本文關(guān)鍵詞：糖基化終末產(chǎn)物通過氧化應(yīng)激及內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)SH-SY5Y細(xì)胞凋亡的研究，由筆耕文化傳播整理發(fā)布。

        研究背景阿爾茨海默病(AD)是老年人常見的疾病之一,嚴(yán)重影響人類的身心健康,給患者家庭及社會帶來了沉重的負(fù)擔(dān)。近年來越來越多的研究顯示,2型糖尿病與AD的發(fā)生及發(fā)展密切相關(guān),Luchsinger等對1262名老年人群調(diào)查顯示1/3的癡呆與糖尿病有關(guān),相關(guān)流行病學(xué)調(diào)查發(fā)現(xiàn),DM患者發(fā)生AD的危險性是非DM患者的2倍。糖尿病導(dǎo)致AD發(fā)生的機(jī)制與胰島素細(xì)胞信號轉(zhuǎn)導(dǎo)異常、高膽固醇血癥及高血糖狀態(tài)下非酶糖基化反應(yīng)生成的糖基化終末產(chǎn)物(advanced glycation end products, AGEs)有關(guān)。糖的醛基和蛋白質(zhì)的氨基經(jīng)過復(fù)雜的非酶糖基化反應(yīng)(Maillard反應(yīng))生成一類不可逆聚合物,即糖基化終末產(chǎn)物,AGEs在糖尿病視網(wǎng)膜病變、動脈硬化、糖尿病腎病等靶器官損害中發(fā)揮重要作用,而AD患者腦內(nèi)老年斑和神經(jīng)元纖維纏結(jié)中均發(fā)現(xiàn)有AGEs的聚積,Takeuchi和Maczurek等研究也指出,AGEs在糖尿病所致的認(rèn)知功能障礙和AD病理變化中起著非常重要的作用,是引起老年人認(rèn)知功能下降的危險因素之一。AGEs能夠促進(jìn)蛋白的聚積與交聯(lián),直接抑制蛋白的正常功能,并能夠通過促進(jìn)活性氧(reative oxygen species, ROS)的產(chǎn)生對機(jī)體造成生物損傷,除此之外還能與其特異性受體(主要是RAGE)結(jié)合進(jìn)而通過一系列信號轉(zhuǎn)導(dǎo)通路發(fā)揮其間接毒性作用。AD的典型病理改變是在特定腦區(qū)內(nèi)出現(xiàn)老年斑(senile plaques, SPs)、神經(jīng)元纖維纏結(jié)(neurofibrillarytangles, NFFs)、神經(jīng)元及突觸丟失。神經(jīng)元進(jìn)行性減少是AD的重要病理特征,其中,細(xì)胞凋亡起了主導(dǎo)作用。凋亡是程序性的細(xì)胞死亡過程,外源性死亡受體途徑、內(nèi)源性線粒體凋亡通路以及內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的細(xì)胞凋亡過程目前是公認(rèn)的細(xì)胞凋亡的主要通路,近年來研究發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激性凋亡途徑在多種疾病如糖尿病、腦梗塞、阿爾茨海默病的發(fā)病中扮演了重要角色,我們以往研究提示AGEs參與APP異常代謝及Aβ的生成,而內(nèi)質(zhì)網(wǎng)是生成Aβ1-42的主要場所,在APP的代謝中起著重要作用。內(nèi)質(zhì)網(wǎng)內(nèi)蛋白質(zhì)折疊功能障礙、鈣平衡的破壞及氧化反應(yīng)的異常等都會觸發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)。AGEs是否通過觸發(fā)氧化應(yīng)激及內(nèi)質(zhì)網(wǎng)應(yīng)激進(jìn)而導(dǎo)致細(xì)胞凋亡參與AD發(fā)生發(fā)展尚不清楚,為此,我們設(shè)計該課題,選擇非常接近正常神經(jīng)細(xì)胞形態(tài)及生理功能的人神經(jīng)母細(xì)胞瘤細(xì)胞株SH-SY5Y,以此為研究對象,以非酶糖基化修飾的牛血清白蛋白(AGE-bovine serum albumin, AGE-BSA)處理細(xì)胞,并分別以抗RAGE中和抗體(RAGE-antibody, RAGE-Ab)、抗氧化劑α-硫辛酸(alpha lipoic acid, ALA)及NADPH氧化酶抑制劑二亞苯基碘(diphenyleneiodonium, DPI)預(yù)處理細(xì)胞探討AGEs是否通過氧化應(yīng)激、內(nèi)質(zhì)網(wǎng)應(yīng)激促進(jìn)細(xì)胞凋亡,揭示AGEs參與阿爾茨海默病發(fā)生發(fā)展的可能相關(guān)機(jī)制。目的本實驗通過研究AGEs對體外培養(yǎng)的SH-SY5Y細(xì)胞凋亡的影響及氧化應(yīng)激和內(nèi)質(zhì)網(wǎng)應(yīng)激在該過程中的作用,探討AGEs參與AD發(fā)生發(fā)展的可能機(jī)制。方法選擇體外培養(yǎng)的SH-SY5Y細(xì)胞為模型,分別以不同濃度(0、50、100、200、300μg/mL)的糖基化修飾的牛血清白蛋白(AGE-BSA)處理SH-SY5Y細(xì)胞48h,選取AGE-BSA敏感濃度200μg/mL處理細(xì)胞不同時間(24、36、48h),流式細(xì)胞儀(FCM)分別檢測各組細(xì)胞凋亡率,確定AGE-BSA誘導(dǎo)SH-SY5Y凋亡最佳濃度及時間；將細(xì)胞隨機(jī)分為6組：正常對照組、BSA對照組(200μg/mL BSA)、AGE-BSA組(200μg/mLAGE-BSA)、AGE-BSA+RAGE-Ab組：預(yù)先用抗RAGE-Ab (10μg/mL)干預(yù)細(xì)胞1h,再加入AGE-BSA (200μg/mL); AGE-BSA+ALA組：預(yù)先用ALA(終濃度200μmol/L)干預(yù)細(xì)胞1h,再加入AGE-BSA(200gg/mL)；AGE-BSA+DPI組：先加DPI(30nmo1/L)干預(yù)1h,加入AGE-BSA(200gg/mL)。