PC12细胞缺氧后细胞凋亡与DNA损伤相关基因表达关系的研究

目的 探讨PC12细胞缺氧后细胞凋亡与DNA损伤的关系。方法 采用TUNEL法,结合应用流式细胞术观察PC12细胞缺氧培养不同时间点细胞凋亡现象,以及DNA损伤相关基因表达的改变。结果 在缺氧0.5h开始出现凋亡细胞,引时P53、P21^waf1/cip1蛋白表达开始增高。至缺氧1h凋亡细胞达高峰,此时P53、P21蛋白表达最高（P＜0.05）。至缺氧6－12h,则以坏死为主,此时以上基因表达均减弱。结论 PC12细胞在缺氧早期（0.5-2h）主要出现以凋亡为主的细胞死亡,伴有DNA损伤相关基因表达的动脉改变,提示缺氧后PC12细胞凋亡部分是由于DNA损伤严重、损伤不能及时修复所致。     

灯盏细辛对缺氧复氧损伤的PC12细胞保护机制的研究

目的 探讨灯盏细辛对缺氧复氧（HR）损伤的PC12细胞保护机制。方法 将体外培养的PC12细胞分为正常对照（NC）组、HR组、灯盏细辛10μmol／L（E1）组、20μmol／L（E2）组、30μmol／L（E3）组。将HR组及E1～E3组细胞经HR处理后,测定各组细胞的存活率;通过Western Blot方法观察各组细胞Bcl-2和Bax蛋白含量的变化。结果 （1）与NC组比较,HR组的细胞存活率明显下降（P〈0．01）;与HR组比较,E1-E3组细胞存活率明显升高（均P〈0．01）,且呈剂量依赖性（P〈0．01）。（2）与NC组比较,HR组PC12细胞Bcl-2蛋白表达明显下降,Bax蛋白表达明显增加（均P〈0．01）;与HR组比较,E1-E3组PC12细胞Bcl-2表达明显增加,Bax表达明显下降,并呈剂量依赖性（均P〈0．01）。结论 灯盏细辛对HR损伤的PC12细胞保护作用是通过增加Bcl-2表达、抑制Bax表达的抗凋亡作用实现的。     

P^38激酶在PC12细胞缺氧／再给氧损伤中的作用

目的：探讨ＰＣ１２细胞缺氧／再给氧损伤的信号转导机理。方法：培养的ＰＣ１２细胞先缺氧（９５％Ｎ２／５％ＣＯ２）６ｈ,然后重新给氧,预测不同时间点细胞的存活率和ｃａｓｐａｓｅ－３的活性;用ＭＴＴ法测存活率,ｃａｓｐａｓｅ－３检测试剂盒测ｃａｓｐａｓｅ－３活性。用ｐ３８拮抗剂ＳＢ２０３５８０孵育细胞２ｈ,之后缺氧／再给氧,观察ＳＢ２０３５８０对细胞存活率和ｃａｓｐａｓｅ－３活性的影响。结果：ＰＣ１２细胞缺氧／再给氧后ｃａｓｐａｓｅ－３活性的影响。结果：ＰＣ１２细胞缺氧／再给氧后ｃａｓｐａｓｅ－３活性明显增加并使细胞存活率下降,ＳＢ２０３５８０明显降低缺氧／复氧后ｃａｓｐａｓｅ－３的活性并使细胞死亡减少。结论：ＰＣ１２细胞缺氧／再给氧后至少可以通过激活ｐ３８、ｃａｓｐａｓｅ－３信号分子诱导ＰＣ１２细胞死亡。     

