發(fā)布時間：2018-05-24 05:36

  本文選題：糖尿病腦病 + 認(rèn)知　； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景長期高血糖狀態(tài)可導(dǎo)致中樞神經(jīng)系統(tǒng)損害,進(jìn)而引起學(xué)習(xí)、記憶等能力減退,大腦神經(jīng)生理和結(jié)構(gòu)的改變,以及腦信號傳導(dǎo)異常,統(tǒng)稱為糖尿病腦病。近年來的研究證據(jù)表明,糖尿病是AD的獨立危險因素,且糖尿病可加速AD患者的病情進(jìn)展。目前,糖尿病腦病的發(fā)病機制復(fù)雜且尚不明確。研究發(fā)現(xiàn),海馬中胰島素受體(insulin receptor,IR)/胰島素樣生長因子1受體(insulin like growth factor-1 receptor,IGF-1R)及其下游信號通路障礙誘導(dǎo)了神經(jīng)元的凋亡,參與了糖尿病腦病的發(fā)生發(fā)展。生長因子受體結(jié)合蛋白10 (Growth factor receptor bound protein 10, Grb10)對該信號通路起著重要的負(fù)性調(diào)節(jié)作用,其SH2和BPS區(qū)可以結(jié)合酪氨酸激酶受體(如IR、IGF-1R)。研究表明,經(jīng)由IR/IGF-1R信號通路的Grb10具有生長抑制功能,在胰島素及IGF-1介導(dǎo)的生長發(fā)育乃至神經(jīng)元生長發(fā)育中均起著重要作用。另有研究發(fā)現(xiàn),Grb10和GYF相互作用蛋白1(Grb10 Interacting GYF Protein 1,GIGYF1)通過綁定到Grb10 N末端后迅速地結(jié)合到IGF-1R上,增加了IGF-1刺激的受體酪氨酸磷酸化,進(jìn)而調(diào)節(jié)IGF-1R信號通路。然而,GIGYF1協(xié)同Grb10調(diào)節(jié)IGF-1R信號通路影響糖尿病腦病大鼠認(rèn)知功能的分子機制尚不明確。目的觀察糖尿病腦病大鼠的行為學(xué)、海馬組織超微結(jié)構(gòu)和病理學(xué)的變化,以及IGF-1R信號通路相關(guān)分子在海馬組織中的表達(dá)；并觀察下調(diào)海馬組織中Grb10的表達(dá),對糖尿病腦病大鼠行為學(xué)、海馬組織超微結(jié)構(gòu)和病理學(xué)以及IGF-1R信號通路相關(guān)分子表達(dá)的影響,探討GIGYF1協(xié)同Grb10調(diào)節(jié)IGF-1R信號通路影響糖尿病腦病大鼠認(rèn)知功能的分子機制。方法健康雄性SD大鼠(7-8周齡,體質(zhì)量200-250g),適應(yīng)性喂養(yǎng)1W,禁食12h后,以pH為4.5的0.1mol/L檸檬酸鈉緩沖液稀釋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飲8h以上,取大鼠尾靜脈血檢測空腹血糖值,將空腹血糖18mmol/L的大鼠定為糖尿病大鼠,剔除血糖未達(dá)標(biāo)者。建模成功1周后,利用立體定位技術(shù)將攜帶Grb 10-shRNA的慢病毒顆粒定點注射到海馬組織中,將糖尿病大鼠隨機分為：糖尿病對照組(DM組)、糖尿病假干預(yù)組(DM+0組)和糖尿病干預(yù)組(DM+shRNA組),每組10只；正常大鼠隨機分為：正常對照組(con組)、正常假干預(yù)組(con+0組)和正常干預(yù)組(con+shRNA組),每組10只。在實驗過程中,每周監(jiān)測各組大鼠體重和血糖值。手術(shù)3個月后,通過水迷宮實驗檢測大鼠的行為學(xué)變化、電鏡和光鏡觀察大鼠海馬組織CA1區(qū)超微結(jié)構(gòu)和病理學(xué)的變化,以及qRT-PCR和western blot檢測各組海馬組織中IGF-1R信號通路相關(guān)分子的表達(dá)。結(jié)果1.水迷宮結(jié)果顯示：手術(shù)前和手術(shù)后,各組間的逃避潛伏期、平臺穿越次數(shù)和目的象限內(nèi)的時間均無明顯改變(P0.05)；手術(shù)3個月后,DM組逃避潛伏期較con組、DM+shRNA組明顯增加(p0.01),而平臺穿越次數(shù)和目的象限內(nèi)的時間均較con組、DM+shRNA組明顯減少(p0.01)。2.海馬CA1區(qū)電鏡結(jié)果顯示：糖尿病腦病大鼠海馬神經(jīng)元腫脹,神經(jīng)纖維纏結(jié),突觸小體明顯減少、形態(tài)和結(jié)構(gòu)異常；下調(diào)DM組大鼠海馬組織Grb10的表達(dá),上述變化明顯減少。3.海馬CA1區(qū)HE染色結(jié)果顯示：糖尿病腦病大鼠海馬神經(jīng)元數(shù)量明顯減少且排列紊亂,并出現(xiàn)胞體腫脹、胞膜皺縮、胞質(zhì)深染、胞核大且結(jié)構(gòu)不清,以及神經(jīng)膠質(zhì)大量增生；下調(diào)DM組大鼠海馬組織Grb10的表達(dá),上述變化明顯減輕,與正常大鼠海馬CA1區(qū)形態(tài)和結(jié)構(gòu)類似。免疫組織化學(xué)結(jié)果顯示：Grb 10的表達(dá)聚集于細(xì)胞膜上；DM組海馬組織中Grb10表達(dá)較con組和DM+shRNA組明顯增多(p0.01)。4.qRT_PCR結(jié)果顯示：與con組、DM+shRNA組相比,DM組大鼠海馬組織中Grb10、GIGYF1的mRNA表達(dá)水平明顯升高(P0.01),而IGF-1R的mRNA表達(dá)水平明顯降低(P0.05)。5.Western Blot結(jié)果顯示：DM組大鼠海馬組織中Grb10、GIGYF1蛋白表達(dá)水平與con組、I)M+shRNA組相比明顯升高(P0.01)；p-IRS1、p-IRS2、p-IGF-1R、p-Akt和p-Erkl/2蛋白表達(dá)水平與con組、DM+shRNA組相比明顯降低(P0.01 or P0.05)。結(jié)論1.腹腔一次性注射STZ,可成功建立糖尿病大鼠模型；長期高血糖狀態(tài)可導(dǎo)致糖尿病大鼠認(rèn)知功能障礙,下調(diào)Grb10的表達(dá)可明顯改善其認(rèn)知功能障礙,表明糖尿病腦病大鼠的認(rèn)知功能障礙與海馬區(qū)Grb 10過表達(dá)相關(guān)。2.持續(xù)高血糖狀態(tài)可導(dǎo)致大鼠海馬組織Grb10、GIGYF1過表達(dá),而(Grb10、-shRNA慢病毒顆�？上抡{(diào)糖尿病大鼠海馬Grb10的表達(dá),且Grb10表達(dá)減少的同時并伴有GIGYF1表達(dá)的減少,表明GIGYF1協(xié)同Grb10調(diào)節(jié)IGF-1R及其下游信號通路。3.糖尿病大鼠海馬組織Grb10表達(dá)水平的降低,可引起胰島素受體底物IRS1和IRS2磷酸化水平的增加,表明下調(diào)Grb10的表達(dá)可通過活化IRS1和IRS2來調(diào)節(jié)下游信號通路。4.糖尿病大鼠海馬組織Grb10表達(dá)水平的降低,可引起IGF-1R、Akt和Erk磷酸化水平的增加,表明下調(diào)Grb10的表達(dá)可促進(jìn)IGF-1R及其下游信號通路(PI3K/Akt和Erkl/2信號通路)。綜上所述,長期高血糖狀態(tài)可導(dǎo)致大鼠海馬組織內(nèi)Grb10過表達(dá),IGF-1R及其下游信號通路受到抑制,從而造成了神經(jīng)元形態(tài)學(xué)的改變、功能受損、能量代謝障礙以及加速了神經(jīng)元的凋亡,進(jìn)而導(dǎo)致糖尿病腦病大鼠認(rèn)知功能障礙；而早期下調(diào)糖尿病大鼠Grb 10的表達(dá),GIGYF1可通過綁定于Grb10的N-末端,與IGF-1R間接結(jié)合,增加IGF.1R磷酸化表達(dá)水平,活化的IGF-1R通過活化受體底物(IRS)進(jìn)一步激活下游P13K/Akt和Erk1/2信號通路,進(jìn)而改善糖尿病腦病大鼠的認(rèn)知功能障礙。Grb10在糖尿病腦病神經(jīng)功能的調(diào)節(jié)中起著重要的作用,因此,通過靶向定點干預(yù)Grb10的表達(dá)可為糖尿病腦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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳同度,張昌穎;素食大鼠的貧血現(xiàn)象[J];營養(yǎng)學(xué)報;1957年04期

