老年小鼠内耳中巨噬细胞迁移因子调控血管周常驻巨噬细胞凋亡及其机制研究

Hearing loss related to aging is the most common sensory disorder among elderly individuals. And macrophage migration inhibitory factor (MIF) is a multi-functional molecule which plays an important role in the inner ear of mice. In our recent study, we found that the intrastrial fluid–blood barrier includes a large number of perivascular cells called PVM/Ms which were demonstrated having a critical role for hearing. In addition to this , we still found these cells declined in elder mice and also display typical pattern of morphological changes of apoptosis. However these two contribute to age-related hearing loss has not been reported. So here we put forward the hypothesis that MIF can control the process of apoptosis in PVM/Ms , and so that leads to the hearing loss. To prove it, we use in vitro and invo models to observe proliferation, cell cycle ,apoptosis of PVM/Ms and to detect the expression of Akt,CDK4 and other factors under different conditions. Finnally we aim to prove in elder mice ,MIF can control the process of apoptosis in PVM/Ms by regulating MAPK and(or)PI3K/Akt pathway. This will provide a new idea for clinic therapy of age-related hearing loss.

老年性聋是老年人中常见的一种神经性聋，而MIF是存在于内耳多个组织中与老年性聋相关的重要因子。我们的研究发现和证实了在小鼠内耳血管纹中存在PVM/Ms，其在维持听力中具有重要作用；且老年小鼠内耳中PVM/Ms减少并有类似凋亡的形态表现。但迄今对老年性聋中MIF如何影响PVM/Ms及其机制仍知之甚少。因此我们提出假说，老年小鼠中MIF可能通过某种信号途径引起PVM/Ms凋亡进而引起小鼠听力下降。为了验证这一假说，我们通过分别建立PVM/Ms细胞系及老年小鼠模型，采用免疫荧光染色、流式细胞仪及RNA干扰等手段，从分子、细胞、组织及动物整体水平多方面探讨MIF在老年性聋中引起PVM/Ms凋亡的重要作用，并明确其凋亡机制可能为MAPK和（或）PI3K/Akt信号途径。本研究将从PVM/Ms凋亡及其机制研究的新视点为揭示老年性聋发病机制奠定基础，为其防治提供一条新思路。

巨噬细胞迁移抑制因子（MIF）在听觉功能中起重要作用，但其作用机制尚不未明确。本研究分别从幼年和老年小鼠耳蜗外侧壁血管纹中分离出血管周常驻巨噬细胞（PVM），并检测MIF mRNA和蛋白表达水平；使用MTT法和流式细胞检测技术分析在不同条件下，如MIF体外敲除、体外过表达、体内转染等PVM的存活率和凋亡情况；通过听性脑干反应测试不同条件下小鼠听力。结果表明，老龄小鼠巨噬细胞移动抑制因子（MIF）表达明显下调。在老龄小鼠中，血管周常驻巨噬细胞（PVM）的存活率显著降低，而凋亡数目明显增加。 MIF敲除可使PVM存活率下降并诱导细胞凋亡，MIF 过度表达则显示出相反的结果。进一步的研究表明，MIF在幼年小鼠中被敲除将导致严重的听力损失，但MIF在老年小鼠中过度表达后则使其恢复了听力。 Si-MIF抑制幼年小鼠PVM的活性和诱导细胞凋亡，而AD MIF诱导老龄小鼠PVM的存活和抑制凋亡。此外，MIF有效地改变了CDK1、BRAF、P- ERK1/2、p-PI3K和P -Akt的表达水平.与此同时，ERK抑制剂PD98059或PI3K抑制剂LY294002显著逆转小鼠Si-MIF对幼年小鼠PVM的影响，而ERK激活剂EGF或PI3K激活剂IGF显著逆转Ad-MIF对老龄小鼠PVM的影响。综上所述，MIF通过MAPK和/或PI3K/Akt通路至少部分地介导了PVM/Ms的存活和凋亡。