miR-205在C1型尼曼-匹克病小鼠髓鞘形成中的作用机制研究

Dysmyelination is one of the key factors leading to disorders in NPC1 nervous system, which seriously affecting the patient’s life. Until now, valid clinical treatments for those patients are still not found. Our previous study showed that miR-205 is involved in myelination of Npc1-/- mouse, but the regulatory mechanism is still not clear. According to the results of small RNA sequencing and IPA disease and function analysis, the target gene of miR-205 including Csf1, Abcd1, Fam126a, and Lpar1 is involved in myelination, possible through the regulation of miR-205. In present study, luciferase assay will be performed to confirm the targets of miR-205. Then, we will analyze the effection of miR-205 on myelination in vitro. Furthermore, in order to investigation of target genes in the myeliantion, target genes will be overexpressed or down-regulated in Npc1-/- mouse using brain stereotaxic injection, immunofluorescence, qRT-PCR and Western blot. Finally, rescued and in vivo experiment will be performed to elucidate the mechanism of miR-205 in myelination. This project will give us a better understanding for mechanisms of the myelination, and also provide a possible clinical treatment for demyelinating disease in nervous system.

髓鞘形成障碍是导致C1型尼曼-匹克病患者神经系统受损的重要因素之一，严重影响患者的生活质量，目前尚无有效的治疗方法。本课题组前期研究证实miR-205参与了Npc1-/-小鼠髓鞘形成过程，但其调控机制尚不清楚。结合Small RNA测序及IPA功能与疾病分析，我们推测miR-205调控靶基因Csf1、Abcd1、Fam126a和Lpar1参与Npc1-/-小鼠髓鞘形成。因此，本项目拟采用双荧光素酶报告基因系统验证miR-205的靶基因；进而体外分析miR-205对髓鞘形成的影响；在此基础上构建靶基因超表达和干扰表达载体，利用脑立体定位注射、免疫荧光技术、qRT-PCR和Western blot等研究靶基因对髓鞘形成的调控；最后通过功能拯救和体内实验，阐释miR-205调控靶基因参与髓鞘形成的分子机制，这将为理解髓鞘形成过程提供新的线索，进而为神经系统脱髓鞘性疾病治疗提供新的基因及药物靶点。

本课题通过双荧光素酶报告基因系统、免疫荧光组织化学染色和Western blot等技术，研究mi-205对髓鞘形成的调控，阐释miR-205调控靶基因参与髓鞘形成的分子机制。通过我们的研究发现：（1）miR-205的靶基因是Fam126a和Abcd1；（2）Abcd1和Fam126a分别仅在P9、P65的Npc1-/-小鼠海马中表达显著降低（P<0.05，P<0.01）；（3）Npc1-/-小鼠脑不同部位MBP蛋白表达量随着年龄增长均显著下降，且各部位呈现不同的表达模式；（4）建立成熟的皮层和海马少突胶质细胞分离培养方法。另外，我们还发现：（5）联合应用蛋白芯片和Small RNA测序，构建miRNA调控神经炎症的网络；（6）证实Npc1基因突变导致肝脏的炎症反应，引起肝脏细胞凋亡和肝功能受损;（7）证实Npc1基因突变引起睾丸和附睾结构异常、精子发生异常和生殖细胞凋亡等，导致雄性小鼠不育；（8）证实Npc1-/-和Npc1+/+小鼠生长繁殖和血液生理生化指标存在差异。综合以上结果，这将为理解NPC1的髓鞘形成过程提供新的线索，进而为充分利用Npc1-/-小鼠开展和治疗NPC1的研究奠定重要的基础。