本文選題：甲基苯丙胺(METH) + α-突觸核蛋白��； 參考：《南方醫(yī)科大學(xué)》2012年博士論文

【摘要】：研究背景和目的： 甲基苯丙胺(Methamphetamine, METH)是一種新型毒品,由于合成技術(shù)簡單、合成原料容易獲得,已成為世界上廣泛濫用、快速蔓延、危害最重的毒品之一。因此,對METH毒性損傷作用及其機(jī)制的研究已成為世界面臨的重大課題和研究熱點(diǎn)。 METH具有很強(qiáng)的中樞興奮作用且易形成藥物依賴性,對心、肝、腎等多種組織器官均有毒性作用,特別是對中樞神經(jīng)系統(tǒng)的毒性。研究顯示,METH可致動物大腦多巴胺能神經(jīng)末梢損傷,引起紋狀體內(nèi)多巴胺耗竭、含量下降、多巴胺轉(zhuǎn)運(yùn)體降低、酪氨酸羥化酶活性減少、多巴胺攝取位點(diǎn)及單胺囊泡轉(zhuǎn)運(yùn)體2的缺失。目前,關(guān)于METH的神經(jīng)毒性機(jī)制盡管國內(nèi)外進(jìn)行了大量研究,但結(jié)果尚未完全明確,主要集中于以下3種機(jī)制：多巴胺系統(tǒng)失常和氧化應(yīng)激損傷、谷氨酸和一氧化氮、線粒體功能失常及神經(jīng)元的凋亡等。 本教研室關(guān)于METH神經(jīng)毒性的前期研究中首次發(fā)現(xiàn)METH大鼠模型組紋狀體、皮質(zhì)、海馬三個腦區(qū)中α-突觸核蛋白(α-synuclein, α-SN)表達(dá)明顯升高,并應(yīng)用western blot進(jìn)行了驗(yàn)證。大量證據(jù)表明,α-SN的異常表達(dá)、聚集和纖維化與帕金森病、阿爾茨海默病等神經(jīng)退行性疾病的發(fā)病密切相關(guān),也是帕金森病等疾病藥物治療的作用靶點(diǎn)。α-SN被發(fā)現(xiàn)與上述疾病中多巴胺遞質(zhì)釋放減少、氧化應(yīng)激損傷、細(xì)胞膜結(jié)構(gòu)破壞、Ca2+超載、線粒體功能損傷和細(xì)胞凋亡等多種損傷機(jī)制密切相關(guān)。已有研究發(fā)現(xiàn)METH作用于SK-N-SH細(xì)胞(神經(jīng)母細(xì)胞瘤細(xì)胞)后α-SN表達(dá)升高。然而,對a-SN在METH致神經(jīng)退行性病變中的作用和機(jī)制的研究還未見有報(bào)道,需要研究證實(shí)。 RNA干擾(RNAi)技術(shù)對于研究基因功能是一種有效的工具,它可在轉(zhuǎn)錄后水平高效特異地抑制目標(biāo)基因的表達(dá)。Sapru MK等應(yīng)用RNAi技術(shù)已經(jīng)成功地對人多巴胺能神經(jīng)細(xì)胞系SH-SY5Y中內(nèi)源性的a-SN進(jìn)行了有效沉默。本研究參照Sapru MK等的實(shí)驗(yàn)方法,通過RNAi技術(shù)沉默α-SN基因,并獲得穩(wěn)定轉(zhuǎn)染a-SN shRNA慢病毒表達(dá)載體的細(xì)胞株,進(jìn)而研究α-SN在多巴胺系統(tǒng)失常、氧化應(yīng)激、線粒體功能損傷以及細(xì)胞凋亡等METH神經(jīng)毒性方面的功能,以期為深入研究METH所致神經(jīng)毒性的作用機(jī)理提供理論基礎(chǔ),并為藥物篩選提供靶點(diǎn)。 方法： 1.不同劑量METH作用SH-SY5Y細(xì)胞后的a-SN表達(dá)變化 10%新生胎牛血清(FBS)的DMEM/F12培養(yǎng)基接種SH-SY5Y細(xì)胞至培養(yǎng)瓶中,在37℃和5%CO2條件下培養(yǎng),待細(xì)胞長至80%匯合時(shí),用終濃度0.5mmol/L、1.5mmol/L、2.5mmol/L、3.5mmol/L、4.5mmol/L,培養(yǎng)24h后處理。收集上述各組細(xì)胞,倒置顯微鏡下觀察細(xì)胞形態(tài)變化,CCK-8法檢測細(xì)胞活力變化,Annexin V-FITC/PI雙染法結(jié)合流式細(xì)胞術(shù)結(jié)檢測細(xì)胞早期凋亡率,實(shí)時(shí)熒光定量PCR、Western Blot分別檢測a-SN在基因和蛋白水平的表達(dá)變化。 2.