P~(38)激酶在PC12细胞缺氧/再给氧损伤中的作用

目的:探讨PC12细胞缺氧/再给氧损伤的信号转导机理。方法:培养的PC12细胞先缺氧(95％N2/5％CO2)6h,然后重新给氧,观测不同时间点细胞的存活率和caspase-3的活性;用MTT法测存活率,caspase-3检测试剂盒测caspase-3活性。用p38拮抗剂SB203580孵育细胞2h,之后缺氧/再给氧,观察SB203580对细胞存活率和caspase-3活性的影响。结果:PC12细胞缺氧/再给氧后caspase-3活性明显增加并使细胞存活率下降,SB203580明显降低缺氧/复氧后caspase-3的活性并使细胞死亡减少。结论:PC12细胞缺氧/再给氧后至少可以通过激活p38、caspase-3信号分子诱导PC12细胞死亡。     

氢气预处理对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响

目的探讨氢气对乳鼠海马神经元细胞缺氧/复氧损伤后细胞活力的影响。方法原代培养的SD乳鼠海马神经元细胞,随机分成对照组,缺氧/复氧组和氢气预处理组。对照组常规培养;缺氧/复氧组在100%N2中培养15min后复氧30min;氢气预处理组在2%H2+98%N2中培养15min后复氧30min。采用MTT法检测乳鼠海马神经元细胞活力。结果氢气预处理能够增强细胞活力(P<0.05)。结论氢气预处理对海马神经元细胞缺氧/复氧损伤具有保护作用,可能与提高细胞活力有关。     

缺氧／复氧对星形胶质细胞AQP4表达的影响

目的 探讨缺氧/复氧对于体外培养星形胶质细胞表达AQP4的影响及缺血性脑血管病脑水肿的发病机制.方法 选取新生24 h Wistar大鼠脑组织行星形胶质细胞原代培养并建立缺氧/复氧模型,应用Western blot和免疫细胞化学法检测星形胶质细胞AQP4的表达变化.结果 与对照组比较,缺氧3 h和6 h星形胶质细胞AQP4蛋白表达减少(P<0.01),复氧后6 h和9 h其表达明显增高(P<0.01).结论 AQP4表达变化与细胞损害相关.     

缺氧诱导因子1α与脑缺血缺氧性损伤

缺氧诱导因子1α(HIF-1α)是近年来研究较多的一种转录调节因子,其在常氧状态下降解,低氧状态下稳定,并从细胞质向细胞核转移,参与对低氧的多种生理应答,通过泛素蛋白酶体系统降解,在缺氧神经细胞培养及脑缺血缺氧损伤模型中表达增加,不同药物干预及高压氧预处理对其表达变化影响不同,产生神经保护或者损伤作用,通过研究其表达特点,为临床缺血缺氧性脑损伤的治疗提供新的思路。     

MPP+诱导PC12细胞损伤的蛋白质组学研究

目的研究Mpp~ 作用PC12细胞48h后蛋白质表达谱的改变,进一步在蛋白质水平上阐明帕金森病的发病机制。方法建立Mpp 诱导的PC12细胞帕金森病模型,提取对照组与实验组的细胞总蛋白,应用荧光差异凝胶电泳(Differential Gel Electrophoresis,DIGE)系统构建双向电泳图,DeCyder软件分析蛋白表达差异信息,运用MALDI-TOF质谱鉴定差异蛋白质。结果与对照组相比,实验组共有32个蛋白点发生变化;鉴定了其中7种结构和功能各异的蛋白。结论本研究鉴定出氧化应激和线粒体损伤相关的蛋白thioredoxin、MPPs,与细胞骨架相关的蛋白NF-L、ezrin,具有分子伴侣活性的蛋白NAC、crystaillin,与免疫炎症相关的蛋白gClqBP。这些蛋白的显著改变可能与PD的发病机制密切相关。     

