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。     

丹参川芎嗪注射液对Aβ损伤的PC12细胞保护作用及机制研究

目的 探讨丹参川芎嗪注射液对Aβ损伤的PC12细胞可能的保护作用及机制。方法 将PC12细胞分为5组:空白对照组(未加任何处理药物)、Aβ诱导组(20 μmol/L Aβ处理组)和预处理组(分别加入浓度为5 ml/L、10 ml/L、20 ml/L的丹参川芎嗪注射液孵育24 h后加20 μmol/L Aβ),通过CCK-8法检测细胞增殖活性,流式细胞术(FCM)检测细胞凋亡率,Hoechst 33258染色观察PC12细胞核的改变,荧光分光光度计测定LDH、SOD、GSH及caspase-3活性水平,免疫组织化学方法观察细胞色素C(Cyt-C)蛋白释放水平,Western Blot检测Bcl-2的表达水平。结果 丹参川芎嗪注射液(5、10、20 ml/L)预处理对Aβ诱导的PC12细胞损伤有较好的保护作用,其保护作用随着药物浓度的增加而增强。它能增加Aβ损伤的PC12细胞增殖活力,减少Aβ诱导的PC12细胞凋亡,降低细胞核凝聚现象,抑制Aβ损伤的PC12细胞LDH释放,增强SOD和GSH活性,促进Cyt-C在细胞内表达,降低caspase-3活性,促进Bcl-2的表达。结论 丹参川芎嗪注射液对Aβ诱导的PC12细胞损伤具有与线粒体通路相关的保护作用,其保护作用与它抑制细胞凋亡、抗氧化应激、维持线粒体正常功能、抑制caspase-3的激活、促进抗凋亡因子Bcl-2的表达有关。     

多巴胺抑制PC12细胞增殖和诱导凋亡作用的研究

目的　研究多巴胺(DA)对PC12细胞的增殖抑制和诱导凋亡的作用,探讨帕金森病(PD)神经元的死亡机制。方法　应用免疫组织化学、流式细胞仪、电镜及电泳技术,研究DA对大鼠嗜铬细胞瘤PC12细胞的增殖抑制及诱导凋亡的作用。结果　适当浓度(0.5mmol/L)DA能显著抑制PC12细胞的生长并诱导其凋亡,在作用时间较短时(<12h)表现为对PC12细胞的生长抑制,此时流式细胞仪检测未见凋亡峰,但细胞周期显示S期细胞明显抑制。此时Bcl-2染色呈强阳性,电镜下细胞形态基本正常,可见线粒体、内质网肿胀及核分裂相减少。当作用时间延长时(>24h),流式细胞仪可见典型亚二倍体凋亡峰,此时电泳可见典型DNA“阶梯状”电泳带,电镜可见核浓缩、染色体边聚等凋亡特征性核结构改变,Bcl-2染色阳性率降低。结论　DA具有抑制PC12细胞增殖和诱导凋亡作用,细胞凋亡参与了PD的病变过程。     

酸敏感离子通道在PC12细胞中的表达

目的：检测酸敏感离子通道（ASICs）在PC12细胞中的表达情况。方法：采用RT-PCR、Westernblot以及单细胞膜片钳记录方法检测ASICs在培养的PC12细胞中的表达情况。结果：PC12细胞中有ASICs蛋白的表达,且ASICs的6个亚基：ASIC1a,ASIC1b,ASIC2a,ASIC2b,ASIC3,ASIC4均有表达,给予酸诱导能产生电流。结论：PC12细胞上存在ASICs电流,且能被ASICs阻断剂阿米洛利所阻断,这为研究ASICs在神经退行性疾病中的作用提供了较为理想的细胞模型。     

6-OHDA诱导PC12细胞凋亡及作用机制

目的 探讨6-羟基多巴胺(6-OHDA)诱导PC12细胞损伤的可能作用机制.方法 不同剂量6-OHDA加入培养的大鼠肾上腺嗜铬细胞瘤细胞(pheochromocytoma cell,PC12)24h后,用四甲基偶氮唑盐法(MTT法)检测细胞的活力,流式细胞仪检测细胞凋亡率以及Bax、Bcl-2的蛋白表达.结果 在加入不同浓度的6-OHDA时PC12细胞活力显著下降,细胞凋亡百分率对照组为8.73±1.09,不同浓度的6-OHDA处理组显著上升,分别为10.97±1.52、25.77±0.95、57.94±1.23,较对照组有显著性差异(P<0.01),并且Bcl-2蛋白表达下降,Bax蛋白表达上升,Bcl-2/Bax比值和正常组比较有显著性差异(P<0.01).结论 6-OHDA能显著诱导PC12细胞损伤,并呈剂量依赖性,其作用机制涉及到促进细胞内bax,以及抑制Bcl-2的表达.     