干擾α-SN表達(dá)的SH-SY5Y細(xì)胞株的建立及沉默效果檢測 (1)根據(jù)文獻(xiàn)報(bào)道的有效siRNA序列和白行設(shè)計(jì)的2條siRNA,結(jié)合pLVTHM載體的結(jié)構(gòu)特點(diǎn),合成3對shRNA,構(gòu)建shRNA表達(dá)質(zhì)粒載體,并通過DNA測序?qū)?gòu)建的質(zhì)粒載體進(jìn)行鑒定。 (2)對上述質(zhì)粒載體進(jìn)行慢病毒包裝和病毒滴度測定后,轉(zhuǎn)染SH-SY5Y細(xì)胞。 (3)用實(shí)時(shí)熒光定量PCR和Western Blot分別檢測α-SN在基因和蛋白水平表達(dá)的抑制效率,篩選出最佳shRNA靶序列,并結(jié)合流式分選,建立穩(wěn)定轉(zhuǎn)染該shRNA重組慢病毒載體的細(xì)胞株。 (4)用最佳shRNA靶序列與不帶綠色熒光蛋白的慢病毒載體pLKO.1重組并轉(zhuǎn)染SH-SY5Y細(xì)胞,Western Blot檢測α-SN蛋白水平的抑制效率。 3.干擾α-SN表達(dá)后的SH-SY5Y細(xì)胞株的毒性損傷反應(yīng) 實(shí)驗(yàn)分6組,未轉(zhuǎn)染的正常細(xì)胞對照組(CON)、空白載體對照組(Empty vector)、α-SN干擾組(RNAi)、CON給藥組(CON+METH)、Empty vector給藥組(Empty vector+METH)、RNAi給藥組(RNAi+METH)。各組細(xì)胞分別接種于96孔板或6孔板或10cm培養(yǎng)皿,當(dāng)細(xì)胞生長至80%匯合時(shí),棄原培養(yǎng)液,處理組加入含3.5mmol/L METH的2%血清培養(yǎng)基,對照組換成等體積不含METH的2%血清培養(yǎng)基,細(xì)胞繼續(xù)培養(yǎng)后,收集細(xì)胞或細(xì)胞培養(yǎng)液進(jìn)行各項(xiàng)實(shí)驗(yàn),檢測以下項(xiàng)目： (1)倒置顯微鏡下觀察各組細(xì)胞形態(tài)學(xué)損傷的變化、CCK-8法觀察各組細(xì)胞細(xì)胞活力的變化。 (2)實(shí)時(shí)熒光定量PCR檢測各組細(xì)胞內(nèi)TH、DAT、VMAT-2在基因水平的表達(dá)變化。 (3) ELISA方法檢測各組細(xì)胞內(nèi)DA含量的改變。 (4)酶化學(xué)方法檢測ROS、NOS、NO的活性變化,分別用ROS熒光檢測試劑盒、NOS活性檢測試劑盒、NOS檢測試劑盒檢測。 (5)通過Annexin V-FITC/PI雙標(biāo)記及流式細(xì)胞技術(shù)檢測細(xì)胞的凋亡率；電子顯微鏡觀察細(xì)胞超微結(jié)構(gòu)的變化。 (6)用Tetramethylrhodamine ethyl ester (TMRE)標(biāo)記線粒體△Ψm(跨膜電位),結(jié)合激光掃描共聚焦顯微鏡檢測各組細(xì)胞△Ψm的變化。 (7)用Calcein標(biāo)記技術(shù)結(jié)合共聚焦顯微鏡檢測線粒體通透轉(zhuǎn)變孔道(mitochondfial permeability transition pore, MPTP)開放的變化。 (8)各組細(xì)胞與Fluo-3/AM共孵育,采用共聚焦顯微鏡觀察細(xì)胞內(nèi)鈣離子熒光強(qiáng)度的變化。 (9) Western blot技術(shù)檢測各組細(xì)胞線粒體內(nèi)和胞質(zhì)內(nèi)Cyt C的變化。 結(jié)果： 1.以未經(jīng)METH處理的SH-SY5Y細(xì)胞為對照組,0.5～4.5mmol/L METH處理的SH-SY5Y細(xì)胞24h后倒置顯微鏡下可見胞體皺縮變圓,變圓細(xì)胞的胞質(zhì)透亮度增加,可見環(huán)形透亮區(qū),突起變短、斷裂、消失,并可見細(xì)胞脫壁漂浮現(xiàn)象,且細(xì)胞損傷隨METH濃度增加而呈增強(qiáng)趨勢。CCK-8法測得SH-SY5Y細(xì)胞存活率隨METH濃度增加逐漸降低,除0.5mmo1/L處理組與對照組相比無顯著性差異(P=0.274)外,其他各濃度處理組與對照組相比均有顯著性差異(P0.001)。細(xì)胞凋亡率隨METH濃度增加而逐漸增加,各濃度處理組與對照組相比均有顯著性差異(P0.001)。實(shí)時(shí)熒光定量PCR結(jié)果顯示：與未經(jīng)METH處理的對照組相比,0.5mmol/L處理組細(xì)胞中α-SN-mRNA的表達(dá)量沒有顯著差異(P=0.936),其它濃度METH處理組α-SN-mRNA均顯著性上升(P≤0.001)。