组蛋白甲基转移酶亚基mDpy-30对缺氧新生鼠星形胶质细胞迁移增殖的调节

Neonatal hypoxic ischemic encephalopathy (HIE) has been a frequently critical situation in clinical practice. As a significant problem there have no specific regimens to avoid high mortality and morbidity, might usually result in severe long period of neurodevelopment outcome or disability. Astrocytes play a main role in centre nerve system repairments after the hypoxic ischemic brain damage of neonetal rats ，and its functions of cell migration and proliferation is foundation for repairing according many reports, but its molecular mechanisms of migration and proliferation is unclear yet. mDpy-30 is an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex. We have previously shown that mDpy-30 also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized guanine nucleotide exchange factor (GEF) BIG1. Moreover, we have found mDpy-30 can affect level of H3K4me3, may play a novel role at the crossroads of endosomal trafficking, and adhesion/migration of glioma cells in vitro, It is only quite recently that we observed some changes about the migration and proliferation of astrocyte in vitro after depleted mDpy-30 with siRNA and shRNA, and the hypoxic injure can increase the expressing of mDpy-30 in astrocytes. We presume that mDpy-30 as a subunit of H3k4MT can affect the genes transcription of some important proteins which involved in migration, division and proliferation of nerve cells such as Rho and Cyclin proteins; Because BIG1 is one kind of GEFs, mDpy-30/BIG1 complex may have function to manipulate Rho proteins activities by catalyzing Rho-GDP altered to Rho-GTP forms; Otherwise, as the research of crystal structure showed that mDpy-30 has a RⅡ like domain just like regulatory subunit of PKA we suppose that mDpy-30/BIG1 complex may possess PKA function to activate ROCKⅡ. Our research will adopt siRNA and cDNA transfection to deplete or overexpress level of protein mDpy-30 in astrocytes, then we will observe the ability of migration and proliferation of astrocytes, furthermore, we will detect the changes of proteins which mentioned in our presume either their quantities or/and distributions in astrocytes. And we will detect the function of mDpy-30 on astrocyte migration and proliferation in hypoxic injure model of astrocytes and hypoxic ischemic brain damage model of neonatal rats. This research not only will be very helpful to reveal the molecular mechanisms which mDpy-30 manipulate migration and proliferation of nerve cells but also may bring new clue in clinical protect and improve nerve system development of newborn infants and potential treatment to HIBD as well.

新生儿缺氧缺血性脑病（HIE）是临床常见危重症，缺乏特异治疗措施，致残率高，是新生儿临床急待解决的难题。研究发现星形胶质细胞（AS）在新生鼠缺氧缺血性脑损伤（HIBD）修复中扮演重要角色，AS迁移和增殖是神经修复的关键，我们曾发现HIBD后AS有明显增加。mDpy-30是一种组蛋白甲基转移酶的结构亚基。我们曾发现mDpy-30能够调控整合素β1合成、运输，调节细胞粘附迁移。我们近期发现缺氧会导致AS表达mDpy-30增加，而mDpy-30对AS迁移、生长有影响。我们尚不清楚mDpy-30是否通过调控Rho、ROCK 、Cyclin等参与控制细胞迁移、分裂的蛋白质的合成、活化，从而调控AS迁移增殖。本研究建立AS缺氧模型，并采用siRNA和cDNA转染调控细胞mDpy-30水平，检测调节AS迁移、增殖的信号分子的改变；探讨mDpy-30对缺氧AS迁移、增殖调节的机制。为治疗HIE寻找新线索。

目的：探索H3K4MT复合体亚基mDpy-30对细胞粘附/迁移能力的影响。.方法：用H3K4MT不同亚基成分的siRNA及cDNA转染细胞，使细胞相应蛋白质的表达降低或增加。用免疫荧光染色技术观测细胞整合素β1转运过程所受的影响。采用Western blot分析、免疫沉淀、质谱分析、GST pull-down分析等多种蛋白分析技术检测相应蛋白质的水平及变化。采用定量PCR分析（q-PCR analysis）技术检测相应mRNA的改变。并对细胞对细胞外基质（ECM）的粘附能力以及迁移能力进行分析。.结果：（1）去除mDpy-30、BIG1或其它H3K4MT亚基会增加神经胶质细胞在体外的粘附/迁移能力。相反，过度表达mDpy-30会降低胶质细胞的粘附/迁移；（2）去除mDpy-30、BIG1或H3K4MT的另一种亚基RbBP5均导致位于细胞突起部位的整合素β1增加，而且，去除mDpy-30, RbBP5均会引起细胞整合素β1蛋白含量和其mRNA水平增加。.结论： mDpy-30以及它的相互作用蛋白可能是一种全新的细胞粘附/迁移调节因子，通过影响整合素β1的表达及其内涵体转运等环节调节细胞的粘附/迁移。