本文選題：鐵 + α-突觸核蛋白　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：帕金森病(Parkinson’s disease,PD)是一種以黑質(zhì)致密帶多巴胺(dopamine,DA)能神經(jīng)元進(jìn)行性缺失為特征的中樞神經(jīng)系統(tǒng)退行性疾病。其臨床表現(xiàn)為運動不能、肌僵直、靜止性震顫及姿勢反射障礙等。盡管研究顯示遺傳、環(huán)境和年齡等因素均可能參與PD發(fā)病,但其確切病因不清。PD的主要病理特征是黑質(zhì)(substantia nigra,SN)致密部多巴胺能神經(jīng)元缺失和路易小體(Lewy body,LB)的形成。α-突觸核蛋白是LB的主要成分,神經(jīng)元中α-突觸核蛋白的異常聚集能夠改變細(xì)胞膜電位、損傷線粒體功能、誘導(dǎo)內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum,ER)應(yīng)激而造成細(xì)胞損傷,其中內(nèi)質(zhì)網(wǎng)應(yīng)激是α-突觸核蛋白的核心毒性作用機(jī)制之一。除了長期以來關(guān)注的其在細(xì)胞內(nèi)的毒性作用之外,α-突觸核蛋白能夠被神經(jīng)元釋放,進(jìn)而被周圍的細(xì)胞(包括神經(jīng)元、膠質(zhì)細(xì)胞等)再攝取,從而能夠在細(xì)胞-細(xì)胞之間、相互聯(lián)系的結(jié)構(gòu)之間進(jìn)行傳遞。鐵沉積是PD另一重要的神經(jīng)病理學(xué)改變,尸檢證據(jù)表明,PD黑質(zhì)區(qū)單個神經(jīng)元內(nèi)的鐵含量增高,黑質(zhì)鐵的水平與PD的進(jìn)程相關(guān)。本課題組前期研究證實鐵能夠促進(jìn)α-突觸核蛋白的聚集,增強(qiáng)α-突觸核蛋白在細(xì)胞內(nèi)的毒性作用,然而鐵沉積與細(xì)胞間傳播的α-突觸核蛋白之間的關(guān)系尚未有研究報道。以往神經(jīng)退行性疾病的研究熱點都集中在特定受損的神經(jīng)元上,占腦體積20%-50%的星形膠質(zhì)細(xì)胞在神經(jīng)系統(tǒng)的發(fā)育、突觸傳遞、離子平衡、神經(jīng)免疫等方面,都起著十分重要的作用,因此星形膠質(zhì)細(xì)胞在神經(jīng)退行性疾病的作用開始受到關(guān)注。星形膠質(zhì)細(xì)胞本身表達(dá)極低水平的α-突觸核蛋白,但其能攝取胞外的α-突觸核蛋白。進(jìn)入到星形膠質(zhì)細(xì)胞內(nèi)的α-突觸核蛋白的聚集促進(jìn)促炎因子和化學(xué)因子的釋放,進(jìn)而導(dǎo)致神經(jīng)元的損傷和小膠質(zhì)細(xì)胞的繼發(fā)性激活。本研究選用C6膠質(zhì)瘤細(xì)胞(星形膠質(zhì)細(xì)胞瘤的細(xì)胞系),原代培養(yǎng)的腹側(cè)中腦(ventral mesencephalon,VM)星形膠質(zhì)細(xì)胞及VM神經(jīng)元為細(xì)胞模型,應(yīng)用蛋白免疫印跡、免疫熒光、熒光實時定量PCR、流式細(xì)胞術(shù)等方法,深入探討細(xì)胞外的α-突觸核蛋白處理后VM星形膠質(zhì)細(xì)胞促炎因子的變化及鐵對此過程的影響,并評價內(nèi)質(zhì)網(wǎng)應(yīng)激是否參與了細(xì)胞外α-突觸核蛋白的毒性作用,并觀察VM神經(jīng)元的損傷變化。研究結(jié)果如下:1.100 nmol/L、300 nmol/L的α-突觸核蛋白單體處理C6膠質(zhì)瘤細(xì)胞24 h后,IL-1β mRNA的升高分別約為對照組的1.83、2.69倍,TNF-αmRNA的升高分別約為對照組的1.35、1.74倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.01);且100 nmol/L組與300 nmol/L組相比差別有統(tǒng)計學(xué)意義(P0.01)。以上結(jié)果說明,α-突觸核蛋白單體能夠引起C6膠質(zhì)瘤細(xì)胞促炎因子表達(dá)增加,且促炎因子的表達(dá)升高對α-突觸核蛋白具有濃度依賴性。2.100 nmol/L、300 nmol/L的α-突觸核蛋白單體處理VM星形膠質(zhì)細(xì)胞24 h后,IL-1βmRNA的升高分別約為對照組的9.58、21.7倍,TNF-αmRNA的升高分別約為對照組的4.80、6.17倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.0001),且100 nmol/L組與300 nmol/L組相比差別有統(tǒng)計學(xué)意義(P0.01)。以上結(jié)果說明,α-突觸核蛋白單體能夠引起VM星形膠質(zhì)細(xì)胞促炎因子表達(dá)增加,且促炎因子的升高對α-突觸核蛋白具有濃度依賴性。3.100μmol/Lα-突觸核蛋白單體37℃恒溫振蕩3 d,制備α-突觸核蛋白纖維體。100nmol/L、300 nmol/L的α-突觸核蛋白纖維體處理VM星形膠質(zhì)細(xì)胞24 h后,IL-1βmRNA的升高分別約為對照組的3.22、7.91倍,TNF-αmRNA的升高分別約為對照組的3.09、4.33倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05),且100 nmol/L組與300 nmol/L組相比差別有統(tǒng)計學(xué)意義(P0.05)。α-突觸核蛋白纖維體引起促炎因子的升高水平低于α-突觸核蛋白單體組,與α-突觸核蛋白單體組相比,差別有統(tǒng)計學(xué)意義(P0.05)。以上結(jié)果說明,α-突觸核蛋白纖維體也能夠引起VM星形膠質(zhì)細(xì)胞促炎因子表達(dá)增加,且促炎因子的升高對α-突觸核蛋白具有濃度依賴性;但α-突觸核蛋白單體對VM星形膠質(zhì)細(xì)胞的毒性要高于α-突觸核蛋白纖維體。4.100 nmol/L的α-突觸核蛋白單體處理C6膠質(zhì)瘤細(xì)胞24 h后,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白CHOP、ATF-4、XBP-1s的表達(dá)升高,分別約為對照組的1.33、2.05、1.24倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05)。300 nmol/L的α-突觸核蛋白組,CHOP、ATF-4、XBP-1s的表達(dá)升高,分別約為對照組的1.50、2.10、1.25倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05)。