隨著METH濃度增加,α-SN-mRNA表達(dá)量逐步上升。Western Blot也證實(shí)α-SN蛋白的表達(dá)隨著METH濃度增加而逐步上升。 2.篩選確認(rèn)沉默效果最佳的shRNA,成功構(gòu)建α-SN-shRNA-pLVTHM重組慢病毒載體,轉(zhuǎn)染SH-SY5Y細(xì)胞,可抑制α-SN基因mRNA表達(dá)最高達(dá)到84%。用最佳shRNA與不帶綠色熒光蛋白的慢病毒載體pLKO.1重組,獲得穩(wěn)定轉(zhuǎn)染α-SN-shRNA-pLKO.1的細(xì)胞株。Western Blot實(shí)驗(yàn)證實(shí)上述兩個RNAi細(xì)胞株中的a-SN蛋白表達(dá)均顯著下降。 3. Empty vector組(轉(zhuǎn)染空白載體的細(xì)胞)、RNAi組(轉(zhuǎn)染α-SN-shRNA的細(xì)胞)與CON組(未轉(zhuǎn)染的細(xì)胞)相比,細(xì)胞活性均無顯著性差異(P0.05)。與未經(jīng)METH處理的CON組細(xì)胞相比,3.5mmol/L METH處理的CON組可見大量死亡漂浮的細(xì)胞、細(xì)胞活性顯著性下降(P≤0.001), DAI、TH、 VMAT-2mRNA表達(dá)顯著性下降(P0.001,P0.001,P0.001),細(xì)胞內(nèi)DA含量顯著下降(P≤0.001), ROS、NOS、NO水平顯著性升高(P0.001,P0.001,P0.001)。而RNAi+METH組與CON+METH組相比,鏡下死亡漂浮的細(xì)胞減少、細(xì)胞活性顯著性升高(P≤0.001)、DAT、TH、VMAT-2mRNA顯著性回升(P0.001,P0.001,P0.001),細(xì)胞內(nèi)DA含量顯著性回升(P≤0.001)、ROS、NOS、NO的表達(dá)水平顯著性回落(P0.001,P0.001,P0.001)。 4.3.5mmol/L METH處理的CON組細(xì)胞早期凋亡率較未經(jīng)METH處理的CON組細(xì)胞顯著升高(P≤0.001)。其中RNAi+METH組早期凋亡率明顯低于CON+METH組(P0.001)。3.5mmol/L METH作用24小時(shí)后,對照組細(xì)胞電鏡下可見：1)壞死的形態(tài)學(xué)特征：內(nèi)質(zhì)網(wǎng)、高爾基體等細(xì)胞器輕度空泡化,核溶解、核仁消失；2)早期凋亡細(xì)胞的形態(tài)學(xué)特征：胞體縮小,胞膜斷裂,染色質(zhì)凝聚成新月狀,附著在核膜周邊；3)吞噬了線粒體的自噬小體；4)發(fā)現(xiàn)多個類似路易小體、多層旋渦狀排列的均勻物質(zhì)。3.5mmol/L METH處理的RNAi組細(xì)胞電鏡下未發(fā)現(xiàn)類似路易小體的結(jié)構(gòu),且壞死、凋亡、白噬等損傷變化較對照組明顯減少。 5.熒光標(biāo)記結(jié)合激光掃描共聚焦顯微鏡觀察發(fā)現(xiàn)：1)METH作用2小時(shí)后,TMRE標(biāo)記檢測顯示給藥處理后的細(xì)胞胞質(zhì)內(nèi)紅色熒光強(qiáng)度較METH作用前減弱,但RNAi+METH組細(xì)胞胞質(zhì)內(nèi)熒光強(qiáng)度明顯高于METH處理的正常細(xì)胞和空白載體組。表明METH可導(dǎo)致線粒體膜電位降低,而RNAi+METH組線粒體膜電位降低程度低于CON組,提示干擾α-SN表達(dá)可一定程度上抑制線粒體膜電位下降。2)Calcein和CoCl2標(biāo)記檢測發(fā)現(xiàn)METH作用后細(xì)胞胞質(zhì)內(nèi)綠色熒光強(qiáng)度明顯減弱,表明METH使線粒體通透性轉(zhuǎn)換孔道開放程度增加。RNAi+METH組線粒體通透性轉(zhuǎn)換孔道開放程度低于CON組,說明干擾α-SN表達(dá)能夠抑制線粒體通透性轉(zhuǎn)換孔道開放。3)Fluo-3標(biāo)記細(xì)胞質(zhì)內(nèi)游離鈣離子顯示METH作用后細(xì)胞胞質(zhì)內(nèi)綠色熒光強(qiáng)度明顯增強(qiáng),表明細(xì)胞內(nèi)游離鈣離子濃度增高,其中RNAi+METH組細(xì)胞內(nèi)鈣離子濃度增高程度顯著低于CON組,說明干擾a-SN表達(dá)可抑制細(xì)胞外鈣離子內(nèi)流。 