6—羟基多巴胺诱导PC12细胞凋亡及其可能分子机制

探讨神经毒素 6 羟基多巴胺 (6 OHDA)诱导PC12细胞死亡本质及其可能分子机制。不同剂量 6 OHDA处理PC12细胞 2 4h后 ,分别用流式细胞仪、透射电镜、TUNEL染色和DNA电泳检测细胞凋亡 ;5 0 μmol/L 6 OH DA处理PC12细胞 2 0h后 ,用RT PCR检测caspase 3表达 ;用westernblot检测Caspase 3p32蛋白酶原的切割。结果如下 :①流式细胞仪检测PC12细胞凋亡百分率 ,对照组为 1.10 %± 1.14% ,30 ,4 0 ,5 0 μmol/L 6 OHDA处理组分别为 4 .73 %± 1.0 6% ,10 .15 %± 1.2 1%和 19.94 %± 2 .5 1% ,较对照组均有显著性差异 (P <0 .0 1)。 4 0 ,5 0 μmol/L 6 OHDA处理组透射电镜、TUNEL染色均证实存在大量凋亡细胞 ;5 0 μmol/L 6 OHDA处理组DNA电泳呈现一定间隔的“梯状”条带。② 5 0 μmol/L 6 OHDA处理组caspase 3mRNA水平增高约一倍 ,并且检测到Cas pase 3p2 0活性片段。本研究提示 6 OHDA能诱导PC12细胞凋亡 ,并呈剂量依赖性 ;激活Caspase 3蛋白酶可能参与 6 OHDA诱导PC12细胞凋亡过程     

1—甲基—4—苯基吡啶诱导PC12细胞凋亡的研究

为探讨帕金森病(PD)黑质多巴胺神经元的死亡方式,本文用DNA未端标记法,借助荧光显微镜观察黑质神经毒素MPP~+对儿茶酚胺细胞株PC12细胞凋亡的影响。结果发现,低浓度的MPP~+(20～80uM)可诱导PC12细胞凋亡,高浓度时(10～200uM)则主要引起细胞坏死。本文提示凋亡可能参与了PD多巴胺神经元死亡的早期阶段。     

活化素A对百草枯所诱导的PC12细胞损伤的保护作用

目的:观察重组人活化素A(rhAct)对百草枯所诱导的PC12细胞损伤的保护作用。方法:将百草枯、活化素A、Ldeprenyl加入体外培养的PC12细胞中,用四甲基偶氮唑盐(MTT)法检测细胞活力的变化;免疫细胞化学法和RTPCR评价细胞的酪氨酸羟化酶和Bcl2蛋白及mRNA表达水平的变化,脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测细胞凋亡的变化,比较各组的差异。结果:预先给予活化素A和Ldeprenyl的两组细胞活力明显高于百草枯损害组,酪氨酸羟化酶和Bcl2蛋白及mRNA的表达强于损害组,同时两组的凋亡细胞明显减少,活化素A和Ldeprenyl两组间无显著差异。结论:活化素A和Ldeprenyl通过上调Bcl2的表达,抑制凋亡的发生,而对百草枯所诱导的PC12细胞损伤具有保护作用。     

6羟多巴胺诱导PC12细胞凋亡的研究

为探讨帕金森病(PD)黑质多巴胺神经元的死亡机制。本文用DNA末端标记法,借助荧光显微镜观察6羟多巴胺对儿茶酚胺细胞株PC12细胞凋亡的影响。结果发现:低浓度范围内的6羟多巴胺可诱导PC12细胞凋亡,而单胺摄取抑制剂脱甲丙咪嗪则可抑制其诱导的细胞凋亡。本文提示凋亡可能参与了PD早期病变过程。     

L—dopa诱导PC12细胞凋亡及Bcl—2、Bax表达的改变

目的：探讨L－dopa治疗帕金森病（PD）疗效减退的机制及其毒性作用机制。方法：以PC12细胞为多巴胺神经元的细胞模型,利用PI／HO33342双染结合荧光显微镜技术、电镜技术、流式细胞术及免疫荧光技术检测不同浓度的L-dopa对CPC12铁凋亡诱导作用及凋亡相关基因Bcl-2、Bax表达的改变。结果50、100、150μmol/L不同浓度L－dopa处理组凋亡率分别为12．4％、24．4％、37．2％、PI／HO33342双染可区别凋亡,坏死和正常细胞,且可以见到染色质碎裂;电镜下可见早期凋亡细胞和晚期凋亡细胞,给予L-dopa处理后,Bcl-2的表达量减少,与凋亡率呈显著负相关;Bax的表达量增加,与凋亡率呈显著正相关。结论：L－dopa诱导PC12细胞凋亡且呈量效关系,提示L－dopa可能是通过凋亡途径损害多巴胺神经元导致疗效减退,其机制可能是通过改变Bcl-2/Bax的比值来介导细胞凋亡。     

