鐵導(dǎo)致的alpha-突觸核蛋白聚集在帕金森病中的作用研究.pdf

1、鐵導(dǎo)致的alpha一突觸核蛋白聚集在帕金森病中的作用研究摘要帕金森病(Parkinson’Sdisease，PD)是繼阿爾茨海默病之后的第二大神經(jīng)系統(tǒng)退行性疾病。在我國，65歲以上的人群帕金森病的患病率為17％。PD的神經(jīng)病理學(xué)基礎(chǔ)是黑質(zhì)(substantianigra，SN)致密帶多巴胺(dopamine，DA)能神經(jīng)元的變性脫失以及由此而導(dǎo)致的紋狀體DA含量降低。帕金森病的特征性病理結(jié)構(gòu)是路易小體，其主要成分是聚集的alpha突觸核

2、蛋白，另外還有鐵，蛋白酶體亞單位，泛素，熱休克蛋白等成分。在家族性或散發(fā)性帕金森病中，中腦黑質(zhì)內(nèi)的alpha突觸核蛋白mRNA水平均有增高。這些結(jié)果提示alpha突觸核蛋白與上述路易小體內(nèi)的成分相互作用可能參與了多巴胺能神經(jīng)元的選擇性死亡。越來越多的證據(jù)表明鐵在PD的發(fā)病中是一個(gè)關(guān)鍵因素，PD病人黑質(zhì)變性的神經(jīng)元內(nèi)均發(fā)現(xiàn)鐵含量的增高與路易小體的形成。近年來人們發(fā)現(xiàn)鐵能夠促進(jìn)alpha突觸核蛋白的聚集，但具體機(jī)制還不清楚。血紅素加氧酶1(

3、Hemeoxygenase1，HO1)作為熱休克蛋白32，存在于路易小體中。近來的研究發(fā)現(xiàn)，細(xì)胞內(nèi)過表達(dá)HO1能夠通過激活泛素蛋白酶體系統(tǒng)而促進(jìn)alpha一突觸核蛋白的降解。在HO1基因編碼區(qū)的啟動(dòng)子內(nèi)存在激活蛋白1(activtorprotein1AP一1)序列，被稱作抗氧化應(yīng)激元件(antioxidantresponsesdements，ARE)，它能夠受到具有氧化還原活性的核轉(zhuǎn)錄因子(NFE2regulatedfactor2，Nm

4、)的精密調(diào)節(jié)。已證實(shí)Nrf2／HO1途徑是細(xì)胞內(nèi)抗氧化應(yīng)激的一條重要途徑。那么在鐵引起的alpha突觸核蛋白聚集過程中是否有Nrf2／HO一1途徑的參與呢本實(shí)驗(yàn)中，我們運(yùn)用Fd或具有氧化還原活性的Fe2作用于SKN—SH細(xì)胞，運(yùn)用甲基噻唑基四唑(methylthiazolyltetrazolium，MTT)法，流式細(xì)胞術(shù)、PCR、小干涉RNA、硫磺素S染色以及電鏡等技術(shù)，探討鐵導(dǎo)致alpha突觸核蛋白的聚集及其毒性反應(yīng)機(jī)制。結(jié)果發(fā)現(xiàn)：1

5、細(xì)胞內(nèi)過表達(dá)alpha突觸核蛋白對(duì)SKNSH細(xì)胞存活率沒有明顯的影響，而干涉掉alpha一突觸核蛋白48h后細(xì)胞存活率下降10％。與對(duì)照組相比，在過表達(dá)alpha突觸核蛋白的細(xì)胞中，F(xiàn)e”(1oopmol／L500lamol／L，1mmoYL，10mmol／L)孵育24h進(jìn)一步加重了細(xì)胞內(nèi)高鐵導(dǎo)致的細(xì)胞存活率下降，而同樣濃度的Fe3孵育干涉掉alpha突觸核蛋白的細(xì)胞則可阻斷存活率的下降。2Fe2(100lxmol／L)孵育過表達(dá)alp

6、ha突觸核蛋白的SKNSH細(xì)胞4h或Fe3(100pmol／L1mmol／L)孵育24h，與對(duì)照組相比，細(xì)胞內(nèi)線粒體跨膜電位差(mitochondrialtransmembranepotential，AWm)I拘T降及活性氧物質(zhì)(reactiveoxygenspecies，ROS)生成的增多更為明顯，而同樣濃度的vd或Fe3孵育干涉掉alphaExperimentalstudyofiron—inducedalpha—synucleina

7、ggregationinthepathogenesisofParkinson’sdiseaseAbstractParkinson’Sdisease(PD)isthesecondmostcommonneurodegenerativedisorderafterAlzheimer’Sdiseaseaffecting17％ofpopulationOVertheageof65inChinaPDispathologicallycharactized

8、byaselectivelossofdopaminergicneuronsinthesubstantianigra(SN)andthesubsequentdopamine(DA)depletioninthestriatumThepathologicalhallmarkofPDareintracellularinclusions，termedLewybodies(LBs)，whicharemainlycomposedbyamixtureo

9、fproteinsincludingaggregatedalphasynuclein，iron，proteasomesubunits，ubiquitin，heatshockproteins(HSPs)，etcIncreasedlevelsofalpha—synucleinmRNAinthemidbrainofpatientsarebothfoundinsporadicPDandfamilialPDThedepositionofthese

10、componentstoformtheLBsmayprovideUSsomenewinsightsintothemechanismsunderlyingneurodegenerationinPDMountingevidencedemonstratethatironplaysacrucialroleinthepathogenesisofPDIncreasedironcontentandLBsformationarefoundindegen

11、erativedopaminergicneuronsinPDStudieshavefoundthatironcouldpromotealphasynucleinaggregation，buttheexactmechanismislargelyunknownHemeoxygenase1(HO一1)，alsoknownasHSP32，ispresentinLBsArecentstudyreportsthatoverexpressionofH

12、O一1triggersintracellularproteasomaldegredationofalphasynucleinAnewclassofactivatorprotein1(AP1)relatedsequencesfoundinthepromoterregionofgenesencodingHOIWastermedantioxidantresponseselements(ARE)，whicharetightlyregulated

13、bytheredox—sensitivetranscriptionfactorNF—E2regulatedfactor2(Nrf2)Nrf2／HO一1pathwayhasbeenprovedtobeallimportantintracellularanti—oxidativemechanismWhetherNrf2／HOIpathwayisinvolvedinironinducedalphasynucleinaggregationisn

14、otclearUsingMTTassayflowcytometryPCR，siRNA，thioflavinSstainingelectronmicroscopyandothermethods，inthepresentstudyweinvestigatethepossiblemechanismunderlyingironinducedalphasynucleinaggregation，aswellasthesubsequenttoxice

15、ventsbyexposingSKNSHcellstoironTheresultsareasfollows：1Alpha—synucleinoverexpressionhadnoeffectsonSK—N—SHcellsviabilitywhiletherewasa10％reductioninthecellviabilitywimalphasynuleinknockdownFerriciron(100tmol／L，500lxmol／L，