本文選題：七氟烷 + 認知功能��； 參考：《天津醫(yī)科大學》2017年博士論文

【摘要】：七氟烷(Sevoflurane)是新生兒及兒童臨床上最常應用的全身麻醉藥(General Anesthetics)[1,2],其對小兒發(fā)育期大腦的神經(jīng)毒性受到廣泛關(guān)注,但具體機制尚不清楚。研究表明,兒童期多次暴露于全身麻醉藥物更易造成其發(fā)育期大腦的神經(jīng)毒性,從而影響其遠期學習記憶能力(Learning ability)[3-5],而Tau蛋白磷酸化在其認知功能損傷的發(fā)生發(fā)展中具有重要的作用[6-9]。近年來,有研究指出,非微管結(jié)合Tau蛋白(Microtubule-unbound Tau,MUT),可能是Tau蛋白代謝異常(磷酸化及異常聚集)的早期標志,而能量及Nuak1(Nuak family SNF1-like kinase 1)對其有調(diào)控作用[10,11]。本課題以Nuak1/Tau信號通路為切入點,深入研究七氟烷多次麻醉對幼年及成年小鼠認知功能及神經(jīng)損傷的影響,并探討其分子機制,同時尋求相關(guān)腦保護方法。實驗一:七氟烷多次麻醉對不同年齡小鼠認知功能改變及Tau蛋白表達的影響目的:七氟烷多次麻醉可致幼年小鼠遠期認知功能障礙,Tau蛋白過度磷酸化可造成神經(jīng)損傷,而非微管結(jié)合Tau蛋白可能是Tau蛋白早期代謝的標志。本實驗擬探討七氟烷多次麻醉對幼年(P6)及成年(P60)小鼠遠期認知功能改變及不同類型Tau蛋白及其磷酸化位點表達的影響。方法:出生6 d(P6)及出生60 d(P60)小鼠各52只隨機分為4組(n=26/組):幼年對照組(P6+Control)、幼年七氟烷組(P6+Sevoflurane)、成年對照組(P60+Control)、成年七氟烷組(P60+Sevoflurane)。其中七氟烷組給予3%七氟烷+60%O2處理,每天2 h,連續(xù)3 d,對照組只給予60%O2處理,每天2 h,連續(xù)3 d。七氟烷連續(xù)處理后22 d,對不同組小鼠(P30,P84)進行Morris水迷宮實驗檢測其認知功能;七氟烷連續(xù)處理后0 d(P8,P62),取小鼠海馬及皮層組織,應用ELISA檢測total Tau表達、RT-PCR檢測Tau m RNA表達、Western blot檢測total Tau(Tau46),T22(Tau蛋白早期聚合物,可溶性低聚體,oligomers)Tau-PS202/PT205(PHF-Tau),Tau-PS356(R4)表達、微管結(jié)合實驗檢測不同類型Tau蛋白(微管結(jié)合Tau蛋白及非微管結(jié)合Tau蛋白)表達、熒光染色檢測Tau蛋白早期聚集(T22)、串聯(lián)質(zhì)譜分析檢測Tau蛋白不同磷酸化位點表達。結(jié)果:1.七氟烷多次麻醉可致幼年小鼠遠期認知功能損傷,而對成年小鼠沒有影響;2.幼年小鼠皮層和海馬組織中Tau m RNA及total Tau蛋白表達均高于成年小鼠;3.幼年小鼠皮層及海馬組織T22及Tau-PS356表達明顯高于成年小鼠,七氟烷處理可使幼年小鼠皮層及海馬Tau-PS202/PT205表達增加,而成年小鼠則沒有差異;4.幼年小鼠海馬及皮層主要為非微管結(jié)合Tau蛋白,而成年小鼠主要為微管結(jié)合Tau蛋白;5.幼年小鼠腦片中可在其大腦皮層發(fā)現(xiàn)Tau蛋白早期聚集(可溶性低聚體,T22),而成年小鼠腦片不見聚集;6.七氟烷多次麻醉可致幼年小鼠皮層大部分Tau蛋白位點磷酸化增強,而對成年小鼠影響不大。結(jié)論:幼年小鼠與成年小鼠相比,其發(fā)育期大腦更易遭受到七氟烷多次麻醉的打擊,造成Tau蛋白多位點磷酸化增加,從而影響其遠期認知功能,其機制與非微管結(jié)合Tau蛋白表達密切相關(guān)。實驗二:Nuak1在七氟烷多次麻醉所致發(fā)育期小鼠大腦神經(jīng)毒性中的關(guān)鍵作用目的:2016年,一項新研究指出,Nuak1(Nuak family DNF1-like kinase 1,又叫做AMPK related protein 5,ARK5)可通過選擇性磷酸化位于Tau蛋白重復序列(R4)上的Ser356位點,從而調(diào)節(jié)Tau蛋白代謝。本實驗擬通過探討Nuak1對Tau蛋白的調(diào)控作用,通過應用Nuak1特異性阻滯劑HTH-01-015,擬證明Nuak1在七氟烷多次麻醉所致發(fā)育期小鼠大腦神經(jīng)毒性中的關(guān)鍵作用。方法:出生6 d(P6)及出生60 d(P60)小鼠各12只隨機分為4組(n=6/組):幼年對照組(P6+Control)、幼年七氟烷組(P6+Sevoflurane)、成年對照組(P60+Control)、成年七氟烷組(P60+Sevoflurane),相關(guān)處理同實驗一。多次麻醉后0 d(P8,P62)后立即處死小鼠,提取其海馬及皮層組織,應用RT-PCR及Western blot檢測不同年齡組小鼠大腦皮層及海馬Nuak1基因及蛋白表達、應用串聯(lián)質(zhì)譜(MS/MS)分析Nuak1不同磷酸化位點表達;另選80只P6小鼠,隨機分為4組(n=20/組):對照+溶劑組(Control+Vehicle)、七氟烷+溶劑組(Sevoflurane+Vehicle)、對照+Nuak1阻滯劑組(Control+HTH-01-015)、七氟烷+Nuak1阻滯劑組(Sevoflurane+HTH-01-015)。七氟烷及氧氣處理同上,所有P6小鼠于七氟烷麻醉及氧氣處理前30 min,腹腔注射(i.p.)Nuak1特異性阻滯劑HTH-01-015(10 mg/kg)或者溶劑(生理鹽水+DMSO)100μL/次,連續(xù)注射3 d,并于七氟烷處理后22 d(P30)通過Morris水迷宮實驗檢測認知功能;于七氟烷及氧氣處理后0 d,取皮層及海馬組織,通過Western blot實驗檢測Nuak1、total Tau(Tau46)、T22、Tau-PS356及Tau-PS202/PT205表達、通過免疫熒光染色檢測T22蛋白聚集情況、通過微管結(jié)合實驗檢測不同類型Tau蛋白(微管結(jié)合Tau蛋白及非微管結(jié)合Tau蛋白)表達。結(jié)果:1.幼年小鼠大腦皮層及海馬Nuak1蛋白表達明顯高于成年小鼠,而Nuak1基因表達(m RNA)與成年小鼠相比沒有統(tǒng)計學意義;2.串聯(lián)質(zhì)譜分析得出,幼年小鼠Nuak1只有2個磷酸化位點(Nuak1-PS389,Nuak1-PS446)表達,并且幼年小鼠皮層組織Nuak1磷酸化表達明顯低于成年小鼠;3.給予Nuak1特異性阻滯劑HTH-01-015后,對于幼年小鼠,七氟烷多次處理并沒有造成其遠期認知功能損傷;4.給予Nuak1特異性阻滯劑后,幼年小鼠大腦皮層及海馬Nuak1及total Tau表達沒有差異,而Tau-PS356、Tau-PS262/PT205表達與對照+溶劑組相比均降低,且七氟烷處理并不能使其增加;5.