GIGYF1协同Grb10调节IGF-1R信号通路影响糖尿病脑病大鼠认知功能的机制研究

发布时间：2018-05-24 05:36

本文选题：糖尿病脑病 + 认知　；参考：《重庆医科大学》2015年硕士论文

【摘要】：背景长期高血糖状态可导致中枢神经系统损害,进而引起学习、记忆等能力减退,大脑神经生理和结构的改变,以及脑信号传导异常,统称为糖尿病脑病。近年来的研究证据表明,糖尿病是AD的独立危险因素,且糖尿病可加速AD患者的病情进展。目前,糖尿病脑病的发病机制复杂且尚不明确。研究发现,海马中胰岛素受体(insulin receptor,IR)/胰岛素样生长因子1受体(insulin like growth factor-1 receptor,IGF-1R)及其下游信号通路障碍诱导了神经元的凋亡,参与了糖尿病脑病的发生发展。生长因子受体结合蛋白10 (Growth factor receptor bound protein 10, Grb10)对该信号通路起着重要的负性调节作用,其SH2和BPS区可以结合酪氨酸激酶受体(如IR、IGF-1R)。研究表明,经由IR/IGF-1R信号通路的Grb10具有生长抑制功能,在胰岛素及IGF-1介导的生长发育乃至神经元生长发育中均起着重要作用。另有研究发现,Grb10和GYF相互作用蛋白1(Grb10 Interacting GYF Protein 1,GIGYF1)通过绑定到Grb10 N末端后迅速地结合到IGF-1R上,增加了IGF-1刺激的受体酪氨酸磷酸化,进而调节IGF-1R信号通路。然而,GIGYF1协同Grb10调节IGF-1R信号通路影响糖尿病脑病大鼠认知功能的分子机制尚不明确。目的观察糖尿病脑病大鼠的行为学、海马组织超微结构和病理学的变化,以及IGF-1R信号通路相关分子在海马组织中的表达；并观察下调海马组织中Grb10的表达,对糖尿病脑病大鼠行为学、海马组织超微结构和病理学以及IGF-1R信号通路相关分子表达的影响,探讨GIGYF1协同Grb10调节IGF-1R信号通路影响糖尿病脑病大鼠认知功能的分子机制。方法健康雄性SD大鼠(7-8周龄,体质量200-250g),适应性喂养1W,禁食12h后,以pH为4.5的0.1mol/L柠檬酸钠缓冲液稀释STZ,避光并快速以6Omg/kg量一次性腹腔注射建立糖尿病大鼠模型。3天后,造模大鼠隔夜禁食不禁饮8h以上,取大鼠尾静脉血检测空腹血糖值,将空腹血糖18mmol/L的大鼠定为糖尿病大鼠,剔除血糖未达标者。建模成功1周后,利用立体定位技术将携带Grb 10-shRNA的慢病毒颗粒定点注射到海马组织中,将糖尿病大鼠随机分为：糖尿病对照组(DM组)、糖尿病假干预组(DM+0组)和糖尿病干预组(DM+shRNA组),每组10只；正常大鼠随机分为：正常对照组(con组)、正常假干预组(con+0组)和正常干预组(con+shRNA组),每组10只。在实验过程中,每周监测各组大鼠体重和血糖值。手术3个月后,通过水迷宫实验检测大鼠的行为学变化、电镜和光镜观察大鼠海马组织CA1区超微结构和病理学的变化,以及qRT-PCR和western blot检测各组海马组织中IGF-1R信号通路相关分子的表达。结果1.水迷宫结果显示：手术前和手术后,各组间的逃避潜伏期、平台穿越次数和目的象限内的时间均无明显改变(P0.05)；手术3个月后,DM组逃避潜伏期较con组、DM+shRNA组明显增加(p0.01),而平台穿越次数和目的象限内的时间均较con组、DM+shRNA组明显减少(p0.01)。2.海马CA1区电镜结果显示：糖尿病脑病大鼠海马神经元肿胀,神经纤维缠结,突触小体明显减少、形态和结构异常；下调DM组大鼠海马组织Grb10的表达,上述变化明显减少。3.海马CA1区HE染色结果显示：糖尿病脑病大鼠海马神经元数量明显减少且排列紊乱,并出现胞体肿胀、胞膜皱缩、胞质深染、胞核大且结构不清,以及神经胶质大量增生；下调DM组大鼠海马组织Grb10的表达,上述变化明显减轻,与正常大鼠海马CA1区形态和结构类似。免疫组织化学结果显示：Grb 10的表达聚集于细胞膜上；DM组海马组织中Grb10表达较con组和DM+shRNA组明显增多(p0.01)。4.qRT_PCR结果显示：与con组、DM+shRNA组相比,DM组大鼠海马组织中Grb10、GIGYF1的mRNA表达水平明显升高(P0.01),而IGF-1R的mRNA表达水平明显降低(P0.05)。5.Western Blot结果显示：DM组大鼠海马组织中Grb10、GIGYF1蛋白表达水平与con组、I)M+shRNA组相比明显升高(P0.01)；p-IRS1、p-IRS2、p-IGF-1R、p-Akt和p-Erkl/2蛋白表达水平与con组、DM+shRNA组相比明显降低(P0.01 or P0.05)。结论1.腹腔一次性注射STZ,可成功建立糖尿病大鼠模型；长期高血糖状态可导致糖尿病大鼠认知功能障碍,下调Grb10的表达可明显改善其认知功能障碍,表明糖尿病脑病大鼠的认知功能障碍与海马区Grb 10过表达相关。2.持续高血糖状态可导致大鼠海马组织Grb10、GIGYF1过表达,而(Grb10、-shRNA慢病毒颗粒可下调糖尿病大鼠海马Grb10的表达,且Grb10表达减少的同时并伴有GIGYF1表达的减少,表明GIGYF1协同Grb10调节IGF-1R及其下游信号通路。3.糖尿病大鼠海马组织Grb10表达水平的降低,可引起胰岛素受体底物IRS1和IRS2磷酸化水平的增加,表明下调Grb10的表达可通过活化IRS1和IRS2来调节下游信号通路。4.糖尿病大鼠海马组织Grb10表达水平的降低,可引起IGF-1R、Akt和Erk磷酸化水平的增加,表明下调Grb10的表达可促进IGF-1R及其下游信号通路(PI3K/Akt和Erkl/2信号通路)。综上所述,长期高血糖状态可导致大鼠海马组织内Grb10过表达,IGF-1R及其下游信号通路受到抑制,从而造成了神经元形态学的改变、功能受损、能量代谢障碍以及加速了神经元的凋亡,进而导致糖尿病脑病大鼠认知功能障碍；而早期下调糖尿病大鼠Grb 10的表达,GIGYF1可通过绑定于Grb10的N-末端,与IGF-1R间接结合,增加IGF.1R磷酸化表达水平,活化的IGF-1R通过活化受体底物(IRS)进一步激活下游P13K/Akt和Erk1/2信号通路,进而改善糖尿病脑病大鼠的认知功能障碍。Grb10在糖尿病脑病神经功能的调节中起着重要的作用,因此,通过靶向定点干预Grb10的表达可为糖尿病脑病的防治提供新的思路和方向。
[Abstract]:Background chronic hyperglycemia can lead to damage to the central nervous system, which leads to impairment of learning, memory, and neurophysiological and structural changes in the brain, as well as abnormal brain signals, known as diabetic encephalopathy. Recent evidence suggests that diabetes is a independent risk factor for AD, and diabetes can accelerate the condition of AD patients. Progress. At present, the pathogenesis of diabetic encephalopathy is complex and not clear. The study found that the insulin receptor (IR) / IR / insulin like growth factor receptor (insulin like growth factor-1 receptor, IGF-1R) and the downstream signal pathway barriers induce neuronal apoptosis and participate in the development of diabetic encephalopathy. The growth factor receptor binding protein 10 (Growth factor receptor bound protein 10, Grb10) plays an important negative regulatory role in the signaling pathway, and its SH2 