以上結(jié)果說明,細(xì)胞外的α-突觸核蛋白能夠誘導(dǎo)C6膠質(zhì)瘤細(xì)胞出現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激。自噬溶酶體途徑(autophagy-lysosome pathway,ALP)抑制劑氯喹(40μmol/L)或泛素蛋白酶體系統(tǒng)(ubiquitin-proteasome system,UPS)抑制劑MG132(20μmol/L)單獨處理細(xì)胞24 h后,CHOP、ATF-4的表達(dá)升高,提示抑制ALP或UPS均可誘導(dǎo)細(xì)胞出現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激。氯喹與α-突觸核蛋白共孵育后與單獨氯喹處理組相比沒有顯著差別,提示進(jìn)入細(xì)胞內(nèi)的α-突觸核蛋白能夠經(jīng)UPS降解。而MG132與α-突觸核蛋白共孵育后與單獨MG132處理組相比明顯下降,差別有統(tǒng)計學(xué)意義(P0.0001)。以上結(jié)果說明,進(jìn)入到細(xì)胞內(nèi)的 α-突觸核蛋白可以誘導(dǎo)C6膠質(zhì)瘤細(xì)胞出現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激;細(xì)胞外進(jìn)入到細(xì)胞內(nèi)的α-突觸核蛋白可以經(jīng)UPS和ALP降解,且ALP在UPS被抑制的情況下更容易被細(xì)胞外的α-突觸核蛋白誘導(dǎo)增強(qiáng)。5.100 nmol/L的α-突觸核蛋白單體處理VM星形膠質(zhì)細(xì)胞24 h后,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白CHOP、ATF-4、XBP-1s的表達(dá)升高,分別約為對照組的1.96、2.23、1.29倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05)。300 nmol/L的α-突觸核蛋白組,CHOP、ATF-4、XBP-1s的表達(dá)升高,分別約為對照組的2.02、2.32、1.48倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05)。以上結(jié)果說明,α-突觸核蛋白能夠誘導(dǎo)VM星形膠質(zhì)細(xì)胞出現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激。6.10μmol/L、100μmol/L枸櫞酸鐵銨(ferric ammonium citrate,FAC)處理C6膠質(zhì)瘤細(xì)胞24 h后,10μmol/L FAC組促炎因子表達(dá)量較對照組沒有變化,與100nmol/L的α-突觸核蛋白單體共孵育細(xì)胞24 h后,促炎因子的表達(dá)量較α-突觸核蛋白組沒有顯著變化。100μmol/L FAC組促炎因子表達(dá)量較對照組增加,IL-1β、TNF-αmRNA的升高約為對照組的1.72、1.37倍,差別有統(tǒng)計學(xué)意義(P0.05),與100 nmol/L的α-突觸核蛋白單體共孵育細(xì)胞24 h后,促炎因子的表達(dá)量較α-突觸核蛋白組也沒有顯著變化。以上結(jié)果說明,C6膠質(zhì)瘤細(xì)胞內(nèi)鐵水平升高對細(xì)胞外α-突觸核蛋白的毒性作用沒有明顯影響。7.10μmol/L FAC處理VM星形膠質(zhì)細(xì)胞24 h后,促炎因子IL-1β、TNF-α的mRNA表達(dá)水平有升高的趨勢,但無統(tǒng)計學(xué)意義(P㧐0.05),與100 nmol/L的α-突觸核蛋白單體共孵育VM星形膠質(zhì)細(xì)胞24 h后,可誘導(dǎo)α-突觸核蛋白引起的促炎因子釋放量增加,與對照組相比,IL-1β、TNF-αmRNA分別約升高11.92、4.95倍,差別有統(tǒng)計學(xué)意義(P0.0001);與α-突觸核蛋白組相比,IL-1β、TNF-αmRNA分別約升高26%、18%,差別有統(tǒng)計學(xué)意義(P0.05)。100μmol/L FAC處理VM星形膠質(zhì)細(xì)胞24 h后,促炎因子IL-1β、TNF-α的mRNA表達(dá)水平也有升高的趨勢,且與對照組相比,TNF-αmRNA的升高有統(tǒng)計學(xué)意義(P0.05),與100 nmol/L的α-突觸核蛋白單體共孵育細(xì)胞24 h后,可誘導(dǎo)α-突觸核蛋白引起的促炎因子表達(dá)量增加,與對照組相比,IL-1β、TNF-αmRNA分別約升高11.59、6.05倍,差別有統(tǒng)計學(xué)意義(P0.0001);與α-突觸核蛋白組相比,IL-1β、TNF-αmRNA分別約升高22%、44%,差別有統(tǒng)計學(xué)意義(P0.01)。以上結(jié)果說明,原代培養(yǎng)的VM星形膠質(zhì)細(xì)胞內(nèi)鐵水平升高增強(qiáng)細(xì)胞外α-突觸核蛋白的毒性作用。8.100μmol/L FAC處理VM星形膠質(zhì)細(xì)胞24 h后,CHOP、ATF-4、XBP-1s的表達(dá)有升高的趨勢,且與對照組相比,ATF-4的升高有統(tǒng)計學(xué)意義(P0.01),100 nmol/L的α-突觸核蛋白與100μmol/L FAC共孵育VM星形膠質(zhì)細(xì)胞24 h后,CHOP、ATF-4、XBP-1s的表達(dá)升高,與對照組相比,分別約升高1.61、1.93、2.26倍,差別有統(tǒng)計學(xué)意義(P0.001);與α-突觸核蛋白組相比,CHOP、ATF-4、XBP-1s分別約升高1.24、1.25、1.76倍,差別有統(tǒng)計學(xué)意義(P0.05)。以上結(jié)果說明,原代培養(yǎng)的VM星形膠質(zhì)細(xì)胞內(nèi)鐵水平升高增強(qiáng)細(xì)胞外α-突觸核蛋白誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激。9.100 nmol/L、300 nmol/L的α-突觸核蛋白單體處理VM神經(jīng)元24 h后,與對照組相比ΔΨm分別下降12%、15%,差別有統(tǒng)計學(xué)意義(P0.001)。100 nmol/L的α-突觸核蛋白單體處理VM神經(jīng)元24 h后,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白CHOP、ATF-4、XBP-1s的表達(dá)升高,分別約為對照組的1.62、1.26、1.21倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05)。