缺氧诱导因子-1在缺氧幼鼠脑组织中的表达及意义

目的探讨缺氧窒息幼鼠脑组织中缺氧诱导因子-1α(hypoxia-inducible factor-1alpha,HIF-1α)表达水平的变化。方法取50只SD幼鼠,随机分为模型组(n=25)和对照组(n=25)2组,模型组做缺氧处理,对照组不作缺氧处理,于缺氧后48h测定幼鼠脑组织中HIF-1αmRNA表达的相对含量。结果 2组幼鼠脑组织中HIF-1αmRNA表达含量的差异有统计学意义(P<0.05)。HIF-1αmRNA表达的相对含量缺氧组[(1.00±0.02)ng/L]比正常组[(0.040.01)ng/L]显著增高(P<0.05),正常组HIF-1α表达含量相对极少。结论 HIF-1α是在缺氧条件下产生的一种转录因子,通过调控一系列与适应缺氧有关的基因表达来减缓细胞缺氧所带来的损伤。     

稳定表达人CNTF诱导PC12细胞分化作用的研究

目的:探讨CNTF对PC12细胞的诱导分化作用。方法:应用转基因技术将pcDNA-S-hCNTF质粒转入COS7细胞进行表达,并对CNTF表达进行检测,然后将hCNTF修饰过的COS7细胞与PC12细胞进行联合培养,4天后对CNTF作用后的PC12进行形态学观察,β-tubulin免疫细胞化学染色及不同培养时间4天,5天后阳性细胞的指数进行分析,同时通过MTF实验对诱导培养后的PC12细胞内增殖进行检测。结果:PC12细胞与hCHTF修饰过的COS7细胞经过4天的联合培养,细胞大部分形态呈现出类似于神经元的形态,胞体立体感较强,并且细胞长出较长的突起。另外细胞呈现β-tubulin反应阳性,并且随培养时间延长,β-tubulin阳性细胞的数目有增加的趋势(从76.6％增加到88.3％),最后,反映细胞增殖程度的OD值为0.328±0.019,明显低于对照组0.586±0.028,0.597±0.032。结论:hCNTF对PC12细胞向神经元分化有明显的促进作用。     

硫化氢对PC12细胞存活素表达的影响及其神经保护作用

目的 探讨硫化氢(H2s)对PC12细胞存活素表达的影响及其神经保护作用.方法 应用Western blot检测不同浓度(50～800 μmol/L)的H2S供体硫氢化钠(NaHS)处理PC12细胞不同时间(0～180min)诱导PC12细胞存活素表达的量效和时效关系;细胞计数Kit-8(CCK-8)比色法检测细胞的存活率.结果应用不同浓度NaHS处理PC12细胞30 min后.在50～200 μmol/L浓度范围内呈浓度依赖性地促进存活素表达:但随着NaHS浓度的增加,存活素表达逐渐下降,当NaHS 浓度达800 μmol/L时存活素表达低于正常.应用400 μmo/L NariS处理PC12细胞不同时间,在0～60min时间范围内呈时间依赖性地促进PC12细胞存活素的表达,但随着处理时间的继续延长,存活索的表达逐渐下降.另一方面.400 μmo/L NariS预处理还能增强氯化钴(COCl2)对存活素表达的促进作用.并可显著减轻CoCl2对PC12细胞的损伤作用,使细胞存活率增加.结论 在一定浓度和时间范围内H2S可上调存活素的表达,此作用可能与其保护PC12细胞对抗化学性缺氧损伤有关.     

Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation

Reactive oxygen species (ROS) are supposed to play an important role in hypoxia- and ischemia/reperfusion-mediated neuronal injury with the characteristics of apoptosis. There are many reports showing that cobalt chloride (CoCl(2)) could mimic the hypoxic responses in some aspects including production of ROS in cultured cells. The cytotoxicity of CoCl(2) and its molecular mechanisms have yet to be elucidated. We report that CoCl(2) triggered neuronal PC12 cells apoptosis in a dose- and time-dependent manner. Apoptosis was demonstrated by morphological changes and DNA fragmentation, and was dependent on macromolecular synthesis. Apoptosis was also confirmed by the decrease of the expression of Bcl-X(L). To our knowledge, this is the first documentation of the apoptotic induction of CoCl(2) on PC12 cells. Furthermore, ROS production in PC12 cells was increased during CoCl(2) treatment. Antioxidants, which could inhibit ROS production, significantly blocked CoCl(2)-induced apoptosis, suggesting that apoptosis is mediated by ROS production. We also observed a significant increase of the DNA-binding activity of AP-1 in response to CoCl(2) and this increase was blocked by antioxidants, showing that CoCl(2)-induced apoptosis is accompanied by ROS-activated AP-1. CoCl(2)-treated PC12 cells may serve as an in vitro model for studies of molecular mechanisms in ROS-linked neuronal disorders.     