and BPS regions can be combined with tyrosine kinase receptors (such as IR, IGF-1R). Growth and development and neuronal growth play an important role in both growth and development. Other studies have found that Grb10 and GYF interaction protein 1 (Grb10 Interacting GYF Protein 1, GIGYF1) quickly bind to IGF-1R by binding to the N terminal of Grb10, increasing tyrosine phosphorylation of IGF-1 stimulated receptor tyrosine, and then regulating the IGF-1R signal pathway. The molecular mechanism of IGYF1 with Grb10 to regulate the cognitive function of diabetic encephalopathy in rats is not clear. Objective To observe the behavior of the diabetic encephalopathy, the ultrastructure and pathology of the hippocampus, and the expression of the IGF-1R signaling pathway related molecules in the hippocampus, and to observe the Grb1 in the hippocampus in the down-regulation of the hippocampus. 0 expression, the influence of the behavior of diabetic encephalopathy, the ultrastructure and pathology of the hippocampus and the expression of IGF-1R signaling pathway, and to explore the molecular mechanism of GIGYF1 synergistic Grb10 regulating IGF-1R signaling pathway to affect the cognitive function of diabetic encephalopathy rats. Methods healthy male SD rats (7-8 weeks of age, body mass 200-250g), adaptation After feeding 1W, after fasting 12h, the 0.1mol/L citrate buffer solution of pH 4.5 was diluted STZ, and the diabetic rat model was established by intraperitoneal injection of 6Omg/kg at one time to establish the diabetic rat model.3 days later, and the rats were given the night fasting to drink 8h above, and the rat tail vein blood was taken to detect the fasting blood glucose value, and the rats with fasting blood sugar 18mmol/L were determined to be diabetes big. After 1 weeks of successful modeling, the rats were injected with Grb 10-shRNA lentivirus particles into the hippocampus, and the diabetic rats were randomly divided into diabetic control group (group DM), diabetic false intervention group (group DM+0) and glycuria intervention group (group DM+shRNA), with 10 rats in each group. It was divided into two groups: normal control group (Group con), normal intervention group (group con+0) and normal intervention group (group con+shRNA), with 10 rats in each group. During the experiment, the weight and blood sugar of each group were monitored every week. After 3 months of operation, the behavior changes of rats were detected by water maze test, and the ultrastructure of the hippocampus CA1 area was observed by electron microscope and light microscope. The changes in pathology, and the expression of IGF-1R signaling pathway related molecules in the hippocampus of each group were detected by qRT-PCR and Western blot. Results 1. water maze showed that the escape latency of each group before and after operation, the time of crossing the platform and the time in the target quadrant were not significantly changed (P0.05); after 3 months of operation, the DM group escaped. The incubation period was higher than that of the con group (P0.01), and the time in the number of platform traversing and the target quadrant were all more than that in the con group, and the DM+shRNA group decreased significantly (P0.01) in the.2. hippocampal CA1 area. The results showed that the hippocampal neurons were swollen, the nerve fibers were tangled, the synapses were significantly reduced, the morphological and structural abnormalities were reduced, and the DM group was down. The expression of Grb10 in the hippocampus of rats was significantly reduced by the above changes in the hippocampus CA1 region HE staining results showed that the number of hippocampal neurons in the hippocampus of