利用分子雷达研究吗啡对PC12细胞的作用机制

分子雷达是一种能够在分子层次对活细胞内生物过程进行实时、原位观察的探测技术。本文利用分子雷达研究了吗啡对PC12细胞的作用机制。实验结果表明，吗啡对PC12的作用具有时间和浓度依赖性，吗啡能引起细胞内[Ca^2 ]瞬时升高和波动，并且能导致细胞核浓缩和线粒体膜电势丧失。     

毛细管电泳检测神经细胞凋亡

建立了毛细管电泳检测凋亡细胞DNA片段的方法.以DNA相对分子质量标准品为溶质,考察了分离条件(电压、进样时间、温度、聚合物浓度)对分离的影响.在优化条件下,利用毛细管无胶筛分电泳对缺氧缺血过程中不同时间点神经PC12细胞DNA片段进行了分析,并与流式细胞仪结果比较,研究缺氧缺血时胶质细胞凋亡过程.     

Chaperones Are Necessary for the Expression of Catalytically Active Potato Apyrases in Prokaryotic Cells

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5′-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.     

Gene Expression Analysis in MCF-7 Breast Cancer Cells Treated with Recombinant Bromelain

The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p?相似文献   

Neurite Outgrowth-Promoting Compounds from the Petals of Paeonia lactiflora in PC12 Cells

Isorhamnetin-3-O-glucoside and astragalin, flavonol glucosides, were isolated from the petals of Paeonia lactiflora as neurite outgrowth-promoting compounds. Isoquercitrin, formed by demethylating the B ring of isorhamnetin-3-O-glucoside or by adding a hydroxyl group to the B ring of astragalin, was evaluated for neurite outgrowth-promoting activity and was compared with the activities of isorhamnetin-3-O-glucoside and astragalin. The activities of isorhamnetin, kaempferol, and quercetin, aglycones corresponding to isorhamnetin-3-O-glucoside, astragalin, and isoquercitrin, respectively, were also evaluated. Isorhamnetin-3-O-glucoside and astragalin showed much stronger neurite outgrowth-promoting activities than the activities of the other tested flavonoids. They exhibited relatively weak anti-oxidant activities and moderate AChE inhibitory activities compared to the activities of the other tested flavonoids. Isorhamnetin-3-O-glucoside and astragalin promoted morphological neurite outgrowth and the expression of neurofilaments induced by NGF in PC12 cells. Isorhamnetin-3-O-glucoside and astragalin might be candidate compounds as neural differentiation agents in peripheral nerves and functional food ingredients preventing cognitive decline.     

Protective Effect of Ferulic Acid against Hydrogen Peroxide Induced Apoptosis in PC12 Cells

Ferulic Acid (FA) is a highly abundant phenolic phytochemical which is present in plant tissues. FA has biological effects on physiological and pathological processes due to its anti-apoptotic and anti-oxidative properties, however, the detailed mechanism(s) of function is poorly understood. We have identified FA as a molecule that inhibits apoptosis induced by hydrogen peroxide (H₂O₂) or actinomycin D (ActD) in rat pheochromocytoma, PC12 cell. We also found that FA reduces H₂O₂-induced reactive oxygen species (ROS) production in PC12 cell, thereby acting as an anti-oxidant. Then, we analyzed FA-mediated signaling responses in rat pheochromocytoma, PC12 cells using antibody arrays for phosphokinase and apoptosis related proteins. This FA signaling pathway in PC12 cells includes inactivation of pro-apoptotic proteins, SMAC/Diablo and Bad. In addition, FA attenuates the cell injury by H₂O₂ through the inhibition of phosphorylation of the extracellular signal-regulated kinase (ERK). Importantly, we find that FA restores expression levels of brain-derived neurotrophic factor (BDNF), a key neuroprotective effector, in H₂O₂-treated PC12 cells. As a possible mechanism, FA increases BDNF by regulating microRNA-10b expression following H₂O₂ stimulation. Taken together, FA has broad biological effects as a neuroprotective modulator to regulate the expression of phosphokinases, apoptosis-related proteins and microRNAs against oxidative stress in PC12 cells.     

