单胺递质对皮质酮所致的PC12细胞损伤的保护作用

目的　观察单胺递质在抗抑郁剂产生的效应中所起的作用。方法　单胺递质 (5 HT、DA、NE)、去甲丙咪嗪(DIM)及MK80 1与皮质酮 0 2mmol·L-1共孵PC12细胞48h ,并用MTT比色法判断细胞损伤程度。采用Fura 2 /AM荧光标记法测定胞内Ca2 + 浓度。结果　高浓度皮质酮处理PC12细胞后 ,细胞存活率比正常组降低 ,胞内 [Ca2 + ]i升高 ,表明细胞受到损伤或部分死亡。当给予 5 HT ,DA ,NE ,DIM及MK80 1后 ,细胞存活率增高 ,而胞内 [Ca2 + ]i降低 ,细胞损伤减少或已接近正常。结论　与抗抑郁剂一样 ,单胺递质对皮质酮损伤的PC12细胞有明显的保护作用 ,并且能够明显降低皮质酮诱发的PC12细胞胞内Ca2 + 超载。提示单胺递质可能参与或介导了抗抑郁剂的神经元保护作用 ,这可能是抗抑郁剂的作用机制之一     

抗抑郁剂作用机制的研究及其在新药研发中的应用

目的：探讨结构迥异的各类抗抑郁剂可能的共同作用方式与机理，为新药研发提供新策略。方法：MTT比色法及乳酸脱氢酶法检测细胞活性；以FLira-2／AM荧光标记法检测胞内Ca^2 浓度；比色法检测一氧化氮合酶(NOS)活性；流式细胞仪法检测细胞分裂周期；免疫组化法检测小鼠海马神经元再生及脑源性神经营养因子(BDNF)水平；利用基因芯片技术检测药物相关基因。结果：三类经典抗抑郁剂[包括三环类的去甲丙咪嗪(DIM)、5-HT重摄取抑制剂氟西汀(FLU)、单胺氧化酶抑制剂马氯贝胺(MOC)]对N-甲基-D-天冬氨酸(NMDA)300μM处理24h所致．PC12细胞损伤有显抑制作用，并且减弱NMDA诱导的PC12细胞内Ca^2 超载及NOS活性。继药物对抗细胞损伤作用的研究之后，接下来探讨了药物是否可促进细胞分裂与神经元再生。研究表明，NMDA600μM处理3天可显降低：PC12细胞S期百分率，而DIM1，5μM，FLU1，5μM或MOC2，10μM可显逆转这一现象，使S期细胞百分率显提高。进一步研究发现，慢性应激24天小鼠海马齿状回神经元再生低下甚至消失，而DIM 10mg／kg，FLU10mg／k或MOC 40mg／kg(ip)显提高海马神经元再生。同时，抗抑郁剂显提高海马脑源性神经营养因子(BDNF)的水平。本研究室在研新药巴戟天六聚寡糖、槲皮素三糖甙、胍丁胺表现出与上述经典药物一致的作用。利用小鼠2048点表达谱芯片发现，DIM5μM可以诱导皮质酮处理的PC12细胞内259个基因表达改变，其中163个基因表达降低(如葡萄糖激酶等)；96个表达升高(如生长分化因子-9)。以细胞原位杂交验证，结果与基因芯片俭测一致。结论：各类结构不同的经典抗抑郁剂均可对抗NMDA诱导的细胞损伤并海马神经元再生，这可能与其抑制NMDA受体-Ca^2 -NOS通路功能密切相关，其结果导致与神经元营养与再生、生长与分化密切相关基因的表达改变(如BDNF、生长分化因子等)。本研究室在研新药巴戟天六聚寡糖、槲皮素三糖甙、胍丁胺均有显细胞保护和促神经元再生作用，其抗抑郁活性也在多模型中得到证实。     

地昔帕明对皮质酮诱导培养的PC12细胞调亡的拮抗作用(英文)

目的:探讨三环类抗抑郁剂的作用机制.方法:运用DNA电泳法、流式细胞仪法、电镜法检测了皮质酮诱导的PC12细胞凋亡并观察了去甲丙米嗪(DIM)的效应 结果:皮质酮10μmol/L处理5d可显著诱导PC12细胞凋亡,凋亡发生率最高达(28±9)％,DNA电泳图谱则表现出典型的梯状条带,DIM的1和5μmol/L使凋亡发生率明显降低并使梯状条带变浅、减轻,细胞的超微结构也有明显改善.结论:DIM对皮质酮诱导的PC12细胞凋亡有拮抗作用,这可能是其抗抑郁效应的细胞机制之一.     

