沉默信号调控因子1在1型糖尿病模型小鼠角膜和三叉神经节中的表达及意义

背景 糖尿病是引发角膜神经病变的高危因素之一.沉默信号调控因子1(Sirt1)在糖代谢、脂代谢、胰岛素分泌调节中发挥着重要的作用,并与神经系统性疾病密切相关.目前,关于Sirt1与糖尿病性角膜神经病变的关系尚不完全清楚. 目的 探讨Sirt1在1型糖尿病C57BL/6-Ins2Akita/J小鼠角膜及三叉神经节中的表达及其意义,探讨Sirt1与糖尿病性角膜神经病变的关系. 方法 取C57BL/6-Ins2Akita/J雄性小鼠与同窝出生的野生型C57BL/6小鼠各8只,分别作为1型糖尿病模型组和正常对照组.两组小鼠均在12月龄时过量麻醉处死,处死前检测空腹血糖、测体质量.取小鼠三叉神经节和角膜组织,采用苏木精-伊红染色法检测两组小鼠三叉神经节和角膜组织的组织病理学变化,用免疫组织化学法检测三叉神经节和角膜组织中Sirt1蛋白的表达和定位;用荧光定量PCR法检测Sirt1 mRNA在三叉神经节和角膜组织中的表达;采用Western blot检测两组小鼠三叉神经节和角膜组织中Sirt1蛋白的相对表达量,并进行比较.结果 C57BL/6-Ins2Akita/J 小鼠三叉神经节细胞大小不均,细胞排列较为疏松,神经纤维排列紊乱,角膜上皮细胞层数减少,角膜变薄;野生型C57BL/6小鼠神经节细胞排列紧密,细胞形态均一,神经纤维排列整齐.免疫组织化学法检测结果显示,C57 BL/6-Ins2Akita/J小鼠角膜中Sirt1蛋白的表达强度低于野生型C57BL/6小鼠.荧光定量PCR结果显示,Sirt1 mRNA在C57B L/ 6-Ins2Akita/J小鼠角膜表达的灰度值显著低于野生型C57BL/6小鼠(0.56±0.03 vs.0.98±0.13),差异有统计学意义(t=5.853,P=0.010);C57BL/6-Ins2Akita/J小鼠三叉神经节中Sirt1 mRNA表达的灰度值为2.45±0.18,低于野生型C57 BL/6小鼠的2.51±0.22,但差异无统计学意义(t=0.587,P=0.599).Western blot检测结果显示,C57 BL/6-Ins2Akita/J小鼠角膜中Sirt1蛋白的表达显著低于野生型C57BL/6小鼠(0.780±0.017 vs.1.300±0.012),差异有统计学意义(t=33.140,P=0.001);两组小鼠间三叉神经节中Sirt1蛋白的表达差异无统计学意义(1.100±0.015 vs.1.110±0.017) (t=0.430,P=0.709).结论 12月龄C57B L/ 6-Ins2Akita/J小鼠角膜的神经和结构发育异常,Sirt1参与糖尿病角膜病变的发病,有可能是潜在的靶点分子.

Expression and significance of silence signal regulating factor 1 in cornea and trigeminal ganglion in type 1 diabetes model mice

Background Diabetes is one of the risk factors that leads to corneal neuropathy. Silent signal regulatory factor 1 （ Sirtl ） plays an important role in glucose metabolism, lipid metabolism, regulation of insulin secretion and is closely related to the nervous system disease. The relationship between Sirtl and diabetic corneal neuropathy is not fully understood. Objective This study was to detect the expression of Sirtl in cornea and trigeminal ganglion with type 1 diabetes model mice and explore the association of Sirtl expression with diabetic corneal neuropathy. Methods Eight C57BL/6-Ins2Akita/J male mice and eight wild-type C57BL/6 male mice in the same litter were selected as type 1 diabetes model group and control group,respectively. The mice of two groups were sacrificed in overdose anesthesia method at 12-month old. Histological examination of cornea and trigeminal ganglion was performed using hematoxylin and eosin staining. Expression and localization of Sirtl protein in cornea and trigeminal ganglion were detected using immunohistochemistry. Western blot assay and fluorescine quantitative PCR were respectively used to quantitatively analyze the expression of Sirtl protein and Sirtl mRNA. Results Trigeminal ganglion ceils were uneven in size and shape with the loosened cellular arrangement and disorder neurofibrosis alignment,and the corneal epithelial cells were less in the C57BL/6-Ins2Akita/J mice, but the trigeminal ganglion cells and corneal epithelial cells were normal in wild-type C57BL/6 mice. Immunochemisty exhibited that Sirtl protein was expressed mainly in corneal epithelium and the expression of Sirtl protein was stronger in the C57BL/6 mice than that in C57BL/6-Ins2Akita/J mice. Fluorescine quantitative PCR assay showed that the gray scale value of Sirtl mRNA in cornea in C57BL/6-Ins2Akita/J mice was lower than that of the wild-type C57BL/6 mice（0.56±0. 03 vs. 0.98±0.13） with significant difference（t =5. 853 ,P=0.010）. Western blot showed that the expression of Sirtl protein in cornea was lower in C57BL/6-Ins2Akita/J mice than that of the wild-type C57BL/6 mice （0.78±0. 017 vs. 1. 300±0. 012） with significant difference（ t=33. 140 ,P = 0. 001 ）. However, no significant differences were seen in the gray scale value of Sirt 1 mRNA （ 2.45 ±0. 18 vs. 2. 51 ±0. 22 ） （ t = 0. 587, P = 0. 599 ） and protein level （ 1. 100 ±0. 015 vs. 1.110±0. 017 ） （ t = 0. 430, P = 0. 709） in trigeminal ganglion tissues between C57BL/6-Ins2Akita/J mice and wide-type C57BL/6 mice. Conclusions The corneal nerve and structure is abnormal in 12-month-old C57BL/6-Ins2Akita/J mouse. Sirtl is involved in the pathogenesis of diabetic keratoneuropathy,suggesting that it may be a potential target.