the.3. rats was significantly reduced and disorderly, and the swelling of the cells, the membrane contraction, the deep cytoplasm, the large nucleus and the abnormal structure of the nucleus, and the proliferation of the glia in the hippocampus of the DM group, and the hippocampus of the DM group of rats were reduced. The expression of Grb10 was significantly reduced, similar to the morphology and structure of CA1 area in the hippocampus of normal rats. The immunohistochemical results showed that the expression of Grb 10 was aggregated on the cell membrane, and the expression of Grb10 in the hippocampus of DM group was significantly higher than that in the con group and the DM+shRNA group (P0.01).4.qRT_PCR results showed that the DM group was larger than the con group and DM+shRNA group. The mRNA expression level of Grb10 and GIGYF1 in rat hippocampus increased significantly (P0.01), while the mRNA expression level of IGF-1R decreased significantly (P0.05).5.Western Blot results showed that the Grb10 in the hippocampus of DM group was significantly higher than that of the IGF-1R group. The expression level was significantly lower than that in group con and group DM+shRNA (P0.01 or P0.05). Conclusion a diabetic rat model can be successfully established by 1. intraperitoneal injection of STZ, and long-term hyperglycemia can lead to cognitive dysfunction in diabetic rats. The expression of Grb10 can obviously improve the cognition dysfunction, indicating the cognition of diabetic encephalopathy rats. Dysfunction associated with overexpression of Grb 10 in the hippocampus.2. sustained hyperglycemia can lead to the expression of Grb10 and GIGYF1 in the hippocampus of rats. (Grb10, -shRNA lentivirus particles can downregulate the expression of Grb10 in the hippocampus of diabetic rats, and the decrease of Grb10 expression is accompanied by a decrease in GIGYF1 expression, indicating that GIGYF1 synergistic Grb10 regulates IGF-1R and its lower levels. The decrease of Grb10 expression level in hippocampus of.3. diabetic rats induced the increase of insulin receptor substrate IRS1 and IRS2 phosphorylation level, indicating that the down regulation of Grb10 can be regulated by activating IRS1 and IRS2 to regulate the decrease of Grb10 expression in the hippocampus of the lower signal pathway of.4. diabetic rats, which can cause IGF-1R, Akt and Erk. The increase of phosphorylation level indicates that down regulation of Grb10 expression can promote IGF-1R and its downstream signaling pathway (PI3K/Akt and Erkl/2 signaling pathway). To sum up, long-term hyperglycemia can lead to Grb10 overexpression in the hippocampus of rats and the inhibition of IGF-1R and its downstream signal pathways, resulting in morphological changes of neurons and impairment of function. The disturbance of energy metabolism and the acceleration of neuronal apoptosis and the cognitive impairment in diabetic rats, and the early downregulation of the expression of Grb 10 in diabetic rats, GIGYF1 can be bound to the N- terminal of Grb10 and indirectly combined with IGF-1R to increase the level of phosphorylation of IGF.1R, and the activated IGF-1R passes through the activated receptor substrate (IRS). One step to activate the downstream P13K/Akt and Erk1/2 signaling pathway, and then improve the cognitive dysfunction.Grb10 in diabetic encephalopathy, plays an important role in the regulation of the neurological function of diabetic encephalopathy. Therefore, the intervention of the target targeted Grb10 expression can provide new ideas and directions for the prevention and control of diabetic encephalopathy.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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