處理48h后FCM檢測各組細(xì)胞凋亡率,Hoechst33258熒光染色觀察凋亡細(xì)胞核形態(tài)變化；應(yīng)用活性氧熒光探針DCFH-DA檢測AGE-BSA干預(yù)SH-SY5Y細(xì)胞不同時間(0、12、24、48h)后活性氧(ROS)水平,及不同藥物處理48h后細(xì)胞ROS水平；通過免疫細(xì)胞熒光化學(xué)及免疫蛋白印跡方法觀察AGEs處理細(xì)胞不同時間(0、12、24、48h)后GRP78、p-eIF2α、Caspase-12的蛋白表達(dá)情況；選取各蛋白表達(dá)高峰時間點(diǎn),應(yīng)用免疫蛋白印跡方法檢測各不同處理組SH-SY5Y細(xì)胞內(nèi)GRP78、p-eIF2α、Caspase-12蛋白表達(dá)變化。結(jié)果1.FCM測細(xì)胞凋亡率1.1正常細(xì)胞凋亡率為(2.23±0.08)%,50、100、200、300μg/mLAGE-BSA作用48h后凋亡率分別為(2.98±0.67)%、(8.23±0.42)%、(16.8±1.27)%、(19.6±2.89)%；200μg/mL AGE-BSA作用24、36、48h后凋亡率分別為(7.54±1.08)%、(10.75±1.19)%、(16.8±1.27)%,提示AGE-BSA能誘導(dǎo)細(xì)胞凋亡,且凋亡率隨AGE-BSA濃度增加及時間延長而增高。1.2分別用RAGE中和抗體、ALA、DPI預(yù)處理細(xì)胞后再加入AGE-BSA200μg/mL作用48h,細(xì)胞凋亡率分別為(5.18±0.16)%、(5.94±0.10)%、(7.26±1.22)%,與AGE-BSA組相比凋亡率分別下降69.1%、64.6%、56.8%,差異顯著(P<0.01或P<0.05),BSA對照組與正常對照組比較差異無統(tǒng)計學(xué)意義。2. Hoechst33258熒光染色觀察凋亡細(xì)胞核形態(tài)：不同處理組細(xì)胞經(jīng)Hoechst33258染色在熒光顯微鏡下觀察形態(tài),正常對照組和BSA對照組細(xì)胞核呈彌散均勻的藍(lán)色熒光,無核濃聚,AGE-BSA組可見大量散在凋亡樣改變的細(xì)胞核,表現(xiàn)為亮藍(lán)色,出現(xiàn)核碎裂,熒光強(qiáng)度比正常細(xì)胞明顯增強(qiáng),抗RAGE-Ab、ALA、DPI預(yù)處理組均可見細(xì)胞熒光強(qiáng)度比AGE-BSA組降低,凋亡細(xì)胞數(shù)明顯減少。3. DCFH-DA檢測SH-SY5Y細(xì)胞ROS水平3.1AGE-BSA干預(yù)0h的細(xì)胞內(nèi)僅有少量ROS, AGE-BSA干預(yù)細(xì)胞12h可見細(xì)胞內(nèi)ROS水平顯著升高,接近0h組細(xì)胞的4倍,并且隨AGE-BSA作用時間的延長,ROS水平不斷增高,與0h組細(xì)胞比較差異具有統(tǒng)計學(xué)意義(P<0.01)。3.2AGE-BSA及不同預(yù)處理48h檢測ROS水平：與正常組比較,AGE-BSA組細(xì)胞內(nèi)ROS明顯增多(P<0.01),是正常組的6.95倍,而NC組與BSA對照組比較無顯著差異,RAGE-Ab組、ALA組、DPI組細(xì)胞內(nèi)ROS水平較AGE-BSA組分別下降56.2%、46.5%、54.3%,差異均有統(tǒng)計學(xué)意義(P<0.01),提示AGEs可能通過與其受體RAGE結(jié)合進(jìn)而激活NADPH氧化酶促進(jìn)ROS的產(chǎn)生。4.免疫細(xì)胞熒光化學(xué)及免疫蛋白印跡方法觀察SH-SY5Y細(xì)胞中的GRP78、p-eIF2α、Caspase-12蛋白表達(dá)并進(jìn)行半定量分析4.1免疫細(xì)胞熒光化學(xué)方法觀察GRP78、p-eIF2α、Caspase-12在SH-SY5Y細(xì)胞的表達(dá),AGE-BSA處理后熒光染色陽性細(xì)胞數(shù)明顯增多,GRP78主要在胞漿表達(dá),而p-eIF2α全細(xì)胞都有表達(dá),但胞核熒光強(qiáng)度更高,免疫蛋白印跡方法結(jié)果顯示AGE-BSA分別處理細(xì)胞0、12、24、48h, GRP78、p-eIF2α、 Caspase-12蛋白表達(dá)水平呈時間依賴性增高,GRP78、p-eIF2α在AGEs處理24h后出現(xiàn)表達(dá)峰值,Caspase-12在48h出現(xiàn)表達(dá)高峰。4.2按分組要求給予細(xì)胞不同處理24h,免疫蛋白印跡結(jié)果提示AGEs組GRP78、p-eIF2α蛋白表達(dá)水平分別為正常對照組的3.56倍、6.39倍,而預(yù)先用抗RAGE-Ab、DPI、ALA分別處理細(xì)胞可以顯著降低GRP78、p-eIF2α的表達(dá),GRP78下降率分別為39.5%、14.1%、19.3%,p-eIF2α下降率分別為28%、41%、33%,差異有統(tǒng)計學(xué)意義(P<0.05或P<0.01)。處理48h后檢測Caspase-12蛋白表達(dá)水平,Caspase-12在正常細(xì)胞和BSA組弱表達(dá),AGE-BSA乍用于細(xì)胞可上調(diào)Caspase-12蛋白表達(dá),與正常組和BSA組比較差異均有顯著性(P<0.01),而應(yīng)用抗RAGE-Ab、ALA和DPI分別預(yù)處理細(xì)胞均可部分阻斷AGE-BSA導(dǎo)致的Caspase-12上調(diào),下降率分別為35.9%、43.7%、50.5%,差異顯著(P<0.01)。結(jié)論1. AGEs通過與其受體RAGE結(jié)合,進(jìn)而激活SH-SY5Y的NADPH氧化酶促進(jìn)促進(jìn)ROS產(chǎn)生。2. AGEs可能通過促進(jìn)ROS的產(chǎn)生引發(fā)GRP78表達(dá)增加及PERK-eIF2a途徑的內(nèi)質(zhì)網(wǎng)應(yīng)激,進(jìn)而誘導(dǎo)神經(jīng)細(xì)胞凋亡,參與AD的發(fā)病。3.阻斷AGEs的受體RAGE及DPI、α-硫辛酸抗氧化干預(yù)能通過降低氧化應(yīng)激及內(nèi)質(zhì)網(wǎng)應(yīng)激減少神經(jīng)細(xì)胞凋亡,可望成為防治AD的新的治療靶點(diǎn)。

