【作者】 陈东；

【导师】 王海波；

【作者基本信息】 山东大学 ， 耳鼻咽喉科学（专业学位）， 2013， 博士

【摘要】 第一部分单纯疱疹病毒1型致小鼠面瘫中脑干面神经核团基质金属蛋白酶9的动态表达研究研究目的：建立1型单纯疱疹病毒(herpes simplex virus type1, HSV-1)致小鼠面瘫的动物模型,探讨基质金属蛋白酶9(matrix metalloproteinase-9, MMP9)在小鼠脑干面神经核团中表达的变化规律。实验方法：将160只四周龄Balb/c雄性小鼠随机分为病毒接种组(121只),生理盐水对照组(30只)和空白对照组(9只)。通过将HSV-1接种至小鼠耳廓背面皮肤的方法建立面瘫动物模型。用蛋白质印迹法(western blot)、real-time PCR、免疫荧光技术检测小鼠脑干面神经核团中MMP9的表达变化规律。实验结果：病毒接种组小鼠中有63只(52.07%)于接种后2-5天出现不同程度的面瘫症状,面瘫症状持续3-7天自发恢复正常。其中33只面瘫小鼠进入研究课题的第一部分。MMP9mRNA在生理盐水组小鼠脑干面神经核团表达处于低水平,与空白组相比无统计学差异(P>0.05)。在面瘫组,面瘫后6h可见MMP9表达升高,2d达到高峰,与空白对照组相比差异有统计学意义(P<0.01),此后MMP9mRNA表达逐渐降低。生理盐水组小鼠MMP9蛋白表达在脑干面神经核团表达处于低水平,与空白组相比无统计学差异(P>0.05),而在面瘫组小鼠,6h表达开始升高,2d达到高峰,与空白对照组相比差异有统计学意义(P<0.01)。激光共聚焦显微镜观察显示,面瘫后2天MMP9蛋白在小鼠脑干面神经核团中表达较空白对照组明显增强。研究结论：提示MMP9参与了HSV-1致小鼠面瘫的病理过程,并在其中起到重要作用。第二部分糖皮质激素对基质金属蛋白酶9表达的抑制作用研究研究目的：在建立HSV-1致小鼠面瘫的动物模型基础上,研究糖皮质激素对小鼠脑干面神经核团中MMP9表达的抑制作用。实验方法：在第一部分实验中造模成功的面瘫小鼠30只进入本部分实验,干预药物为甲基泼尼松龙丁二酸钠(methylprednisolone sodium succinate, MPSS)和糖皮质受体阻断剂(RU486)。将面瘫小鼠随机分为MPSS干预组面瘫小鼠和MPSS+RU486干预组面瘫小鼠。MPSS干预组在面瘫发生后的2天内每天腹腔注射MPSS一次,MPSS+RU486干预组每天腹腔注射MPSS和RU486各一次。用western blot技术检测小鼠脑干面神经核团中MMP9的表达变化。正常对照组小鼠和无药物干预组面瘫小鼠材料为第一部分实验中的标本。实验结果：HSV-1致小鼠面瘫2天后,MPSS干预组中MMP9蛋白的表达受到抑制,并且这种抑制作用可以被RU486阻断,即MPSS干预组与无干预组面瘫小鼠相比以及MPSS干预组与MPSS+RU486干预组面瘫小鼠相比,MMP9蛋白表达的差异均有统计学意义(P<0.01),无干预组与MPSS+RU486干预组相比,MMP9蛋白的表达差异无统计学意义(P>0.05)。研究结论：提示在HSV-1致小鼠面瘫中,MPSS可能通过抑制MMP9的表达减轻脑干面神经核团的损伤。更多还原

【Abstract】 PartⅠThe Alterations of Matrix Metalloproteinase-9in Mouse Brainstem During Herpes Simplex Virus Type1-induced Facial PalsyObjectives:To establish an animal model of facial nerve palsy in mice induced by herpes simplex virus type1(HSV-1) infection so as to explore the changes of matrix metalloproteinase-9(MMP9) in the mouse brainstem during the development of facial paralysis induced by HSV-1.Methods:160four-weeks Balb/c male mice were randomly divided into virus inoculated group (121mice), saline group (30mice) and normal control group (9mice). HSV-1was inoculated into the surface of posterior auricle of mouse to establish a paralyzed animal model. Western blot and Immunofluorescent staining were used to detect the level of MMP9protein, real-time PCR was used to measure the level of MMP9mRNA.Results:Of the121mice subjected to retroauricular inoculation with HSV-1,63(52.07%) developed unilateral facial paralysis between2and5days. In the condition without any administration, the facial paralysis recovered spontaneously within a period of3-7days. Of the63mice,33mice were involved in experiment Part1as facial paralysis group. In the saline group, the expressions of MMP9mRNA in the facial nucleus of brainstem were at low levels, and compared with the control group there were no significant difference (P>0.05). In facial paralysis group, the expression of MMP9mRNA increased at6h post-induction of facial paralysis, and peaked at2d, showing a significant difference compared with control group (P<0.01), and then followed by a decline in the tendency to normal level. MMP9protein expression in the saline group was the same as mRNA expression. In facial paralysis group, the expression of MMP9protein increased at6h, and peaked at2d, showing a significant difference compared with control group (P<0.01). Confocal laser scanning microscopy showed that MMP9was present at a low level in normal control group, while strongly expressed in paralyzed group at the time point of2days post-induction of facial paralysis.Conclusions:Our findings suggest that MMP9in mouse brainstem is involved in the evolution of facial palsy induced by HSV-1and may play an important role in the pathogenesis of this disease. Part ⅡInhibitory Effect of Methylprednisolone Sodium Succinate on Matrix Metalloproteinase-9Expression in Brainstem of Paralyzed MiceObjectives:To explore the inhibitory effect of methylprednisolone sodium succinate (MPSS) on MMP9expression in the mouse brainstem during the development of facial paralysis induced by HSV-1.Methods:30mice with facial paralysis induced by HSV-1were randomly divided into two groups. In group one, mice were injected daily with MPSS for2days; in group two, mice were injected daily with combination of MPSS and glucocorticoid receptor blocker (RU486) for2days. In the two groups, western blot was used to detect the level of MMP9protein at the time point of2days post-induction of facial paralysis. MMP9protein was also be detected in paralyzed group without any administration at the time point of2days and in normal control group as same as in experiment Part1.Results:Expression of MMP9of paralyzed mice was inhibited by MPSS, and the inhibition could be blocked by RU486. At the time point of2days, expressions of MMP9protein in paralyzed mice with administration of MPSS were lower significantly than those in paralyzed mice without any administration and those with combination of MPSS and RU486(P<0.01), and were higher significantly than normal control (P<0.01).Conclusions:MPSS might effectively relieve HSV-1-mediated damages by inhibitory effect on expression of MMP9in HSV-1-induced facial paralysis.更多还原