給予Nuak1阻滯劑后,幼年小鼠皮層及海馬中的非微管結(jié)合Tau蛋白明顯減少,Tau蛋白早期聚集明顯降低。結(jié)論:Nuak1通過選擇性磷酸化位于重復序列R4上的Tau-PS356,使Tau蛋白與微管解離,從而增加非微管結(jié)合Tau蛋白含量,七氟烷多次麻醉可刺激非微管結(jié)合Tau蛋白,從而激活GSK3β,正反饋形成超磷酸化,從而產(chǎn)生神經(jīng)毒性,以致遠期認知功能障礙。而Nuak1含量受自身磷酸化調(diào)節(jié),其在腦中的蛋白表達與其自身磷酸化成反比。實驗三:能量不足(Energy deprivation)在七氟烷多次麻醉致小鼠發(fā)育期大腦神經(jīng)毒性中的作用目的:研究指出,能量不足(Energy deprivation)和氧化應激(Oxidative stress)可能是導致Tau蛋白磷酸化聚集的主要原因[12]。本實驗通過應用維生素K2(一種能量補充劑),擬證明能量在七氟烷所致小鼠發(fā)育期大腦神經(jīng)毒性中的作用。方法:出生6 d(P6)及出生60 d(P60)小鼠各12只隨機分為4組(n=6/組):幼年對照組(P6+Control)、幼年七氟烷組(P6+Sevoflurane)、成年對照組(P60+Control)、成年七氟烷組(P60+Sevoflurane),相關(guān)處理同實驗一。多次麻醉后0d(P8,P62)處死小鼠,提取皮層組織,利用ATP檢測試劑盒檢測不同年齡組小鼠ATP表達;另選80只P6小鼠,隨機分為4組(n=20/組):對照+玉米油組(Control+Corn oil)、七氟烷+玉米油組(Sevoflurane+Corn oil)、對照+維生素K2組(Control+Vitamin K2)、七氟烷+維生素K2組(Sevoflurane+Vitamin K2)。七氟烷組小鼠給予3%七氟烷+60%O2處理,每天2 h,連續(xù)3 d,對照組只給予60%O2處理,每天2 h,連續(xù)3 d。所有P6小鼠于七氟烷麻醉及氧氣處理前30 min,腹腔注射(i.p.)Vitamin K2(100 mg/kg)或者玉米油100μL/次,連續(xù)注射3 d,并于七氟烷處理后22 d(P30)通過Morris水迷宮實驗檢測認知功能;于七氟烷及氧氣處理后0 d,取皮層及海馬組織,通過ELISA檢測total Tau表達、通過Western blot實驗檢測Nuak1、total Tau(Tau46)、T22、Tau-PS356,Tau-PS202/PT205表達、通過免疫熒光染色檢測Tau蛋白聚集情況、通過微管結(jié)合實驗檢測不同類型Tau表達。結(jié)果:1.幼年小鼠皮層的ATP表達明顯低于成年小鼠,且七氟烷刺激可使ATP明顯降低;2.給予Vitamin K2后,對于P6小鼠,七氟烷多次處理并沒有造成其遠期認知功能損傷;3.給予Vitamin K2后,P6小鼠大腦皮層及海馬total Tau蛋白表達沒有差異,而Nuak1、Tau-PS356、Tau-PS262/PT205表達與對照+玉米油組相比均降低,且七氟烷處理并不能使其增加;4.給予Vitamin K2后,幼年小鼠皮層及海馬中的非微管結(jié)合Tau蛋白明顯減少,Tau蛋白早期聚集(T22)明顯消失。結(jié)論:幼年小鼠大腦皮層及海馬能量表達明顯低于成年小鼠,而能量不足(能量剝奪)本身可以激活AMPK,使得Nuak1表達增加,從而使得Tau蛋白,特別是非微管結(jié)合Tau蛋白的含量增加,造成神經(jīng)脆弱性(Neuronal vulnerability),形成Tau蛋白早期聚集,由此,在七氟烷多次麻醉的刺激下,Tau蛋白大量脫微管,磷酸化甚至超磷酸化,從而造成神經(jīng)發(fā)育損傷,影響遠期認知及學習功能。小結(jié)1.七氟烷多次麻醉可造成幼年小鼠遠期認知功能障礙,對成年小鼠沒有影響;2.幼年小鼠發(fā)育期大腦中主要表達非微管結(jié)合Tau蛋白,而成年小鼠大腦主要表達微管結(jié)合Tau蛋白;3.幼年小鼠發(fā)育期大腦皮層中Tau蛋白早期聚集造成神經(jīng)脆弱性,在七氟烷多次麻醉下形成Tau蛋白大量位點超磷酸化,從而造成神經(jīng)損傷;4.Nuak1通過選擇性磷酸化Tau-PS356以調(diào)節(jié)Tau蛋白代謝,在七氟烷多次麻醉致小鼠發(fā)育期大腦神經(jīng)毒性中起關(guān)鍵作用;5.幼年小鼠大腦ATP含量遠遠低于成年小鼠,能量嚴重不足;6.能量不足可使Nuak1磷酸化降低,從而使Nuak1表達增加,并造成幼年小鼠發(fā)育期大腦神經(jīng)脆弱性,可能是七氟烷多次麻醉致小鼠發(fā)育期大腦神經(jīng)損傷的根本原因。
[Abstract]:Seven fluorane (Sevoflurane) is the most commonly used general anesthetic (General Anesthetics) [1,2] for newborns and children. The neurotoxicity of the brain is widely concerned in the development of children, but the specific mechanism is not clear. It affects its long term learning and memory (Learning ability) [3-5], and Tau protein phosphorylation plays an important role in the occurrence and development of cognitive impairment. In recent years, studies have shown that non microtubules combined with Tau protein (Microtubule-unbound Tau, MUT), can be the early stage of metabolic abnormalities of Tau proteins (phosphorylation and abnormal aggregation). Mark, energy and Nuak1 (Nuak family SNF1-like kinase 1) have a regulatory effect on [10,11]. this topic with Nuak1/Tau signaling pathway as the breakthrough point, in-depth study of the effects of seven halothane multiple anaesthesia on cognitive function and nerve damage