    Background Alzheimer’s disease is one of the frequently encountered disease, it seriously affected physical and mental health of humen, and it has brought a heavy burden to patients family and social. Recent studies have pointed out that T2DM closely associated with the occurrence and development of AD. Luchsinger investigated1262elderly people and the results demonstrate that1/3of dementia related to diabetes. Relevant epidemiological survey found that the risk of DM patients suffer from AD was2times of nondiabetic. Mechnisms about DM caused occurrence of AD related to abnormal insulin signal transduction, hypercholesterolemia and advanced glycation end products produced in non-enzymetic glycosylation. Advanced glycation end products (AGEs) are structurally diverse irreversible polymer to which the amino sugar and protein deposits through a series of complex non-enzymatic reaction (Maillard response). AGEs played an important part in diabetic retinopathy, arteriosclerosis and diabetic nephropathy and other target organ damage.There were accumulation of AGEs in senile plaque and neurofibrillary tangles in AD patients. Takeuchi and Maczurek’s research indicated that AGEs played an important part in cognitive dysfunction resulted from DM and AD, AGEs is one of the risk factors cause cognitive decline of elderly people. AGEs may promote protein crosslinking and accumulation, inhibit the functional proteins, stimulate the generation of reactive oxygen species (ROS) directly caused biological damage. AGEs can also bind to the receptors for AGEs (RAGE), further to play indirect toxic effects through a series of signal transduction pathways.Pathological characteristic of AD is the formation of senile plaques, neurofibrillary tangles and neuronal and synaptic loss in specific brain regions. An significant pathologic feature of AD is progressive and specific neurons loss, and apoptosis holds a prominent place in cells death. Apoptosis is programmed cell death, and it has been known that there are3pathways can result cell apoptosis, including death receptors pathway, mitochondria pathway and endoplasmic reticulum stress (ERS)-mediated apoptosis. Recent studies found that ERS-mediated apoptosis play an important role in occurrence of many diseases such as diabetes and AD et al. Our previous studies indicated AGEs participated in abnormal metabolism of APP and production of Ap, ER is the main place which produced Aβ1-42and play an important part in metabolism of APP. Dysfunction of protein folding in ER, destructed calcium balance and abnormal oxidation reaction both can trigger ERS. It is not very explicitly that whether AGEs lead to apoptosis.through triggering oxidative stress and ERS and whether it involved in occurrence and development of AD. SH-SY5Y cell is closely similar to normal nerve cell in morphology and physiological functions. Therefore, we design this subject and choose SH-SY5Y as research object. We use AGE-bovine serum albumin to treat SH-SY5Y cells, and respectively pretreat cells with RAGE-antibody, alpha lipoic