300 nmol/L的α-突觸核蛋白組,CHOP、ATF-4、XBP-1s的表達(dá)升高,分別約為對照組的1.33、1.25、1.28倍,與對照組相比差別有統(tǒng)計學(xué)意義(P0.05)。以上結(jié)果說明,α-突觸核蛋白能夠誘導(dǎo)VM神經(jīng)元出現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激。以上結(jié)果表明,細(xì)胞外的α-突觸核蛋白單體能夠引起C6膠質(zhì)瘤細(xì)胞和VM星形膠質(zhì)細(xì)胞促炎因子IL-1β、TNF-α的mRNA表達(dá)水平的升高,并且α-突觸核蛋白纖維體也引起促炎因子的升高,但是升高水平低于α-突觸核蛋白單體組,表明細(xì)胞外進(jìn)入細(xì)胞內(nèi)的α-突觸核蛋白能夠引起VM星形膠質(zhì)細(xì)胞促炎因子表達(dá)水平增加,且α-突觸核蛋白單體的毒性要高于α-突觸核蛋白纖維體。進(jìn)入到細(xì)胞內(nèi)的α-突觸核蛋白可以經(jīng)UPS和ALP途徑降解,且ALP在UPS被抑制的情況下激活更明顯。細(xì)胞外的進(jìn)入到細(xì)胞內(nèi)的α-突觸核蛋白可誘導(dǎo)C6膠質(zhì)瘤細(xì)胞和VM星形膠質(zhì)細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白CHOP、ATF-4、XBP-1s的表達(dá)升高。細(xì)胞內(nèi)高鐵會進(jìn)一步增強(qiáng)細(xì)胞外的α-突觸核蛋白誘導(dǎo)VM星形膠質(zhì)細(xì)胞促炎因子表達(dá)增加和內(nèi)質(zhì)網(wǎng)應(yīng)激水平。本實驗在星形膠質(zhì)細(xì)胞中,探討了鐵沉積與細(xì)胞間傳播的α-突觸核蛋白之間的關(guān)系,為進(jìn)一步闡明PD發(fā)病過程中鐵與α-突觸核蛋白相互作用的機(jī)制及干預(yù)措施,提供了新的實驗依據(jù)。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is a neurodegenerative disease of the central nervous system characterized by the progressive loss of dopamine (dopamine, DA) neurons in the dense black mass. Its clinical manifestations are movement inability, muscle stiffness, static tremor, and postural reflex, although studies show that genetic, environmental and age factors are possible. The main pathogeny of PD is not clear, but its exact pathogeny is not clear. The main pathological features of.PD are the loss of dopaminergic neurons in the dense part of substantia nigra (SN) and the formation of the Louis corpuscle (Lewy body, LB). Alpha synuclein is the main component of LB. The abnormal aggregation of alpha synuclein in neurons can change the cell membrane potential and damage mitochondrial work. It can induce endoplasmic reticulum (ER) stress and cause cell damage, in which endoplasmic reticulum stress is one of the core toxic mechanisms of alpha synuclein. In addition to its long-term toxicity in cells, the alpha synuclein can be released by the deity element, and then the surrounding cells (including neurons, The iron deposition is another important neuropathological change in PD. Autopsy evidence suggests that the iron content in a single neuron in the PD substantia nigra region is higher and the level of the ferrous iron is related to the process of PD. Promoting the aggregation of alpha synuclein and enhancing the toxic effect of alpha synuclein in the cell, however, the relationship between iron deposition and intercellular alpha synuclein has not been reported. The previous research focuses on neurodegenerative diseases are focused on the specific damaged Shen Jing Yuan, which accounts for astrocytes of the brain volume 20%-50%. It plays a very important role in the development of the nervous system, synaptic transmission, ion balance, neuroimmunity and so on. Therefore, astrocytes are concerned about the role of neurodegenerative diseases. Astrocytes themselves