in young and adult mice, and explore its molecular mechanism, and seek related brain protection methods. Experiment 1: Seven The effects of multiple anaesthesia on cognitive function changes and Tau protein expression in mice of different ages: seven fluoroalkanes can cause long-term cognitive impairment in young mice. Excessive phosphorylation of Tau protein can cause nerve damage, while non microtubule combined with Tau protein may be a marker of early metabolism of Tau protein. This experiment is to discuss seven fluoroalkanes. The effect of secondary anesthesia on the long-term cognitive function changes and the expression of different types of Tau protein and its phosphorylation sites in young (P6) and adult (P60) mice. Methods: 52 mice born 6 d (P6) and 60 d (P60) mice were randomly divided into 4 groups (n=26/ group): juvenile control group (P6+ Control), juvenile seven fluorane group (P6+Sevoflurane), adult control group (P60+Control), In the adult seven fluorane group (P60+Sevoflurane), the seven halothane group was given 3% seven fluorothane +60%O2 treatment, 2 h a day and 3 D continuously. The control group was given only 60%O2 treatment, 2 h a day, and 22 D continuously after 3 d. seven Fluoroalkane continuous treatment, and the cognitive function was detected in the Morris water maze test for different groups of mice (P30, P84); 0 d (seven fluorothane) after continuous treatment. The expression of total Tau was detected by ELISA, and Tau m RNA expression was detected by RT-PCR, Western blot detected total Tau (Tau46). The expression of non microtubule combined with Tau protein, the early aggregation of Tau protein (T22) was detected by fluorescence staining, and the expression of different phosphorylation sites of Tau protein was detected by tandem mass spectrometry. Results: 1. seven halothane multiple anaesthesia could cause long-term cognitive impairment in young mice, but no effect on adult mice; 2. the Tau m RNA and total T in the cortex and hippocampus of young mice. The expression of Au protein was higher than that of adult mice; the expression of T22 and Tau-PS356 in the cortex and hippocampus of 3. young mice was significantly higher than that of adult mice. Seven halothane treatment could increase the expression of Tau-PS202/PT205 in the cortex and hippocampus of young mice, but there was no difference in adult mice. 4. of the hippocampus and cortex of young mice were mainly non microtubules combined with Tau protein, and adult mice were small. The rats were mainly microtubules combined with Tau protein; 5. young mice brain slices could find early aggregation of Tau protein (soluble oligomer, T22) in its cerebral cortex, while the brain slices of adult mice were not clustered; 6. seven fluoroalkanes could increase the phosphorylation of most of the Tau protein loci in young mice, but little effect on adult mice. Conclusion: young young mice are small. Compared with adult mice, the brain of the adult mice is more susceptible to the attack of seven