【作者】 孙忠文；

【导师】 朱梅佳；

【作者基本信息】 山东大学 ， 神经病学， 2013， 硕士

【摘要】 目的肿瘤坏死因子α诱导蛋白8样因子2(tumor necrosis factor-α induced protein-8-like-2, TIPE2)和肿瘤坏死因子α诱导蛋白8(tumor necrosis factor-α induced protein-8, TNFAIP8)属于TNFAIP8家族成员,是近年新发现的蛋白质,它们能够负性调节免疫反应和细胞凋亡,维持机体内环境稳定。糖尿病脑血管病具有发病率高、卒中预后差的特点；糖尿病能够显著促进阿尔茨海默病的病程进展,严重危害人类生命健康和生活质量。目前研究认为,高血糖本身和糖尿病诱导的淀粉样蛋白Aβ1-40沉积均可以诱导脑组织炎症水平增高,免疫反应介导的炎症损伤和细胞凋亡在糖尿病脑组织损伤中发挥重要作用,但其具体机制尚不明确。因此,探讨高血糖诱导的脑内炎症反应过程,对于理解糖尿病脑损伤的发病机制具有重要意义。为此,本课题主要目的是通过观察TIPE2和TNFAIP8在2型糖尿病大鼠脑组织中的表达特征及其与Aβ1-40沉积的相关性,初步探讨TIPE2和TNFAIP8是否参与糖尿病脑组织损伤的发生发展。方法(1)采用自发性2型糖尿病大鼠(Goto-Kakizaki rat, GK rat)大鼠为2型糖尿病动物模型(GK组),Wistar大鼠为非糖尿病动物模型(Control组);每组8只大鼠。(2)运用免疫组织化学染色法检测脑组织TIPE2、TNFAIP8和Aβ1-40的表达及空间分布；RT-PCR、Western blot法分别检测脑组织TIPE2和TNFAIP8的mRNA水平和蛋白含量；ELISA法检测脑组织Aβ1-40蛋白含量。(3)所有计量资料以均数±标准差((?)±s)表示,采用SPSS17.0软件包对数据进行亡检验和两变量间Person相关性分析,P<0.05认为差异具有统计学意义。结果(1)TIPE2主要表达于皮层和皮层下组织结构的神经胶质细胞和微／小血管管壁。与Control组相比,GK组大鼠脑组织TIPE2蛋白、mRNA.阳染血管数和阳染神经胶质细胞数均显著增高(P均<0.05)。(2)TNFAIP8主要分布于皮层和皮层下组织结构的神经元和血管管壁。与Control组相比,GK组大鼠脑组织TNFAIP8蛋白、mRNA.阳染血管数和阳染神经细胞数均显著增高(P均<0.05)。(3)A β1-40主要分布于皮质和海马等组织结构的神经元、神经胶质细胞和血管壁。与Control组相比,GK组大鼠脑组织A β1-40蛋白、阳染血管数和阳染细胞数均显著增高(P均<0.05)。(4)脑组织TIPE2蛋白表达与Aβ1-40蛋白水平在Control组(r=0.814)和GK组(r=0.744)均呈正相关(P均<0.05)。(5)脑组织TNFAIP8蛋白表达与Aβ1-40蛋白水平在Control组(r=0.739)和GK组(r=0.756)均呈正相关(P均<0.05)。结论(1)正常大鼠脑组织TIPE2和TNFAIP8呈低水平表达,提示TIPE2和TNFAIP8可能参与维持正常脑组织损伤内环境稳态。(2)糖尿病诱导大鼠脑组织TIPE2和TNFAIP8表达增多,提示TIPE2和TNFAIP8可能参与减轻糖尿病脑组织炎症损伤。(3)糖尿病大鼠脑组织TIPE2和TNFAIP8表达均与Aβ1-40沉积呈正相关,提示糖尿病诱导的Aβ1-40沉积可能启动反馈调节通路,上调TIPE2和TNFAIP8基因转录与蛋白表达。更多还原

【Abstract】 OBJECTIVETumor necrosis factor alpha induced protein-8-like-2(TIPE2) and tumor necrosis factor alpha induced protein-8(TNFAIP8), two recently been discovered protein of TNFAIP8family, could negatively regulates immune responses and cell apoptosis, and maintain the body’s internal homeostasis. Diabetic cerebrovascular disease, characterized by high incidence and poor prognosis of stroke, induces serious damage to human health and quality of life. Current studies suggest that both high level of beta-amyloid proteins1-40(Aβ1-40) induced by diabetes and high blood sugar itself could induced brain excessive inflammatory response. Immune-mediated inflammatory injury and apoptosis play an important role in diabetic brain damage, but the specific mechanism is still unclear. This study was aimed to investigate the expression pattern of TIPE2and TNFAIP8in the brain of rats with type2diabetes and their correlations with the deposition of Aβ1-40.METHODS(1) Male GK rats were used as a type2diabetes model (GK group) and Wistar rats were selected as control (Control group), with8rats in each group.(2) The expression and localization of TIPE2, TNFAIP8and (Aβ1-40) were detected by immunohistochemical staining of the brain tissues; mRNA and protein levels of TIPE2and TNFAIP8in the brain were assayed by RT-PCR and Western Blotting, respectively. Aβ1-40protein levels in the brain were measured by ELISA. (3) All the data are presented as mean±standard deviation (SD). The SPSS17.0statistical package was used for the analysis. Comparison between two groups was analyzed by t-test, with p<0.05as statistical significance. Person’s correlation analysis was performed to determine the relationship between two groups, with p<0.05as the criterion for testing the significance of the correlation coefficient.RESULTS(1) TIPE2was mainly expressed in neuroglial cells and microvessels of the cortex and subcortical structures. Compared to Wistar rats, GK rats had significantly higher brain levels of TTPE2protein and positively stained blood vessels and cells (p<0.05), and had significantly higher mRNA level of TIPE2(p<0.05).(2) TNFAIP8was primarily observed in neurons and blood vessels of cortex and subcortical structures. Compared to Wistar rats, GK rats had significantly higher brain levels of TNFAIP8protein and positively stained blood vessels and cells (p<0.05), and had significantly higher mRNA level of TNFAIP8(p<0.05).(3) Aβ1-40protein was mainly localized in neurons and neuroglial cells in the cortex and hippocampus, and in the pial arteries, penetrating arteries, and microvessels. Compared to Wistar rats, GK rats had significantly higher brain levels of Aβ1-40protein and positively stained blood vessels and cells (p<0.05).(4) There is a strong positive correlation between TIPE2expression and Aβ1-40levels in the brain of both Wistar rats (r=0.814, p<0.05) and GK rats (r=0.744, p<0.05).(5) There is also a strong positive correlation between TNFAIP8expression and Aβ1-40levels in the brain of both Wistar rats (r=0.739, p<0.05) and GK rats (r=0.756, p<0.05).CONCLUSIONS(1) TIPE2and TNFAIP8express low in non-diabetic brain tissues suggesting their potential involvement in the maintenance of brain homeostasis.(2) Expression of TIPE2and TNFAIP8is significantly heightened in diabetic brain tissues suggesting that they might attenuate inflammation induced by diabetes in GK rat brian.(3) There were strong positive correlations of Aβ1-40deposition with both TIPE2and TNFAIP8expression in GK rats, which suggest that diabetic brain injury induced by the initial insults such as Aβ1-40deposition might trigger a feedback elevation of TIPE2and TNFAIP8expression, which functions to attenuate inflammation-mediated diabetic brain damage.更多还原