铁和细胞外α-突触核蛋白影响体外培养的星形胶质细胞的作用研究

发布时间：2018-06-27 14:43

本文选题：铁 + α-突触核蛋白　；参考：《青岛大学》2017年硕士论文

【摘要】：帕金森病(Parkinson’s disease,PD)是一种以黑质致密带多巴胺(dopamine,DA)能神经元进行性缺失为特征的中枢神经系统退行性疾病。其临床表现为运动不能、肌僵直、静止性震颤及姿势反射障碍等。尽管研究显示遗传、环境和年龄等因素均可能参与PD发病,但其确切病因不清。PD的主要病理特征是黑质(substantia nigra,SN)致密部多巴胺能神经元缺失和路易小体(Lewy body,LB)的形成。α-突触核蛋白是LB的主要成分,神经元中α-突触核蛋白的异常聚集能够改变细胞膜电位、损伤线粒体功能、诱导内质网(endoplasmic reticulum,ER)应激而造成细胞损伤,其中内质网应激是α-突触核蛋白的核心毒性作用机制之一。除了长期以来关注的其在细胞内的毒性作用之外,α-突触核蛋白能够被神经元释放,进而被周围的细胞(包括神经元、胶质细胞等)再摄取,从而能够在细胞-细胞之间、相互联系的结构之间进行传递。铁沉积是PD另一重要的神经病理学改变,尸检证据表明,PD黑质区单个神经元内的铁含量增高,黑质铁的水平与PD的进程相关。本课题组前期研究证实铁能够促进α-突触核蛋白的聚集,增强α-突触核蛋白在细胞内的毒性作用,然而铁沉积与细胞间传播的α-突触核蛋白之间的关系尚未有研究报道。以往神经退行性疾病的研究热点都集中在特定受损的神经元上,占脑体积20%-50%的星形胶质细胞在神经系统的发育、突触传递、离子平衡、神经免疫等方面,都起着十分重要的作用,因此星形胶质细胞在神经退行性疾病的作用开始受到关注。星形胶质细胞本身表达极低水平的α-突触核蛋白,但其能摄取胞外的α-突触核蛋白。进入到星形胶质细胞内的α-突触核蛋白的聚集促进促炎因子和化学因子的释放,进而导致神经元的损伤和小胶质细胞的继发性激活。本研究选用C6胶质瘤细胞(星形胶质细胞瘤的细胞系),原代培养的腹侧中脑(ventral mesencephalon,VM)星形胶质细胞及VM神经元为细胞模型,应用蛋白免疫印迹、免疫荧光、荧光实时定量PCR、流式细胞术等方法,深入探讨细胞外的α-突触核蛋白处理后VM星形胶质细胞促炎因子的变化及铁对此过程的影响,并评价内质网应激是否参与了细胞外α-突触核蛋白的毒性作用,并观察VM神经元的损伤变化。研究结果如下:1.100 nmol/L、300 nmol/L的α-突触核蛋白单体处理C6胶质瘤细胞24 h后,IL-1β mRNA的升高分别约为对照组的1.83、2.69倍,TNF-αmRNA的升高分别约为对照组的1.35、1.74倍,与对照组相比差别有统计学意义(P0.01);且100 nmol/L组与300 nmol/L组相比差别有统计学意义(P0.01)。以上结果说明,α-突触核蛋白单体能够引起C6胶质瘤细胞促炎因子表达增加,且促炎因子的表达升高对α-突触核蛋白具有浓度依赖性。2.100 nmol/L、300 nmol/L的α-突触核蛋白单体处理VM星形胶质细胞24 h后,IL-1βmRNA的升高分别约为对照组的9.58、21.7倍,TNF-αmRNA的升高分别约为对照组的4.80、6.17倍,与对照组相比差别有统计学意义(P0.0001),且100 nmol/L组与300 nmol/L组相比差别有统计学意义(P0.01)。以上结果说明,α-突触核蛋白单体能够引起VM星形胶质细胞促炎因子表达增加,且促炎因子的升高对α-突触核蛋白具有浓度依赖性。3.100μmol/Lα-突触核蛋白单体37℃恒温振荡3 d,制备α-突触核蛋白纤维体。