halothane anaesthesia, resulting in the increase of Tau protein multipoint phosphorylation, which affects its long-term cognitive function. The mechanism is closely related to the non microtubule binding of Tau protein. Experiment two: Nuak1 in the brain neurotoxicity induced by seven fluoroalkanes during multiple anaesthesia. In 2016, a new study indicated that Nuak1 (Nuak family DNF1-like kinase 1, also called AMPK related protein 5, ARK5) can selectively phosphorylate the Ser356 sites on the Tau protein repeat sequence (R4), thus regulating the metabolism of proteins. Nuak1 specific blocker HTH-01-015 was used to prove the key role of Nuak1 in the brain neurotoxicity of mice induced by seven halothane anesthesia. Methods: 12 mice born 6 d (P6) and 60 d (P60) mice were randomly divided into 4 groups (n=6/ group): juvenile control group (P6+ Control), juvenile seven halothane group (P6+Sevoflurane), adult control group (P60+Contr) OL), adult seven fluorane group (P60+Sevoflurane), related treatment with experimental one. After multiple anesthesia 0 d (P8, P62) immediately after the death of mice, extract the hippocampus and cortical tissue, the use of RT-PCR and Western blot to detect the cerebral cortex and hippocampus Nuak1 gene and protein expression in different age groups of mice, using tandem mass spectrometry (MS/MS) to analyze Nuak1 phosphorylation 80 P6 mice were randomly divided into 4 groups (group n=20/): control + solvent group (Control+Vehicle), seven fluorane + solvent group (Sevoflurane+Vehicle), control +Nuak1 blocker group (Control+HTH-01-015), seven fluorothane +Nuak1 blocker group (Sevoflurane+ HTH-01-015). Seven halothane and oxygen treatment, all P6 mice were anesthetized with seven halothane and anaesthesia. 30 min before oxygen treatment, intraperitoneal injection (i.p.) Nuak1 specific blocker HTH-01-015 (10 mg/kg) or solvent (physiological saline +DMSO) 100 mu L/ time, continuous injection of 3 D, and 22 D (P30) after seven fluorane treated by Morris water maze test to detect cognitive function; seven fluorane and oxygen treated 0 d, cortex and hippocampus tissue, through The expression of Nuak1, total Tau (Tau46), T22, Tau-PS356 and Tau-PS202/PT205 was detected. The aggregation of T22 protein was detected by immunofluorescence staining. The expression of different types Tau protein (microtubule binding Tau protein and non microtubule binding Tau protein) was detected by microtubule binding assay. The expression of Nuak1 protein in the cerebral cortex and hippocampus of 1. young mice was obvious. Compared with adult mice, the expression of Nuak1 gene (m RNA) was not statistically significant compared with adult mice. 2. tandem mass spectrometry analysis showed that there were only 2 phosphorylation sites (Nuak1-PS389, Nuak1-PS446) expression of Nuak1 in young mice, and the Nuak1 phosphorylation of the cortical tissue of young mice was significantly lower than that of adult mice; 3. gave Nuak1 specific blocker HTH. After -01-015, for young mice, seven Fluoroalkane multiple treatments did not cause their long-term cognitive impairment. 