髓鞘碱性蛋白（MBPs）在小胶质细胞介导的早产儿脑损伤中的作用及其机制研究

Prematurity is associated with an elevated risk of suffering from brain injuries, and these brain injuries are associated with impaired quality of life due to disorders such as cerebral palsy and behavioral, social, attentional, and cognitive deficits. Hypoxia and ischemia are the major initiating factors of preterm brain injury. Several studies suggested that activated microglial cells may play a crucial role in mediating injury to the preterm brain, but the specific mechanism is obscure. Myelin Basic Proteins (MBPs) is the second most abundant protein in the CNS, and the majority of studies involving them have focused on myelin formation. Growing evidence has revealed that immune cells expressed MBPs both in vivo and in vitro; however, the functional roles of MBPs in microglia have remained uncertain. Our previous study has shown that MBPs expression was significantly increased in activated microglia derived from cell culture and animal models of brain injury using real-time reverse transcription PCR, western blot and immunostaining. In order to clarify the putative functions of MBPs in neuroinflammation, gene knockdown and gene over-expression will be employed to down-regulate and up-regulate MBPs, respectively, in BV-2 cells. In parallel to this, cell proliferation, cell cycle will be assessed by BrdU assay and flow cytometry. Additionally, expression of inducible nitric oxide synthase, IL-1β and TNF-α was determined at mRNA and protein levels. Production of reactive oxygen species and nitric oxide was also assessed by flow cytometry and colorimetric assay, respectively. The specific signaling pathways via which MBP can regulate neuroinflammation remain unclear. This can be investigated in this study which could be confirmed by western blot and immunostaining.

早产儿脑损伤是严重危害新生儿健康的重要疾病。大量研究证明激活的小胶质细胞在其发生发展过程中发挥了重要作用。髓鞘碱性蛋白（MBPs）被视为髓鞘形成细胞的特异性标志物，目前众多的研究主要集中在 MBPs 与髓鞘形成关系和在免疫细胞中的表达方面，但对 MBPs 在小胶质细胞内的作用并不清楚。我们前期研究发现 MBPs 在体内、外激活小胶质细胞中表达明显增高,提示可能与神经炎症相关。因此本项目拟建立早产小鼠脑损伤模型，观察早产小鼠病变脑组织中小胶质细胞内 MBPs的表达变化；同时建立MBPs 基因敲减和过表达的小鼠小胶质细胞模型，对比研究 MBPs 表达水平变化对激活小胶质细胞功能的影响，并利用WB 和ChIP 技术明确 MBPs介导小胶质细胞活化的分子机制。本研究不仅有助与阐明 MBPs 在激活小胶质细胞中的作用及参与神经炎症调控的分子机制，还可为探索早产儿脑损伤的防治策略提供新思路和理论依据。

早产儿脑损伤是严重危害新生儿健康的重要疾病，大量研究证实激活的小胶质细胞其发生发展过程中发挥重要作用。鉴于 MBPs 在体内、外激活小胶质细胞中表达明显增高，本研究探讨 MBP 在活化小胶质细胞中的作用及其调控神经炎症的分子机制。体内实验结果显示 MBPs 在早产小鼠脑损伤模型脑组织中活化的小胶质细胞内表达增高。体外研究：① 选用小鼠小胶质细胞株 BV-2 构建了 MBP 过表达和敲低的稳转细胞株（BV2-Mbp-OE 与BV2-Mbp-KD ）；② MBP 过表达可促进小胶质细胞增殖与迁移，在无糖培养条件下可抑制 S→G2期阻滞，而 MBP 敲减可致相反结果，伴随着 CyclinD1蛋白表达改变；③ 缺氧条件下，MBP 过表达可显著促进小胶质细胞内 iNOS、TNF-α 的 mRNA 和蛋白质表达水平，同时促进小胶质细胞内 ROS 和 NO 的产生，而 MBP 敲减则致相反结果；④ MBP 过表达可显著促进 MAPK 信号通路中 p38、p44/42 及 PI3K-Akt 信号通路中 Akt 分子的磷酸化水平，MBP 敲减则抑制其磷酸化；⑤ mRNA seq 结果提示 Mbp 可通过调控相关基因的可变剪接影响 TGF-β 、Jak/STAT 信号通路活性，上述信号通路可激活 MAPK 信号通路从而促进小胶质细胞活化和神经炎症的发生发展；此外，研究结果提示小胶质细胞内表达的 Mbp 可能与细胞内环核苷酸代谢、脂质代谢及能量代谢有相关。本研究揭示了Mbp 与神经炎症发生发展的相关性，拓展了对小胶质细胞中表达的 Mbp 新功能的认知，为全面深入理解 Mbp 表达对激活小胶质细胞及早产脑组织损伤的影响提供了理论依据。