SIRT1在脂多糖诱导的PC12细胞凋亡中的作用

目的 探讨沉默信息调控因子1(SIRTl)在脂多糖(LPS)诱导的PC12细胞凋亡过程中的作用. 方法 PC12细胞按不同培养方法分为6组:对照组、250 μg/mL组、500 μg/mL组、750μg/mL组、1000 μg/mL组、1250 μg/mL组;对照组常规培养,后5组添加相应浓度的LPS培养.24 h后MTT法测量细胞存活率,同时确定LPS诱导PC12细胞凋亡的适合浓度.再按选定的浓度培养细胞,并根据培养时间的不同分为6组:对照组、1/2 h组、2h组、18h组、24 h组、48 h组,Hoechst染色及流式细胞仪测量细胞凋亡率.Western blotting检测各组细胞中SIRT1的表达情况. 结果 Hoechst染色结果提示PCl2细胞经LPS培养1/2h后出现凋亡,表现为核固缩、核碎裂;18h开始凋亡小体增多,24h达到高峰,48 h后又有所下降.流式细胞仪检测结果显示各实验组细胞凋亡率与对照组相比差异均有统计学意义(P＜0.05),凋亡率变化趋势与Hoechst染色法观察到的结果 一致.Western blotting结果显示对照组SIRTl表达量为1.84±0.04; 1/2 h时SIRT1蛋白表达减低至1.17±0.09;24 h时表达降至最低,为0.62±0.03;48 h组表达量有所回升,达到0.77±0.02;实验组各时间点组与对照组相比差异均有统计学意义(P＜0.05). 结论 LPS可以诱导PC12细胞凋亡,SIRT1在此过程中的表达受到抑制;推测SIRT1在LPS诱导PC12细胞凋亡的过程中起到一定的保护作用.     

Fucosyl-GM1 expression and amyloid-beta protein accumulation in PC12 cells

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are localized primarily in the plasma membrane. For a rat pheochromocytoma cell line, PC12, which has been used frequently as a model for investigating events leading to neuronal differentiation, it is generally thought that GM1 is a major ganglioside, based on reactivity with the probe cholera toxin B subunit (Ctxb). From a series of biochemical studies, however, it has been reported that no GM1 is expressed in PC12 cells. In this study, we have reevaluated GM1 expression and Ctxb reactivity in PC12 cells and a subcloned line, PC12D cells. Flow cytometric analysis with Ctxb revealed that about 30-50% of PC12 cells were reactive with Ctxb. However, a detailed biochemical analysis showed that PC12 cells express abundantly a different ganglioside, fucosyl-GM1, instead of GM1, and the reactivity of Ctxb in the PC12 cells actually arose from its interaction with fucosyl-GM1, which also interacts with this ligand. Because it has been claimed that amyloid-beta protein (Abeta) interacts with GM1 in PC12 cells to provide "seeding" for amyloid to accumulate, we further evaluated this possibility and found that Abeta is mostly likely interacting with fucosyl-GM1 in this cell line. Our data thus suggest that a specific interaction may occur between Abeta and fucosyl-GM1 for the accumulation of amyloid in PC12 cells.     