纳米电极时空分辨监测单个PC12细胞多巴胺量子释放

细胞作为有机体结构与生命活动的基本单位 ,在生命体内的新陈代谢和信息传递等方面起着关键作用 .而细胞受激释放是其参与生命活动的形式之一 ,在整个生命活动中起着非常重要的作用 ,不正常的细胞释放会导致生物体功能紊乱 ,以致产生各种疾病 .对细胞释放进行研究在神经生物学、生物化学、临床、病理和药学等多个学科领域中都具有非常重要的意义[1] .由于细胞的超微体积以及胞内单个囊泡量子释放发生的时间是毫秒级的 ,因此需要一种快速灵敏检测的手段才能对胞内释放情况进行实时监测 .对于多巴胺、肾上腺素、去甲肾上腺素及五羟色胺等具有…     

PC12细胞重离子辐射生物学效应的比较蛋白质组学研究

在太空探索尤其是深空探索之前,必须了解清楚重离子辐射对宇航员健康的威胁。本文使用2Gy剂量的碳离子束辐射PC12细胞,并在继续培养24h之后采用基于定量驱动的比较蛋白质组学方法分析重离子辐射引起的蛋白表达变化。结果显示,重离子辐射后的细胞和对照相比,共有15个蛋白在表达水平上发生显著差异。在这些差异蛋白中,较多蛋白属于热休克蛋白,在生物途径上多数和细胞凋亡有关。这些差异蛋白的鉴定,有助于进一步研究重离子辐射生物学效应的分子机制。     

一种新型化合物的合成及对PC12高分化细胞的神经保护作用

研究了一种新合成化合物(3h8b)的神经保护作用及初步分子机制. 该化合物具有缓解神经毒素6-羟基多巴胺(6-OHDA)和L-谷氨酸(L-Glu)诱导高分化PC12细胞损伤的作用. 可有效改善神经毒素对高分化PC12细胞的损伤, 明显增强细胞活力, 降低细胞核凋亡比率, 抑制细胞内钙离子过载. 经3h8b处理之后, 神经毒素引起的细胞内线粒体膜电位异常显著恢复. 进一步的实验表明, 3h8b可以逆转神经毒素造成的Bcl-2和Bcl-xL抑制性表达. 实验证实3h8b通过线粒体相关途经对高分化PC12细胞起保护作用, 为通过化学合成法获得具有治疗神经退行性病变作用的新型化合物提供了理论依据.     

硒代二半胱氨酸对白血病细胞系的诱导分化研究

硒抑制体外白血病细胞的生长和增殖．并能诱导部分白血病细胞分化成熟。但其药理作用因硒制剂的不同而异．硒代二半胱氨酸抑制V937和K562血病细胞的生长和增殖，半数抑制浓度为3．0μmol／L，经30μmol／L硒代二半胱氨酸作用3天后，U937胞吞噬率以5％上升至14％，K562细胞内血红蛋白含量由0．20增至0．40μg／10^6细胞，说明硒代二半胱氨酸能诱导部分白血病细胞分化成熟。     

High Osmolarity and L‐DOPA Augment Release via the Fusion Pore in PC12 Cells

We have amperometrically measured dopamine release from rat pheochromocytoma cells (PC12 cells) in high osmolarity conditions with and without L ‐3,4‐dihydroxyphenylalanine (L ‐DOPA) treatment. We observe an increase in the number of release events displaying a prespike feature or “foot” when the cells are stimulated in high osmolarity saline. We also see an increase in foot area and duration when cells are stimulated in high osmolarity saline, or high osmolarity saline subsequent to incubation with the dopamine precursor L ‐DOPA in isotonic saline, which serves to increase the vesicle size. The data suggest that membrane biophysics are an important component in defining the rate, duration and amount of neurotransmitter release via the fusion pore.     

Sesquiterpenoids of Illicium jiadifengpi and Their Effects on NGF‐induced Neurite Outgrowth in PC12 Cells

Two new compounds, jiadifenlactone acid monomethyl ester ( 1 ) and jiadifenin ( 3 ), and five known compounds were isolated from the fruits of I. jiadifengpi . Their structures were determined using spectroscopic techniques, including 1D, 2D NMR and HR‐ESI‐MS experiments. The relative stereochemistry of jiadifenlactone acid monometheyl ester ( 1 ) was further confirmed by X‐ray crystallographic data. All isolates were evaluated for their effects on nerve growth factor (NGF)‐mediated neurite outgrowth in pheochromocytoma (PC12) cells and two compounds showed promoting effects.     