4. after Nuak1 specific blockers were given, the expression of Nuak1 and total Tau in the cerebral cortex and hippocampus of young mice was not different, while Tau-PS356, Tau-PS262/PT205 expression was lower than that of the control + solvent group, and seven fluorothane treatment did not make it possible. It increased; 5. after the Nuak1 blocker was given, the non microtubule binding Tau protein in the cortex and hippocampus of the young mice decreased obviously, and the early aggregation of Tau protein decreased obviously. Conclusion: Nuak1 dissociates the Tau protein from the microtubule by selectively phosphorylation of Tau-PS356 on the repeated sequence R4, and increases the content of the non microtubule binding Tau protein, and the seven Fluoroalkane multiple times. Anesthesia stimulates non microtubule binding Tau protein, thus activating GSK3 beta, positive feedback forms hyper phosphorylation, resulting in neurotoxicity, resulting in long-term cognitive dysfunction. The Nuak1 content is regulated by autophosphorylation and its protein expression in the brain is inversely proportional to its own phosphorylation. Three: Energy deprivation (Energy deprivation) is more than seven fluoroalkanes. The role of secondary anesthesia in the brain neurotoxicity of mice during development: the study indicated that Energy deprivation and oxidative stress (Oxidative stress) may be the main cause of the aggregation of Tau protein phosphorylation. This experiment was used to prove that the energy was induced by seven halothane in mice by using vitamin K2 (a energy supplement). Methods: 6 d (P6) and 60 d (P60) mice were randomly divided into 4 groups (n=6/ group): young control group (P6+Control), juvenile seven fluorane group (P6+Sevoflurane), adult control group (P60+Control), adult seven fluorane group (P60+Sevoflurane), related treatment and experiment one. 0d (P8, P62) after multiple anesthesia were executed. In mice, the cortex tissues were extracted and the ATP detection kit was used to detect ATP expression in different age groups. 80 P6 mice were randomly divided into 4 groups (group n=20/): control + corn oil group (Control+Corn oil), seven fluorane + corn oil group (Sevoflurane+Corn oil), control + vitrein K2 group (Control+Vitamin K2), seven halothane + vitamin K2 group Amin K2). Seven fluorane group mice were treated with 3% seven fluorane +60%O2 treatment, 2 h a day, 3 D. The control group was given only 60%O2 treatment, 2 h per day, 3 d. in 3 d. and 30 min before seven Fluoroalkane. The cognitive function was detected by the Morris water maze test. The expression of total Tau was detected by ELISA and 0 d after seven Fluoroalkane and oxygen treatment. The expression of Nuak1, total Tau (Tau46), T22, expression were detected by Western blot experiment. The aggregation of protein was detected by immunofluorescence staining and combined with microtubule through immunofluorescence staining. The expression of different types of Tau was detected in the experiment. Results: the expression of ATP in the cortex of 1. young mice was significantly lower than that of adult mice, and the stimulation of seven halothane decreased the ATP obviously. 