谷氨酸对分化PC12细胞的氧化损伤及人参皂苷Rg1的保护作用

目的观察不同浓度的谷氨酸对PC12细胞的氧化损伤,探讨人参皂苷Rg1对谷氨酸兴奋性毒性的拮抗作用。方法用MTT法检测细胞活性及MDA、GSH、GSH-Px测定,检测谷氨酸对PC12细胞的损害,用人参皂苷Rg1做保护研究。结果谷氨酸可导致PC12细胞活力明显下降,MDA、GSH、GSH-Px测定结果与正常对照组和人参皂苷Rg1保护组有明显差异(P<0．01)。结论人参皂苷Rg1对谷氨酸所致的氧化损伤有保护作用。     

7-Ketocholesterol enhances 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells

Summary. The present study investigated the promoting effect of oxysterol 7-ketocholesterol against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP⁺) in differentiated PC12 cells. 7-Ketocholesterol significantly enhanced the MPP⁺-induced nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, ascorbate, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of MPP⁺ in the presence of 7-ketocholesterol. The results indicate that 7-ketocholesterol shows a synergistic effect against the cytotoxic effect of MPP⁺. 7-Ketocholesterol may enhance the MPP⁺-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of reactive oxygen species and depletion of GSH. The findings suggest that 7-ketocholesterol as a promoting agent for the formation of mitochondrial permeability transition may enhance the toxic neuronal cell injury.     

Decreased levels of nerve growth factor receptor on dexamethasone-treated PC12 cells

Treatment of PC12 cells with dexamethasone leads, in a period of days, to a 60% decrease in the binding of (125I)nerve growth factor. The decrease was maximal after 3 days of treatment with 1 microM dexamethasone, but some decrease was seen after 6 hr and at concentrations as low as 10 nM. The effect was specific for the glucocorticosteroids. Scatchard plots confirmed the overall loss of nerve growth factor binding, and studies with trypsin digestion and Triton X-100 extraction indicated that the decrease in binding was largely due to a decrease in the number of low-affinity receptors. Nerve growth factor-induced changes, such as the induction of ornithine decarboxylase and the generation of neurites, were inhibited, but only minimally, in dexamethasone-treated cells.     

Mechanisms whereby nerve growth factor increases diacylglycerol levels in differentiating PC12 cells

We previously showed indirectly that the increase in diacylglycerol (DAG) levels caused by exposing differentiating PC12 cells to nerve growth factor (NGF) must derive mainly from de novo synthesis and, to a lesser and transient extent, from the hydrolysis of [3H]phosphatidylinositol (PI). To explore further the biochemical mechanisms of this increase, we measured, in PC12 cells, DAG synthesis from glycerol or various fatty acids; its liberation from phosphatidylcholine (PC); and the activities of various enzymes involved in DAG production and metabolism. Among cells exposed to NGF (0-116 h), the labeling of DAG from [3H]glycerol peaked earlier than that of [3H]PC, and the specific radioactivity of [3H]glycerol-labeled DAG was much higher than those of the [3H]phospholipids, indicating that [3H]DAG synthesis precedes [3H]phospholipid synthesis. NGF treatment also increased (by 50-330%) the incorporation of monounsaturated ([3H]oleic acid) and polyunsaturated ([14C]linoleic acid or [3H]arachidonic acid) fatty acids into DAG, and, by 15-70%, into PC. NGF treatment increased the activities of long chain acyl-CoA synthetases (LCASs), including oleoyl-CoA synthetase and arachidonoyl-CoA synthetase, by 150-580% over control, but cholinephosphotransferase activity rose by only 60%, suggesting that the synthesis of DAG in the cells was increased to a greater extent than its utilization. NGF did not promote the breakdown of newly formed [3H]PC to [3H]DAG, nor did it consistently affect the activities of phospholipase C or D. NGF did increase phospholipase A2 activity, however the hydrolysis catalyzed by this enzyme does not liberate DAG. Hence the major source of the increased DAG levels in PC12 cells exposed to NGF appears to be enhanced de novo DAG synthesis, probably initiated by the activation of LCASs, rather than the breakdown of PC or PI.