Nogo-A与NG-R在神经细胞早期分化过程的作用

目的探讨在神经细胞早期生长发育过程中Nogo-A与Nogo-A受体(NG-R)的表达及意义。方法体外培养PC12细胞,实验组以神经生长因子(NGF)诱导其分化,对照组不进行诱导。倒置显微镜下选取20个随机视野进行观察,计数轴突生长和细胞增殖情况。采用免疫荧光染色、逆转录酶聚合酶链反应(RT-PCR)等方法检测PC12细胞生长及分化过程中Nogo-A与NG-R m RNA及蛋白的表达与变化。结果对照组PC12细胞未检出Nogo-A与NG-R的表达。在实验组NGF诱导PC12细胞向交感神经细胞分化过程中,Nogo-A与NG-R表达量逐渐增高(P0.05)。结论在神经细胞发育早期,Nogo-A与NG-R可能发挥促进神经轴突生长的作用。     

重组人胰高血糖素样肽-1衍生物诱导PC12细胞的分化

Glucagon-like peptide-1 (GLP-1) and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases.Its short half-life has limited the application of GLP-1 in the clinic.We generated a mutated form of human GLP-1 (mGLP-1) using site-directed mutagenesis and gene recombination techniques,and found that these modifications significantly prolonged the biological half-life of GLP-1 compared with native GLP-1 (nGLP-1).This study investigated the role of mGLP-1 on inducing PC12 cell differentiation.mGLP-1 induced PC12 cell differentiation with neurite outgrowth and increased the expression of growth-associated protein-43 and neuronal class III-tubulin,and significantly increased cyclic adenosine monophosphate level.No significant difference was found between mGLP-1 and nGLP-1.The results indicate that mGLP-1 activates the GLP-1 receptor,induces PC12 cell differentiation,and has neurotrophic effects.     

Melatonin inhibition of nicotine-stimulated dopamine release in PC12 cells

Melatonin, a pineal hormone, modifies numerous physiologic processes including circadian rhythms and sleep. In specific tissues, melatonin appears to have an inverse relationship with dopamine. To examine this relationship, a pheochromocytoma cell line (PC12) was used to determine the extent of melatonin's ability to inhibit nicotine-stimulated dopamine release. Multiple experiments were conducted that examined: (1). the dose response of acute melatonin (5 min); (2). the effects of chronic melatonin (16 h pre-exposure); (3). the effects of prior nicotine or melatonin exposure (5 min) on melatonin's ability to alter dopamine release from a second 5-min nicotine exposure; and (4). the role of melatonin receptors (by pertussis toxin inhibition) on nicotine-stimulated dopamine release. In the dose response studies, melatonin inhibited nicotine-stimulated dopamine release with an ED50 of 8.6 microM. Chronic exposure to melatonin had no effect on melatonin's acute inhibition of nicotine-stimulated dopamine release. Prior nicotine or melatonin exposure had little effect on subsequent melatonin or nicotine exposure, except that the cells exposed to nicotine were not responsive to a second exposure to nicotine. Blockade of melatonin receptor function by pre-exposure to pertussis toxin (16 h) did not prevent melatonin's inhibition of nicotine-stimulated dopamine release. However, the toxin-treated cells were less inhibited by melatonin when compared to control cells suggesting a partial role for melatonin receptors. These results indicate that melatonin can acutely inhibit nicotine-stimulated dopamine release in PC12 cells. This model system allows detailed examination of melatonin's cellular actions as well as supporting a role for melatonin on neuronal dopamine release.     

Neuregulin found in cultured-sciatic nerve conditioned medium causes neuronal differentiation of PC12 cells

The present work deals with the search and identification of the molecule or combination of molecules, present in a medium conditioned by cultured rat-sciatic nerves (CM), able to cause neuronal differentiation of PC12 cells. The molecular mass range of the active fraction, as well as the thermostability and heparin affinity of the active component found in previous work, all characteristics shared with neuregulin (NRG) family members, led us to search for a NRG protein in the CM. Nerves were previously cultured for 8 days and the CM collected every 24 h, the following 3 days. The CM was concentrated (30,000 NMWL) and fractionated by quaternary ammonium chromatography and Cibacron blue affinity chromatography. The most active fraction B1.2 was further characterized by heparin affinity chromatography, size exclusion HPLC, Western blotting and immunoprecipitation. Results reveal abundance of NRG mRNA in the cultured nerves, presence of a 54 kDa NRG protein in the CM that increases along fractionation, and progressive diminution of fraction B1.2 differentiation activity on PC12 cells by gradual removal of the NRG protein by immunoprecipitation. The abundance of Schwann cells and the lack of axons in the cultured nerves suggest Schwann cells as the main NRG source, to which fibroblasts and perineurial cells might contribute.     