6. Western Blot結(jié)果顯示,METH處理正常細(xì)胞后線粒體內(nèi)細(xì)胞色素C的含量明顯降低,同時(shí)細(xì)胞漿內(nèi)細(xì)胞色素C含量明顯升高,但RNAi+METH組的線粒體內(nèi)細(xì)胞色素C含量下降水平低于對照組,細(xì)胞漿內(nèi)細(xì)胞色素C含量升高程度也低于對照組。 結(jié)論 1.METH可導(dǎo)致SH-SY5Y細(xì)胞形態(tài)學(xué)改變、細(xì)胞活力下降、細(xì)胞凋亡率增加以及α-SN基因和蛋白表達(dá)水平的增加。隨著METH濃度升高,細(xì)胞損傷程度、α-SN的表達(dá)水平均呈增強(qiáng)趨勢。 2.成功構(gòu)建靶向α--SN的重組shRNA慢病毒載體,并顯著下調(diào)了SH-SY5Y細(xì)胞中a-SN基因的表達(dá),獲得穩(wěn)定轉(zhuǎn)染α-SN-shRNA的細(xì)胞株以供長期實(shí)驗(yàn)。 3.干擾α-SN表達(dá)可抑制METH引起的細(xì)胞活力下降、TH、DAT、VMAT-2等基因水平下降、多巴胺耗竭和ROS、NOS、NO水平升高,可能是通過抑制上述多巴胺系統(tǒng)紊亂及氧化應(yīng)激、硝化應(yīng)激損傷,從而拮抗METH引起的細(xì)胞毒性。 4.干擾α-SN表達(dá)能抵抗METH引起的線粒體△Ψm下降、MPTP開放、細(xì)胞外鈣離子內(nèi)流,抑制線粒體的細(xì)胞色素C釋放進(jìn)入胞漿,從而在一定程度上保護(hù)線粒體的正常功能、抵抗METH引起的細(xì)胞壞死和凋亡。
[Abstract]:Research background and purpose:
Methamphetamine (Methamphetamine, METH) is a new drug, because of simple synthesis, synthesis of raw materials are easy to obtain, has become a widespread abuse of the world, one of the most rapid spread of drug harm heavy. Therefore, the research on METH cytotoxicity and its mechanism has become a major issue facing the world and the focus of research.
METH has a strong role in the excitability and easy to form drug dependence on heart, liver, kidney and other organ toxicity of various tissues are particularly toxic to the central nervous system. Research shows that METH can induce animal brain dopaminergic nerve injury, caused by striatal dopamine depletion, decreased dopamine transporter reduction tyrosine hydroxylase activity, reduced loss of dopamine uptake sites and vesicular monoamine transporter 2. At present, about METH at home and abroad despite the neurotoxic mechanism of a large number of studies, but the results are not yet completely clear, mainly focused on the following 3 mechanisms: dopamine system disorder and oxidative stress, glutamate and nitric oxide, mitochondrial dysfunction and neuronal the apoptosis and so on.
