的作用。然而，其作用机制尚不清楚。本研究将从以下三方面来验证我们的机理假说：（1）确定5-HT通过5-HT2受体和ERK1/2信号通路在巨核系祖细胞增殖中发挥作用；（2）确定在巨核细胞中，5-HT通过5-HT2受体，PI3K/AKT信号通路发挥抗凋亡作用；（3）确定在成熟巨核细胞中，5-HT能激活5-HT2受体，ERK1/2信号通路，F-actin重组，最终促进前血小板形成。因此，小分子胺类物质5-HT作为一类非蛋白新的生长因子，可为血小板减少症的治疗和造血干祖细胞的体外扩增发展新方法。同时，5-HT作为神经递质，也为神经系统可能通过神经递质参与造血调控提供新的理论依据。

血小板减低是肿瘤患者接受放疗，化疗或者骨髓移植之后主要的副作用和死因。目前，多种促进血小板生成的药物仍处于临床试验阶段。我们在国际上首次证实了神经递质5-HT能促进巨核细胞和造血干细胞增殖及对巨核细胞抗凋亡的作用，但作用机制尚不明确，因此本研究将验证我们的机理假说：（1）确定5-HT通过5-HT2受体和ERK1/2信号通路在巨核系祖细胞增殖中发挥作用；（2）确定在巨核细胞中，5-HT通过5-HT2受体，PI3K/AKT信号通路发挥抗凋亡作用；（3）确定在成熟巨核细胞中，5-HT能激活5-HT2受体，ERK1/2信号通路，F-actin重组，最终促进前血小板形成。.结果：（i）采用RT- PCR，流式细胞术，免疫荧光和Western Blot四种方法，确定了人类骨髓巨核细胞和巨核细胞株MEG-01，DAMI，CHRF和M07e上存在5-HT2A，2B，2C受体的mRNA和蛋白表达。. （ii）5-HT以剂量依赖性(10-500nM）的形式明显促进人CFU-MK的形成，而5-HT2受体拮抗剂酮舍林可降低这种效果。. （iii）5-HT通过5-HT2R和ERK1/2通路促进CHRF细胞增殖以及通过5-HT2R和AKT途径减少CHRF细胞凋亡。. （iv）在人类骨髓成熟的巨核细胞中，5-HT促进前血小板形成，而5-HT2受体抑制剂酮舍林可以降低这种作用；. （v）在巨核细胞中，5-HT激活5-HT2受体和ERK1/2通路，作用于细胞骨架F-actin重组，从而导致前血小板的形成，加入酮舍林后，该作用被抑制。.科学意义：小分子胺类物质5-HT作为一类非蛋白新的生长因子，可为血小板减少症的治疗和造血干祖细胞的体外扩增发展新方法。同时，5-HT作为神经递质，也为神经系统可能通过神经递质参与造血调控提供新的理论依据。