Dimethylarginine dimethylaminohydrolase 1 regulates nerve growth factor-promoted differentiation of PC12 cells in a nitric oxide-dependent but asymmetric dimethylargenine-independent manner

There are significant morphological and biochemical alterations during nerve growth factor (NGF)-promoted neuronal differentiation, and the process is regulated by molecules, including nitric oxide (NO). Dimethylarginine dimethylaminohydrolase (DDAH) is thought to play a critical role in regulating NO production via hydrolyzing the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus, we tested the role of DDAH in NGF-promoted differentiation of PC12 (pheochromocytoma) cells. The present results show that both mRNA and protein levels of DDAH1 were increased, whereas those of DDAH2 were decreased, during NGF-promoted cell differentiation. Both the DDAH activity and the ADMA level in cultured medium were unchanged in this process. NGF promoted neurite formation and induced the expression of microtubule-associated protein 2 (MAP2), a neuronal marker, which were both significantly repressed by DDAH1 silence with small interfering RNA but not by DDAH2 silence. The expressions of three isoforms of NOS were markedly upregulated after NGF stimulation with a time course similar to that of DDAH1, which were attenuated by DDAH1 silence. Conversely, overexpression of DDAH1 accelerated neurite formation in PC12 cells, concomitantly with upregulating the expression of three NOS isoforms. In summary, our data reveal the critical regulatory effect of DDAH1 on NGF-promoted differentiation of PC12 cells in an NOS/NO-dependent but ADMA-independent manner.     

Alcohols synergize with NGF to induce early differentiation of PC12 cells

The role of alcohols in affecting neuromorphogenesis was investigated in a single cell type, pheochromocytoma (PC12). The effect of ethanol at physiological concentrations in this system leads to enhanced morphological and functional differentiation in combination with nerve growth factor (NGF), PC12 cells treated with a suboptimal concentration of NGF (30 ng/ml) and an alcohol (87 mM) underwent rapid morphological differentiation which was dependent upon the side chain length of the alcohol MeOH < EtOH < PrOH < BuOH. Pyrazole at either 5 or 10 mM had no effect on alcohol induced neurite extension. Assessment of the degree of differentiation promoted by the various alcohols was quantified by an increase in neurite extension, a decrease in the incorporation of [³H]thymidine, an increase in acetylcholine esterase (AChE) activity and immunostaining with neuron specific enolase. Thus, alcohols may function in a specific manner by interacting with transmembrane signalling pathways which promote gene expression and neuronal differentiation.     

Purine-induced alterations of dopamine metabolism in rat pheochromocytoma PC12 cells

Studies with cerebrospinal fluid from subjects with Parkinson's disease suggest that purine abnormalities may be present in this disorder. The effects of purines on dopamine metabolism have not been characterized, though adenosine is known to inhibit dopaminergic neurotransmission. In this study, dopamine, its precursor 3,4-dihydroxyphenylalanine (DOPA), and its degradation products 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured in rat pheochromocytoma PC12 cells following 24-h incubation with 5, 50, and 500 microM adenosine, adenine, guanosine, guanine, hypoxanthine, xanthine, and uric acid. Incubation with adenosine increased DOPA, DOPAC, and HVA, while adenine treatment decreased DOPA. Guanosine (500 microM) decreased DOPA, dopamine, and DOPAC, while lower concentrations increased DOPAC and HVA. Incubation with guanine decreased dopamine, and xanthine decreased dopamine and DOPAC. Hypoxanthine and uric acid exerted minimal effects. These results indicate that purines exert a variety of effects on dopamine metabolism. The influence of purine metabolism on the dopaminergic deficit in the Parkinsonian brain merits further investigation.     

Cocaine inhibition of neuronal differentiation in NGF-induced PC12 cells is independent of ras signaling

In utero exposure to cocaine may result in altered neuronal development. Our previous studies demonstrated cocaine inhibits neurite outgrowth in NGF-induced PC12 cells through dopamine, by activation of D₁ receptors. This study examined where cocaine interferes in the NGF signaling cascade. GSras1 cells that inducibly express activated forms of Ras upon treatment with dexamethasone were used. Morphological differentiation was quantified by counting cells bearing neurite-like processes after 72 h exposure to either dexamethasone or NGF alone, or with cocaine, dopamine or SKF-38393. Cocaine, dopamine, and the D₁ agonist inhibited neurite-like process outgrowth in both dexamethasone and NGF-induced GSras1 cells. GAP-43 expression, used as a measure for biochemical differentiation was severely diminished in NGF and dexamethasone-induced GSras1 cells treated with cocaine. These results suggest that cocaine, dopamine and activation of D₁ receptors affect the NGF signaling downstream, independent of ras expression, leading to altered neuronal differentiation.