A Microchip‐Based System for Immobilizing PC 12 Cells and Amperometrically Detecting Catecholamines Released After Stimulation with Calcium

In this paper, we describe a microchip‐based system for amperometrically monitoring the amount of catecholamines released from rat pheochromocytoma (PC 12) cells. Key to this system is a novel, yet simple method for the immobilization of PC 12 cells in poly(dimethylsiloxane) (PDMS)‐based microchannels. The procedure involves selectively coating microchannels with collagen followed by introduction of PC 12 cells over the PDMS structure, with the cells being immobilized only on the coated portion of the channels. The cell‐coated microchannels can then be reversibly sealed to a glass plate containing electrodes for amperometric detection, resulting in an immobilized cell reactor with integrated microelectrodes. Nafion‐coated microelectrodes made by micromolding of carbon inks were used to measure calcium‐induced catecholamine release from the cells. Varying concentrations of PC 12 cells immobilized in the microchannels led to a catecholamine release ranging from 20 to 160 μM when the cells were stimulated with a calcium solution. This microchip approach leads to a three‐dimensional culture that can be used with this or other cells lines to study the effect of external stimuli on neurotransmitter release.     

Neuroprotective Potency of Tofu Bio-Processed Using Actinomucor elegans against Hypoxic Injury Induced by Cobalt Chloride in PC12 Cells

Fermented soybean products have attracted great attention due to their health benefits. In the present study, the hypoxia-injured PC12 cells induced by cobalt chloride (CoCl₂) were used to evaluate the neuroprotective potency of tofu fermented by Actinomucor elegans (FT). Results indicated that FT exhibited higher phenolic content and antioxidant activity than tofu. Moreover, most soybean isoflavone glycosides were hydrolyzed into their corresponding aglycones during fermentation. FT demonstrated a significant protective effect on PC12 cells against hypoxic injury by maintaining cell viability, reducing lactic dehydrogenase leakage, and inhibiting oxidative stress. The cell apoptosis was significantly attenuated by the FT through down-regulation of caspase-3, caspases-8, caspase-9, and Bax, and up-regulation of Bcl-2 and Bcl-xL. S-phase cell arrest was significantly inhibited by the FT through increasing cyclin A and decreasing the p21 protein level. Furthermore, treatment with the FT activated autophagy, indicating that autophagy possibly acted as a survival mechanism against CoCl₂-induced injury. Overall, FT offered a potential protective effect on nerve cells in vitro against hypoxic damage.     

Determination of neurotransmitters in PC 12 cells by microchip electrophoresis with fluorescence detection

A sensitive fluorescence detection system with an Hg-lamp as the excitation source and a photon counter as the detector for microchip CE (MCE) has been developed. O-Phthaldialdehyde (OPA, lambda(ex) = 340 nm) was employed to label the catecholamine neurotransmitters such as dopamine (DA), norepinephrine (NE), and amino acid neurotransmitters including alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp). The separation of seven derivatized neurotransmitters was successfully performed in MCE and the detection limits (S/N = 3) for DA, NE, Ala, Tau, Gly, Glu, and Asp were 0.85, 0.49, 0.23, 0.15, 0.13, 0.18, and 0.29 fmol, respectively. The system was then successfully applied for separation and determination of neurotransmitters in rat pheochromocytoma (PC 12) cells, and the average amounts of analyte per cell from a cell population were 2.5 fmol for DA, 3.3 fmol for Ala, 8.2 fmol for Tau, 4.0 fmol for Gly, and 1.9 fmol for Glu, respectively. By single-cell injection mode, electrophoresis separation and quantitative measurement of Glu in individual PC 12 cells was obtained. The average value of Glu per cell from single PC 12 cells analysis was found to be 3.5 +/- 3.1 fmol.     

Neuroprotective Effects of a New Derivative of Chlojaponilactone B against Oxidative Damaged Induced by Hydrogen Peroxide in PC12 Cells

A new sesquiterpenoid (1) was obtained by hydrogenating Chlojaponilactone B. The structure of 1 was elucidated according to a combination of NMR, HRESIMS, and NOE diffraction data. The treatment of H₂O₂ in a PC12 cell model was used to evaluate the antioxidant activity of 1. An MMT assay showed that 1 had no cytotoxicity to the PC12 cell and rescued cell viability from the oxidative damage caused by H₂O₂. The treatment of 1 stabilized the mitochondria membrane potential (MMP), which decreased the intracellular ROS level and reduced cell apoptosis in the oxidative stress model. The activities of antioxidant enzyme superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of intracellular glutathione (GSH) were significantly enhanced after the treatment of 1. In addition, the results of qRT-PCR showed that 1 treatment minimized the cell injury by H₂O₂ via the up-regulation of the expression of nuclear factor erythroid 2 (Nrf2) and its downstream enzymes Heme oxygenase 1 (HO-1), glutamate cysteine ligase-modifier subunit (GCLm), and NAD(P)H quinone dehydrogenase 1 (Nqo1). Based on the antioxidant activity of 1, we speculated its potential as a therapeutic agent for some diseases induced by oxidative damage.