木豆素A对皮质酮诱导的PC12细胞损伤的保护作用

利用皮质酮诱导PC12细胞损伤模型,研究木豆素A对皮质酮诱导的PC12细胞的保护作用并探讨相应的保护途径。采用100μmol.L1皮质酮与PC12细胞作用48 h,诱导PC12细胞损伤,然后与不同浓度的木豆素A孵育24 h。检测细胞存活率、LDH渗漏量、细胞内Ca2+浓度及caspase-3活性。结果显示,PC12细胞与皮质酮孵育48 h后细胞存活率明显降低,而LDH漏出量、细胞内Ca2+浓度及caspase-3活性均显著升高;木豆素A(4.0、8.0及16.0μmol.L1)具有改善作用,但量效关系不明显。研究表明,木豆素A对皮质酮诱导的PC12细胞损伤具有明显的保护作用,其保护作用可能是通过降低Ca2+浓度及caspase-3活性来实现的。     

梓醇保护L-谷氨酸和Aβ25-35损伤PC12细胞作用比较作用比较

目的观察梓醇对L-Glu和Aβ25-35损伤PC12细胞的保护作用的异同。方法常规培养神经元样PCI2细胞，预先加入梓醇及对照生理盐水24h后分别加L—Glu 5mmol／L和Aβ25-35 20μmol／L损伤24h，K252a干预组细胞在梓醇前1h加入200nmol／LK252a。MTT法测定细胞存活率，放射配基结合分析测定M2受体密度。结果梓醇100μmol／L明显提高细胞存活率（P〈0．01），10μmol／L、100μmol／L升高M2受体密度（P〈0．05，P〈0．01），K252a部分阻断梓醇对Aβ25-35损伤细胞的保护作用，对梓醇保护L—Glu损伤的阻断作用更强。结论梓醇对L—Glu和Aβ25-35损伤PC12细胞保护作用有差异。     

梓醇保护L-谷氨酸和Aβ25-35损伤PC12细胞作用比较作用比较

目的观察梓醇对L-Glu和Aβ25-35损伤PC12细胞的保护作用的异同。方法常规培养神经元样PCI2细胞，预先加入梓醇及对照生理盐水24h后分别加L—Glu 5mmol／L和Aβ25-35 20μmol／L损伤24h，K252a干预组细胞在梓醇前1h加入200nmol／LK252a。MTT法测定细胞存活率，放射配基结合分析测定M2受体密度。结果梓醇100μmol／L明显提高细胞存活率（P〈0．01），10μmol／L、100μmol／L升高M2受体密度（P〈0．05，P〈0．01），K252a部分阻断梓醇对Aβ25-35损伤细胞的保护作用，对梓醇保护L—Glu损伤的阻断作用更强。结论梓醇对L—Glu和Aβ25-35损伤PC12细胞保护作用有差异。     

眼睛蛇毒心脏毒素对大鼠心肌细胞收缩及细胞内钙离子的作用

目的 研究眼睛蛇毒心脏毒素（Cardiotoxin，CTX）对心肌细胞的形态、收缩幅度和细胞内钙离子（[Ca^2＋]i）的作用。方法 应用荧光计量法（以Fura-2／AM为荧光染料）及光学成像系统来测定单个心肌细胞[Ca^2＋]i和收缩幅度。结果 0．001～1μmol／L的CTX使心肌细胞由杆状变成圆形，药物的作用从第1分钟时开始，到第20分钟时趋于稳定。在电刺激存在的情况下，1μmol／L的CTX最初导致电诱导的[Ca^2＋]i和收缩幅度瞬间增加，接下来[Ca^2＋]i时程延长，最终细胞对电刺激不敏感、突然收缩、[Ca^2＋]i持续增高。在缺乏电刺激的情况下，1μmol／L的CTX可诱导Ca^2＋震荡波、持续性[Ca^2＋]i增高，这种作用与40mmol／L的KCl和10mmol／L咖啡因所引起的[Ca^2＋]i瞬间增加不同。结论 CTX作用初期使[Ca^2＋]i增高，使细胞[Ca^2＋]。超载，同时伴随细胞形状的改变。     

淫羊藿苷抗抑郁及对皮质酮致PC12细胞损伤的保护作用研究

目的：研究淫羊藿苷的抗抑郁及对皮质酮致PC12细胞损伤的保护作用。方法：采用大／小鼠强迫游泳、小鼠悬尾三种实验模型,将动物随机分为对照组、淫羊藿苷低剂量组、淫羊藿苷高剂量组、阿米替林组,观察药物对大／小鼠强迫游泳不动时间、小鼠悬尾不动时间的影响;并在细胞水平建立皮质酮损伤PC12细胞模型,观察淫羊藿苷的细胞保护作用。结果：在大／小鼠强迫游泳和小鼠悬尾实验中,淫羊藿苷可显著缩短大／小鼠的强迫游泳不动时间和小鼠悬尾不动时间,与对照组相比差异有统计学意义（P〈0．05）;在皮质酮损伤PC12细胞的模型上,淫羊藿苷可显著提高PC12细胞的存活率,拮抗皮质酮诱导的细胞损伤作用。结论：淫羊藿苷具有明显的抗抑郁效果,其抗抑郁作用与神经细胞保护作用有关。     