acid (ALA) and diphenyleneiodonium (DPI), in order to explore whether AGEs cause cell apoptosis through triggering oxidative stress and ERS, reveal the possible mechanism of AGEs involved in the occurrence and development of AD.Objective To investigate the effect of AGEs on SH-SY5Y cells apoptosis and the role of oxidative stress and ERS in this process, further to explore the possible mechanism of AGEs cause to AD.Methods As object of this study, the cultured SH-SY5Y cells were treated with different concentrations of AGE-BSA for48h, or treated with AGE-BSA(200ug/ml) for different times, cells apoptosis detected by flow cytometry (FCM) to determine the best concentration and time of AGE-BSA.Divide SH-SY5Y cells into six groups randomly, the normal control group, the BSA group, the AGE-BSA group, the AGE-BSA+anti-RAGE-Ab group, the AGE-BSA+Alpha Lipoic acid(ALA) group, the AGE-BSA+Diphenyleneiodonium (DPI) group. To detect the cells apoptosis by FCM and Hoechst staining after treatment with AGE-BSA and other drugs for48h; The level of ROS evaluated by the2’,7’-dichlorofluorescein diacetate (DCFH-DA) method. The expression of GRP78、p-eIF2α、Caspase-12was analysed by western blotting and immunofluorescence after treatment with AGE-BSA for0-48hours. Then select the peak time of each protein, and use western blotting to estimate the expression of GRP78、p-eIF2α、Caspase-12in different groups.Results1. Cell apoptosis rate measured by FCM1.1Apoptosis rate of normal control group was(2.23±0.08)%, after treatment with50,100.200,300μg/mL AGE-BSA for48hours the rate rised to(2.98±0.67)%,(8.23±0.42)%,(16.8±1.27)%,(19.6±2.89)%, after treatment with200μg/mL AGE-BSA for24hours, the rate was (7.54±1.08)%, it was (10.75±1.19)%for36hours and it was (16.8±1.27)%for48hours. Therefore, AGE-BSA can induce apoptosis, moreover the growth of apoptosis rate was in a time and concentration-dependent manner.1.2After pretreatment with RAGE-Ab, ALA and DPI respectively, the apoptosis rates have declined to (5.18±0.16)%,(5.94±0.10)%,(7.26±1.22)%, the decline rate were69.1%、64.6%、56.8%, the differences were significant compared with AGE-BSA group(P<0.05).There was no significant difference between NC group and BSA group.2. Apoptosis nucleus were detected by Hoechst33258fluorescent staining.Cells of different groups stained with Hoechst33258were observed under fluorescence microscope, the nucleus appeared uniformly dispersed of blue fluorescence, and there are no pyknoticnucleus. There were bright blue apoptosis-like nucleus in AGE-BSA group cells, and nuclear fragmentation appears. Fluorescence intensity was significantly enhanced than the normal cells. The fluorescence intensity of RAGE-Ab group、ALA group and DPI group were lower and apoptotic cells were significantly reduced than AGE-BSA group.3. The level of ROS in SH-SY5Y was detected