express the extremely low level of alpha synuclein, but they can ingest the extracellular alpha synuclein. The aggregation of alpha synuclein in glial cells promotes the release of pro-inflammatory and chemical factors, resulting in neuronal damage and secondary activation of microglia. This study selected C6 glioma cells (astrocytoma cell lines), primary cultured ventral mesencephalon (ventral mesencephalon, VM) astrocytes And VM neurons are cell models, using protein immunoblotting, immunofluorescence, fluorescence real-time quantitative PCR, flow cytometry and other methods, the changes of VM astrocyte pro-inflammatory factors and the effect of iron on the process are discussed in depth after the treatment of extracellular alpha synuclein, and whether endoplasmic reticulum stress is involved in the extracellular alpha synaptic nuclear eggs. The toxicity of white VM neurons was observed. The results were as follows: 1.100 nmol/L, 300 nmol/L alpha synuclein monomer treated C6 glioma cells 24 h, the increase of IL-1 beta mRNA was approximately 1.83,2.69 times of the control group, and the increase of TNF- alpha mRNA was about the 1.35,1.74 times of the control group, and there was a difference between the control group and the control group. The study significance (P0.01), and the difference between the 100 nmol/L group and the 300 nmol/L group was statistically significant (P0.01). The above results indicated that the alpha synuclein monomer could increase the expression of the C6 glioma cell proinflammatory cytokines, and the expression of the proinflammatory cytokines increased to the alpha synuclein with a concentration dependent.2.100 nmol/L, and the 300 nmol/L alpha synaptic nucleus. After 24 h of VM astrocytes were treated with protein monomer, the increase of IL-1 beta mRNA was about 9.58,21.7 times of the control group, and the increase of TNF- alpha mRNA was about 4.80,6.17 times of the control group respectively, and the difference was statistically significant (P0.0001) compared with the control group (P0.0001), and the difference between the 100 nmol/L group and the 300 nmol/L group was statistically significant (P0.01). The above results showed that The expression of alpha synuclein monomer can increase the expression of VM astrocyte proinflammatory factor, and the increase of pro-inflammatory factors has a concentration dependent.3.100 micron mol/L alpha - synuclein monomers at 37 C at 3 D, and the preparation of alpha synuclein fibrous.100nmol/L, and 300 nmol/L alpha synuclein fibers to treat VM After 24 h of astrocytes, the increase of IL-1 beta mRNA was about 3.22,7.91 times of the control group, and the increase of TNF- alpha mRNA was about 3.09,4.33 times of the control group respectively, and the difference was statistically significant compared with the control group (P0.05), and the 100 nmol/L group was statistically significant (P0.05) compared with the 300 nmol/L group. Alpha synuclein fibers caused