2. to P6 mice after Vitamin K2, the multiple treatment of seven Fluoroalkane did not cause its long-term cognitive impairment; 3. Vitamin K2, P6 mice cerebral cortex and hippocampus total Tau. There was no difference in protein expression, but the expression of Nuak1, Tau-PS356 and Tau-PS262/PT205 decreased compared with the control + corn oil group, and seven halothane treatment did not increase it. 4. after Vitamin K2, the non microtubule binding Tau protein in the cortex and hippocampus of young mice decreased obviously, and the early aggregation of Tau egg white (T22) disappeared. Conclusion: the brain of young mice. The energy expression in the cortex and hippocampus is obviously lower than that of the adult mice, and the energy deprivation (energy deprivation) itself can activate AMPK and increase the expression of Nuak1, thus increasing the content of Tau protein, especially the non microtubule combined with Tau protein, causing nervous vulnerability (Neuronal vulnerability), forming the early aggregation of the Tau protein, thus, in seven Fluoroalkane multiple times. Under the stimulation of anesthesia, Tau protein has a large number of microtubules, phosphorylation and even hyper phosphorylation, which can cause neurodevelopmental damage and affect long-term cognitive and learning functions. 1. seven halothane multiple anaesthesia can cause long-term cognitive impairment in young mice and no effect on adult mice; 2. the main expression of non microtubule binding in the brain of young mice. Tau protein, while the adult mouse brain mainly expresses microtubule combined with Tau protein; 3. early accumulation of Tau protein in the cerebral cortex of young mice causes nerve fragility, resulting in the formation of a large number of Tau protein sites hyper phosphorylation under seven halothane anaesthesia, resulting in nerve damage; 4.Nuak1 regulates the Tau protein passage through selective phosphorylation of Tau-PS356. It plays a key role in the brain neurotoxicity of mice during the development of seven fluorothane. 5. the ATP content in the brain of 5. young mice is much lower than that of adult mice, and the energy is seriously insufficient; the deficiency of 6. energy can reduce the phosphorylation of Nuak1, thus increasing the expression of Nuak1 and causing the nervous fragility of the brain in the developmental stage of the young rats, which may be more than seven Fluoroalkane. The underlying cause of brain damage during developmental period is secondary anesthesia.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R614

【相似文獻】

相關(guān)期刊論文 前10條

1 賈洪峰;劉瑤;;七氟烷臨床應用進展[J];航空航天醫(yī)藥;2010年12期

2 楊小霖;Edmond Eger;Manohar Sharma;;七氟烷在小鼠中的代謝研究[J];四川醫(yī)學;2012年11期

3 李麗;呂國義;鄧^，

本文編號：2068003