100nmol/L、300 nmol/L的α-突触核蛋白纤维体处理VM星形胶质细胞24 h后,IL-1βmRNA的升高分别约为对照组的3.22、7.91倍,TNF-αmRNA的升高分别约为对照组的3.09、4.33倍,与对照组相比差别有统计学意义(P0.05),且100 nmol/L组与300 nmol/L组相比差别有统计学意义(P0.05)。α-突触核蛋白纤维体引起促炎因子的升高水平低于α-突触核蛋白单体组,与α-突触核蛋白单体组相比,差别有统计学意义(P0.05)。以上结果说明,α-突触核蛋白纤维体也能够引起VM星形胶质细胞促炎因子表达增加,且促炎因子的升高对α-突触核蛋白具有浓度依赖性;但α-突触核蛋白单体对VM星形胶质细胞的毒性要高于α-突触核蛋白纤维体。4.100 nmol/L的α-突触核蛋白单体处理C6胶质瘤细胞24 h后,内质网应激相关蛋白CHOP、ATF-4、XBP-1s的表达升高,分别约为对照组的1.33、2.05、1.24倍,与对照组相比差别有统计学意义(P0.05)。300 nmol/L的α-突触核蛋白组,CHOP、ATF-4、XBP-1s的表达升高,分别约为对照组的1.50、2.10、1.25倍,与对照组相比差别有统计学意义(P0.05)。以上结果说明,细胞外的α-突触核蛋白能够诱导C6胶质瘤细胞出现内质网应激。自噬溶酶体途径(autophagy-lysosome pathway,ALP)抑制剂氯喹(40μmol/L)或泛素蛋白酶体系统(ubiquitin-proteasome system,UPS)抑制剂MG132(20μmol/L)单独处理细胞24 h后,CHOP、ATF-4的表达升高,提示抑制ALP或UPS均可诱导细胞出现内质网应激。氯喹与α-突触核蛋白共孵育后与单独氯喹处理组相比没有显著差别,提示进入细胞内的α-突触核蛋白能够经UPS降解。而MG132与α-突触核蛋白共孵育后与单独MG132处理组相比明显下降,差别有统计学意义(P0.0001)。以上结果说明,进入到细胞内的 α-突触核蛋白可以诱导C6胶质瘤细胞出现内质网应激;细胞外进入到细胞内的α-突触核蛋白可以经UPS和ALP降解,且ALP在UPS被抑制的情况下更容易被细胞外的α-突触核蛋白诱导增强。5.100 nmol/L的α-突触核蛋白单体处理VM星形胶质细胞24 h后,内质网应激相关蛋白CHOP、ATF-4、XBP-1s的表达升高,分别约为对照组的1.96、2.23、1.29倍,与对照组相比差别有统计学意义(P0.05)。300 nmol/L的α-突触核蛋白组,CHOP、ATF-4、XBP-1s的表达升高,分别约为对照组的2.02、2.32、1.48倍,与对照组相比差别有统计学意义(P0.05)。以上结果说明,α-突触核蛋白能够诱导VM星形胶质细胞出现内质网应激。6.10μmol/L、100μmol/L枸橼酸铁铵(ferric ammonium citrate,FAC)处理C6胶质瘤细胞24 h后,10μmol/L FAC组促炎因子表达量较对照组没有变化,与100nmol/L的α-突触核蛋白单体共孵育细胞24 h后,促炎因子的表达量较α-突触核蛋白组没有显著变化。100μmol/L FAC组促炎因子表达量较对照组增加,IL-1β、TNF-αmRNA的升高约为对照组的1.72、1.37倍,差别有统计学意义(P0.05),与100 nmol/L的α-突触核蛋白单体共孵育细胞24 h后,促炎因子的表达量较α-突触核蛋白组也没有显著变化。以上结果说明,C6胶质瘤细胞内铁水平升高对细胞外α-突触核蛋白的毒性作用没有明显影响。