inflammation. The elevation of factors was lower than that of the alpha synuclein monomer group. Compared with the alpha synuclein monomer group, the difference was statistically significant (P0.05). The above results indicate that the alpha synuclein fiber can also increase the expression of VM astrocyte proinflammatory factor, and the increase of the proinflammatory promoter has a concentration dependence on the alpha synuclein. However, the toxicity of alpha synuclein monomers to VM astrocytes was higher than that of alpha synuclein.4.100 nmol/L, the alpha synuclein monomer treated C6 glioma cells 24 h, and the expression of endoplasmic reticulum stress related protein CHOP, ATF-4, XBP-1s increased, approximately 1.33,2.05,1.24 times that of the control group, as compared with the control group. The expression of alpha synuclein group in P0.05.300 nmol/L, CHOP, ATF-4, XBP-1s expression increased, respectively, about 1.50,2.10,1.25 times in the control group, respectively, and there was a significant difference between the control group and the control group (P0.05). The above results showed that the extracellular alpha synuclein could induce the occurrence of endoplasmic reticulum stress in the C6 glioma cells. Autophagic lysosome path was induced by the extracellular alpha synuclein. Autophagy-lysosome pathway (ALP) inhibitor chloroquine (40 mu mol/L) or ubiquitin proteasome system (ubiquitin-proteasome system, UPS) inhibitor MG132 (20 mol/L) treated cell 24 h alone, CHOP, the expression of ATF-4 is increased, suggesting that the inhibition of endoplasmic reticulum stress in cells can be induced. Chloroquine and alpha synuclein reincubate after incubation. There was no significant difference compared with the single chloroquine treatment group, suggesting that the intracellular alpha synuclein could be degraded by UPS, while the MG132 and the alpha synuclein were significantly decreased compared with the single MG132 treatment group, and the difference was statistically significant (P0.0001). The above results suggest that the alpha synuclein into the cell can induce C 6 glioma cells have endoplasmic reticulum stress; the extracellular alpha synuclein can be degraded by UPS and ALP, and ALP is more likely to be induced by the extracellular alpha synuclein to enhance the.5.100 nmol/L mono nucleoprotein monomer for the treatment of the 24 h of VM astrocytes, and the endoplasmic reticulum stress related protein in the case of UPS inhibition. The expression of CHOP, ATF-4 and XBP-1s increased, which was about 1.96,2.23,1.29 times of the control group respectively. Compared with the control group, the difference was statistically significant (P0.05).300 nmol/L in the alpha synuclein group, the expression of CHOP, ATF-4, XBP-1s increased, respectively, about the 2.02,2.32,1.48 times of the control group, and there was a statistically significant difference compared with the control group (P0.05). The above results were statistically