The former research department on neurotoxicity of METH in rat model group for the first time that METH cortex, striatum, hippocampus in three brain regions of alpha synuclein (alpha -synuclein, alpha -SN) expression was significantly increased, and the application of Western blot was verified. A lot of evidence that the abnormal expression of -SN alpha, aggregation and fibrosis and Parkinson's disease, the incidence of the disease is closely related to Alzheimer's disease and other neurodegenerative effects, but also a target of Parkinson's disease and other diseases therapy. Alpha -SN was found with the disease decreased the release of dopamine, oxidative stress, Ca2+ damage, cell membrane structure overload, various damage mechanism of the damage of mitochondrial function and apoptosis closely related. Studies have found that the effect of METH on SK-N-SH cells (neuroblastoma cells) after alpha -SN expression increased. However, the effect and mechanism of a-SN in METH induced neurodegeneration in The study has not been reported, and it needs to be confirmed.
RNA interference (RNAi) technology is an effective tool for the study of gene function, it can be at the post transcriptional level, specifically inhibits the expression of.Sapru MK gene RNAi technology has been successfully applied to dopaminergic endogenous neural cell line SH-SY5Y a-SN was effectively silent. The method of this research according to Sapru MK the alpha -SN gene silenced by RNAi, and obtain the stable transfection of a-SN shRNA lentiviral vector cell line expression, and study the alpha -SN in the dopamine system disorders, oxidative stress, mitochondrial dysfunction and apoptosis METH neurotoxicity function, in order to provide theoretical basis for further research the mechanism of METH induced neurotoxicity, and provide targets for drug screening.
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