7.10μmol/L FAC处理VM星形胶质细胞24 h后,促炎因子IL-1β、TNF-α的mRNA表达水平有升高的趋势,但无统计学意义(P㧐0.05),与100 nmol/L的α-突触核蛋白单体共孵育VM星形胶质细胞24 h后,可诱导α-突触核蛋白引起的促炎因子释放量增加,与对照组相比,IL-1β、TNF-αmRNA分别约升高11.92、4.95倍,差别有统计学意义(P0.0001);与α-突触核蛋白组相比,IL-1β、TNF-αmRNA分别约升高26%、18%,差别有统计学意义(P0.05)。100μmol/L FAC处理VM星形胶质细胞24 h后,促炎因子IL-1β、TNF-α的mRNA表达水平也有升高的趋势,且与对照组相比,TNF-αmRNA的升高有统计学意义(P0.05),与100 nmol/L的α-突触核蛋白单体共孵育细胞24 h后,可诱导α-突触核蛋白引起的促炎因子表达量增加,与对照组相比,IL-1β、TNF-αmRNA分别约升高11.59、6.05倍,差别有统计学意义(P0.0001);与α-突触核蛋白组相比,IL-1β、TNF-αmRNA分别约升高22%、44%,差别有统计学意义(P0.01)。以上结果说明,原代培养的VM星形胶质细胞内铁水平升高增强细胞外α-突触核蛋白的毒性作用。8.100μmol/L FAC处理VM星形胶质细胞24 h后,CHOP、ATF-4、XBP-1s的表达有升高的趋势,且与对照组相比,ATF-4的升高有统计学意义(P0.01),100 nmol/L的α-突触核蛋白与100μmol/L FAC共孵育VM星形胶质细胞24 h后,CHOP、ATF-4、XBP-1s的表达升高,与对照组相比,分别约升高1.61、1.93、2.26倍,差别有统计学意义(P0.001);与α-突触核蛋白组相比,CHOP、ATF-4、XBP-1s分别约升高1.24、1.25、1.76倍,差别有统计学意义(P0.05)。以上结果说明,原代培养的VM星形胶质细胞内铁水平升高增强细胞外α-突触核蛋白诱导的内质网应激。9.100 nmol/L、300 nmol/L的α-突触核蛋白单体处理VM神经元24 h后,与对照组相比ΔΨm分别下降12%、15%,差别有统计学意义(P0.001)。100 nmol/L的α-突触核蛋白单体处理VM神经元24 h后,内质网应激相关蛋白CHOP、ATF-4、XBP-1s的表达升高,分别约为对照组的1.62、1.26、1.21倍,与对照组相比差别有统计学意义(P0.05)。300 nmol/L的α-突触核蛋白组,CHOP、ATF-4、XBP-1s的表达升高,分别约为对照组的1.33、1.25、1.28倍,与对照组相比差别有统计学意义(P0.05)。以上结果说明,α-突触核蛋白能够诱导VM神经元出现内质网应激。以上结果表明,细胞外的α-突触核蛋白单体能够引起C6胶质瘤细胞和VM星形胶质细胞促炎因子IL-1β、TNF-α的mRNA表达水平的升高,并且α-突触核蛋白纤维体也引起促炎因子的升高,但是升高水平低于α-突触核蛋白单体组,表明细胞外进入细胞内的α-突触核蛋白能够引起VM星形胶质细胞促炎因子表达水平增加,且α-突触核蛋白单体的毒性要高于α-突触核蛋白纤维体。进入到细胞内的α-突触核蛋白可以经UPS和ALP途径降解,且ALP在UPS被抑制的情况下激活更明显。细胞外的进入到细胞内的α-突触核蛋白可诱导C6胶质瘤细胞和VM星形胶质细胞内质网应激相关蛋白CHOP、ATF-4、XBP-1s的表达升高。细胞内高铁会进一步增强细胞外的α-突触核蛋白诱导VM星形胶质细胞促炎因子表达增加和内质网应激水平。本实验在星形胶质细胞中,探讨了铁沉积与细胞间传播的α-突触核蛋白之间的关系,为进一步阐明PD发病过程中铁与α-突触核蛋白相互作用的机制及干预措施,提供了新的实验依据。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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