significant. It is suggested that alpha synuclein can induce VM astrocytes to appear endoplasmic reticulum stress.6.10 mol/L, and 100 mu mol/L citrate (ferric ammonium citrate, FAC) treated C6 glioma cells after 24 h. The expression of pro-inflammatory factors in 10 mu mol/L FAC group was not changed, and 24 of the cells were incubated with the alpha synuclein monomer. The expression of pro-inflammatory factors was not significantly higher than that in the alpha synuclein group. The expression of pro-inflammatory factors in the.100 mol/L FAC group was increased, and the elevation of IL-1 beta and TNF- alpha mRNA was about 1.72,1.37 times in the control group, and the difference was statistically significant (P0.05). The expression of pro-inflammatory factors was expressed after the 24 h of the 100 nmol/L alpha synuclein monomer. There was no significant change in the amount of the alpha synuclein group. The above results showed that the increase in the iron level in C6 glioma cells did not significantly affect the toxicity of extracellular alpha synuclein, and the mRNA expression level of the proinflammatory factor IL-1 beta and TNF- alpha was elevated after.7.10 mol/L FAC treatment of VM astrocytes, but there was no statistical significance. Meaning (P? 0.05), after incubating 24 h of VM astrocytes with the alpha synuclein monomer of 100 nmol/L, the release of pro-inflammatory factors induced by alpha synuclein increased, compared with the control group, IL-1 beta, TNF- alpha mRNA increased approximately 11.92,4.95 times respectively, and the difference was statistically significant (P0.0001); compared with the alpha synuclein group, IL-1 beta, TNF- alpha mRN. A, respectively, increased by 26%, 18%, and the difference was statistically significant (P0.05).100 mu mol/L FAC treated VM astrocytes 24 h, and the expression level of IL-1 beta and TNF- alpha was also elevated, and TNF- alpha mRNA increased statistically compared with the control group, and 24 of the 100 cells were incubated with alpha synuclein monomer after 24 generations. The expression of pro-inflammatory factors induced by alpha synuclein increased. Compared with the control group, IL-1 beta and TNF- alpha mRNA increased by about 11.59,6.05 times respectively, and the difference was statistically significant (P0.0001). Compared with the alpha synuclein group, IL-1 beta, TNF- alpha mRNA increased by 22%, 44%, respectively, with statistical significance (P0.01). The above results indicated that the primary culture was in primary culture. The elevated level of iron in VM astrocytes enhanced the toxic effect of extracellular alpha synuclein, and the expression of CHOP, ATF-4, XBP-1s was elevated after 24 h of VM astrocytes treated by.8.100 micron FAC, and the increase of ATF-4 was statistically significant compared with the control group (P0.01). The 100 nmol/L alpha synuclein was shared with 100 micron. After incubating VM astrocytes 24 h, the expression of CHOP, ATF-4 and