EPB41表达缺失对小鼠骨髓造血红系发育的影响及分子机制

EPB41 gene deletion or mutation is associated with hereditary elliptocytosis. Researches show that EPB41 gene encoding two major isoforms of 80KD and 135KD protein 4.1R during erythropoiesis. 80KD 4.1R is exclusively expressed in mature red blood cell. But in early stage of erythropoiesis, EPB41 encoding 135 KD 4.1R isoform by alternative splicing mechanism. 80KD 4.1R takes important role in maintain red cell membrane stability, but the function of 135 KD 4.1R in nucleated erythrocytes is not fully understood. In our recent study, a stage specific EPB41 gene transcript has been cloned from CFU-E of C57BL/6J mouse, and the protein 135KD 4.1R isoform has been identified by immunoblotting using 4.1R specific antibody. Further investigation shows that the immature red blood cell number in EPB41 knockout mouse peripheral blood increased significantly and chronic anemia was observed in the animal model. The EPB41 knockout mouse has splenomegaly which mimic HE patients. Furthermore, the EPB41 deletion result in the BFU-E and CFU-E colony forming numbers increased in vitro. qRT-PCR show that EPB41 knockout mouse EPO expression increased in kidney but the EPOR expression decreased in bone marrow. These data indicate that EPB41 takes a role in mouse progenitor cell proliferation and differentiation by regulating EPOR mediate signaling pathway. To verify this hypothesis, EPB41 knockout mouse will be used to study the stage specific effects of EPB41 gene deletion and molecular mechanism in murine erythropoiesis. The erythroid lineage culture system, fluorescence activated cell sorting (FACS) and progenitor cell colony forming assay will be used to study EPB41 stage specific expression and function. CFU-E EPOR signal transduction and RNA-seq data will be compared between EPB41 knockout and wild type mice to illuminate the molecular mechanism. This study will not only clarify the stage specific function and regulation mechanism of EBP41 in murine erythropoiesis, but also give new insight on the antenatal diagnosis and individual based genetic treatment.

EPB41是人遗传性椭圆形红细胞增多症HE相关基因，在骨髓造血红系发育进程中主要编码80KD和135KD两种蛋白4.1R分子。成熟红细胞中只表达80KD蛋白4.1R，对维持红细胞膜的稳定性至关重要；135KD蛋白4.1R在有核红细胞中表达，但生物学功能未知。我们在前期研究中发现EPB41基因敲除小鼠外周血未成熟红细胞数量升高，小鼠脾脏肿大，出现类似人HE的溶血性贫血，且红系祖细胞集落形成数量升高，但EPOR表达降低，提示EPB41在小鼠红系发育早期可能通过膜受体EPOR介导的信号转导调控有核红细胞增殖分化，但这方面的研究还未见报道。本课题拟利用EPB41基因敲除小鼠，通过红系祖细胞集落形成、红系发育流式分析、蛋白磷酸化组学、RNA-seq及Western blot技术，研究EPB41表达缺失对小鼠骨髓造血红系发育的影响及分子机制，为HE产前筛查及个体化诊疗奠定基础。

遗传性椭圆形红细胞增多症相关基因EPB41对维持成熟红细胞膜的稳定性至关重要，表达缺失导致红细胞膜结构异常，出现溶血性贫血，但其在红系发育不同阶段有核细胞中的调控功能及分子机制未知。项目利用C57BL/6J-EPB41+/+野生型和C57BL/6J-EPB41-/-基因缺陷型小鼠，从分子、细胞和组学层面研究了EPB41基因在小鼠骨髓造血红系发育不同阶段的生物学功能。结果发现，EPB41+/+小鼠骨髓早期HSPC阶段中EPB41的表达水平较高，在终末分化阶段，EPB41表达水平降低；EPB41-/-小鼠红系相关血常规参数HGB、RBC和HCT均显著降低，血清中EPO的分泌显著增加；EPB41基因缺失对早期干祖细胞阶段的比例没有影响，而导致晚期红系祖细胞CFU-E比例显著增加；检测红系祖细胞BFU-E、CFU-E体外克隆形成能力，发现EPB41基因缺失后，与野生型相比，CFU-E克隆形成能力显著增加，进一步检测红系祖细胞BFU-E、CFU-E的增殖能力，发现EPB41基因缺失后CFU-E的增殖能力显著增加，提示EPB41的调控窗口期为CFU-E细胞阶段；进一步通过转录组学测序生信分析提示EPB41+/+和EPB41-/-小鼠红系祖细胞CFU-E中的差异调控信号通路；最后通过蛋白水平验证差异信号通路中关键节点信号分子的总蛋白及其磷酸化水平，说明EPB41基因参与小鼠骨髓红系发育是通过调控晚期红系祖细胞CFU-E中EPO/EPOR下游Jak2/Stat5信号通路中关键分子的磷酸化水平实现的。研究结果表明EPB41基因通过膜受体介导的细胞信号转导调控晚期红系祖细胞CFU-E的增殖，是CFU-E细胞增殖的负调控因子。

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