2 陳偉強;趙善廣;;自制注射用大鼠固定裝置[J];上海實驗動物科學(xué);1992年04期

3 肖柳英,林培英,馮昭明,張丹;不同周齡的SD大鼠生理、生化及體重的正常值測定[J];中藥新藥與臨床藥理;1996年03期

4 李淑云;簡易大鼠灌胃器的制作[J];錦州醫(yī)學(xué)院學(xué)報;2001年04期

5 楊明智,陳積圣;一種大鼠抓取與固定的新工具介紹[J];上海實驗動物科學(xué);2001年03期

6 戴英,陸群;復(fù)方H_(505)對Wistar大鼠外周血的血液流變學(xué)指標(biāo)的影響[J];中國血液流變學(xué)雜志;2001年01期

7 韋應(yīng)波,孫喜慶,曹新生,姚永杰,馮岱雅,楊長斌;+Gz暴露時間對大鼠記憶功能和行為的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年01期

8 呂學(xué)軍,郭俊生,李敏,周利梅,張永娟;暈船大鼠體內(nèi)鐵含量的變化[J];中國職業(yè)醫(yī)學(xué);2003年04期

9 湯仁仙,王迎偉,王慧,周峰;201A中藥合劑對大鼠抗腎小球基底膜腎炎病變的影響[J];徐州醫(yī)學(xué)院學(xué)報;2003年06期

10 孫同柱,付小兵,翁立新,梁雪梅,陳偉;介紹一種簡易的大鼠保定方法[J];上海實驗動物科學(xué);2004年01期

相關(guān)會議論文 前10條

1 尹音;孫振宇;胡敏;李冬霞;;持續(xù)性高正加速度對大鼠顳頜關(guān)節(jié)損傷的作用[A];第八屆全國顳下頜關(guān)節(jié)病學(xué)及（牙合）學(xué)大會論文匯編[C];2011年

2 祝~=驤;iJ梊霞;洃克琴;崔素英;文允摪;，

本文編號：1927873