XBP-1s increased, which was about 1.61,1.93,2.26 times higher than that of the control group, and the difference was statistically significant (P0.001). Compared with the alpha synuclein group, CHOP, ATF-4, XBP-1s respectively increased the 1.24,1.25,1.76 times respectively, and the difference was statistically significant (P0.05). The above results indicated that the primary culture The increase of iron levels in VM astrocytes enhanced the endoplasmic reticulum stress induced by extracellular alpha synuclein induced endoplasmic reticulum stress.9.100 nmol/L, and 300 nmol/L alpha synuclein monomers treated VM neurons 24 h, and the delta m decreased by 12%, 15% respectively compared with the control group, and the difference was statistically significant (P0.001).100 nmol/L alpha synuclein monomers treated VM nerve After Yuan 24 h, the expression of endoplasmic reticulum stress related protein CHOP, ATF-4, and XBP-1s increased, which was about 1.62,1.26,1.21 times of the control group, respectively. The difference between the control group and the control group was statistically significant (P0.05) the alpha synuclein group of.300 nmol/L, CHOP, ATF-4, XBP-1s, respectively, about the 1.33,1.25,1.28 times of the control group, respectively, compared with the control group. Statistically significant (P0.05). The above results suggest that alpha synuclein can induce endoplasmic reticulum stress in VM neurons. The above results suggest that the extracellular alpha synuclein monomer can cause an increase in the mRNA expression level of the C6 glioma cells and the VM astrocyte IL-1 beta, TNF- alpha, and the alpha synuclein fiber. The level of pro-inflammatory factors increased, but the elevation was lower than the alpha synuclein monomer group, indicating that the alpha synuclein outside the cell could increase the expression level of VM astrocyte proinflammatory cytokines, and the toxicity of alpha synuclein monomers was higher than that of alpha synuclein fibers. The synuclein can be degraded by UPS and ALP pathways, and the activation of ALP is more obvious in the case of inhibition of UPS. The extracellular entry to the intracellular alpha synuclein can induce the expression of C6 glioma cells and the VM astrocyte endoplasmic reticulum stress related protein CHOP, ATF-4, and XBP-1s. The intracellular high iron will further enhance the extracellular matrix. Alpha synuclein induced increased expression of proinflammatory cytokines in VM astrocytes and the level of endoplasmic reticulum stress. In this experiment, the relationship between iron deposition and intercellular transmission of alpha synuclein was investigated in astrocytes, in order to further elucidate the mechanism and intervention measures of the interaction between iron and alpha tactile nucleoprotein in the pathogenesis of PD. A new experimental basis is provided.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R742.5

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