by DCFH-DA3.1There was a small amount of ROS in cells without any AGE-BSA, and after treatment with AGE-BSA for12h, we detected the level of ROS has increased significantly, it was4times what the Oh group. The level of ROS was increased constantly along with treatment time lasting, and the differences were significant (P<0.01).3.2The ROS level of AGE-BSA group was6.95times what the NC group was(P <0.01) and there was no significant difference between NC group and BSA group. The ROS level of RAGE-Ab group, ALA group and DPI group were significantly decreased than AGE-BSA group, the decline rate were56.2%.46.5%,54.3%respectively (P<0.01). The results indicated that AGEs promoted the generation of ROS via the pathway of AGEs-RAGE-NADPH oxidase.4. Expression of GRP78, p-eIF2α, Caspase-12protein detected by immunofluorescence and western blotting4.1Estimated by immunofluorescence and western blotting, treatment with AGEs for0,12,24,48hours, the increased levels of GRP78and p-eEF2α in a time-dependent manner, with peak at24h. The Caspase-12showed the highest level at48h.4.2The levels of GRP78and p-eIF2α were analyzed in the lysates treated with200μg/mL AGE-BSA for24h, the two proteins in AGE-BSA group were respectively3.56times and6.39times what the NC group was, and which could be significantly blocked by the pretreatments of RAGE-Ab, ALA or DPI. The decline rate of GRP78were respectively39.5%,14.1%,19.3%, and that of p-eIF2α were28%,41%,33%, the differences were significant (P<0.05or P<0.01). The level of Caspase-12was measured in the lysates treated with200 μg/mL ASA-BSA for48h and apoptotic initiator marker Caspase-12were similar between the control and BSA groups, however, the level of Caspase-12in AGE-BSA group increased significantly compared with the NC group and BSA group (P<0.01). After pretreatment with RAGE-Ab, ALA or DPI, the level of Caspase-12have fallen by35.9%.43.7%,50.5%respectively(P<0.01).Conclusion1. AGEs could promote the production of ROS through binding to RAGE, further to activate NADPH oxidase.2. AGEs could trigger ERS through stimulating the generation of ROS, and further to cause nerve cells apoptosis, it may be a mechanism of AGEs cause to AD.3. Blocking the binding of AGEs-RAGE, decreasing ROS by ALA or inhibiting NADPH oxidase activity can efficiently prevent oxidative stress and ERS, consequently reduced cell death. It may provide a new method for the treatment of AD.

          糖基化終末產(chǎn)物通過氧化應(yīng)激及內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)SH-SY5Y細(xì)胞凋亡的研究

目錄4-5CONTENTS5-6中文摘要6-11ABSTRACT11-15符合說明16-17前言17-20材料與方法20-31實驗結(jié)果31-34討論34-42結(jié)論42-43附圖43-48參考文獻(xiàn)48-54致謝54-55攻讀碩士學(xué)位期間參與課題及發(fā)表的論文55-56學(xué)位論文評閱及答辯情況表56

  本文關(guān)鍵詞：糖基化終末產(chǎn)物通過氧化應(yīng)激及內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)SH-SY5Y細(xì)胞凋亡的研究，由筆耕文化傳播整理發(fā)布。