血管紧张素Ⅱ对大鼠肾入球小动脉细胞内钙离子浓度的影响及Losartan的拮抗作用

目的探讨血管紧张素Ⅱ（AngⅡ）对肾小球入球小动脉细胞内钙离子浓度（[Ca^2＋]i）的影响与高血压肾小动脉重建的关系及Losartan的治疗作用。方法细胞培养：大鼠肾小球入球小动脉细胞随机分为4组。对照组：不加AngⅡ处理;AngⅡ组：加入终浓度为0.1μmol.L^-1AngⅡ;Losartan组：加入终浓度为50μmol.L^-1Losartan;AngⅡ＋Losartan组：同时加入Losartan 50μmol.L^-1AngⅡ0.1μmol.L^-1负载后上机检测[Ca^2＋]i。结果细胞培养显示AngⅡ引起了细胞收缩变化,表现为胞突变细变短,胞突回缩,胞体变圆,细胞长度与直径均明显缩小。Losartan处理组较AngⅡ组细胞形态变化减轻且Losartan对AngⅡ增高RASMCs内[Ca^2＋]i有明显抑制作用。结论 AngⅡ可引起肾小球入球小动脉细胞内[Ca^2＋]i的上升,导致细胞收缩,因此Ca^2＋超载可能是肾素-血管紧张素系统起作用的重要环节。Losartan可抑制细胞内Ca^2＋超载。     

微矩阵基因芯片筛选抗抑郁剂地昔帕明相关基因的研究

目的　研究抗抑郁剂相关基因 ,探讨其可能作用机制。方法与结果　以高浓度皮质酮 (Cort) 1 0μmol·L-1或Cort与三环类经典抗抑郁剂地昔帕明(DIM) 5 μmol·L-1共同处理PC1 2细胞 3d分别作为对照组和实验组 ,按Trizol一步法提取细胞总RNA并纯化mRNA ,将等量的对照组与实验组mRNA以逆转录法分别标记荧光素cy3和cy5 ,合成cDNA探针并等量混合 ,与小鼠 2 0 48点微矩阵基因芯片杂交 ,扫描荧光信号图像并分析比较二组差异表达基因 ,发现DIM与Cort共孵PC1 2细胞 3d可以诱导2 5 9个基因表达水平发生改变。其中 1 63个基因表达降低 ,如葡萄糖调节蛋白 78ku、葡萄糖激酶(GA)、细胞骨架蛋白、转化性生长因子 β受体结合蛋白、细胞色素C氧化酶、锂盐敏感性内消旋肌醇一磷酸酯酶等 ;有 96个基因表达水平升高 ,如神经元生长分化因子 9(GDF 9)、锌指蛋白 2 1 6、细胞色素P45 0、睾丸特异基因 1等。随机选取二个差异表达基因GA和GDF 9,以细胞原位杂交方法验证 ,取得与基因芯片评价一致的结果。结论　抗抑郁剂DIM作用可能与能量代谢、细胞骨架、营养因子、酶等多层次、多水平的基因表达改变有关。本研究利用基因芯片技术对抗抑郁剂相关基因进行了初步探索 ,为深入研究抗抑郁剂作用机制和药物筛选芯片的研发提供了线索与依     

(-)-Epigallocatechin gallate attenuates glutamate-induced cytotoxicity via intracellular Ca modulation in PC12 cells

1. The effects of (-)-epigallocatechin gallate (EGCG), a green tea polyphenol, on glutamate-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity in PC12 cells were investigated. 2. Changes in [Ca2+]i were measured using Fura-2/AM calcium indicator dye and cellular viabilities were determined by a viable cell count and a 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. 3. Glutamate increased [Ca2+]i in PC12 cells in a dose-dependent manner. (-)-Epigallocatechin gallate attenuated this glutamate (30 mmol/L)-induced [Ca2+]i increase and EGCG (50 micromol/L) increased the viability of PC12 cells against glutamate-induced cytotoxicity. The EGCG effect was also found to be independent of its general anti-oxidant mechanism. In contrast, EGCG directly suppressed both N-methyl-D-aspartate (50 mmol/L)- and kainate (20 mmol/L)-mediated Ca2+ influx, but not metabotropic receptor-mediated Ca2+ release. 4. These results suggest that EGCG reduces the glutamate-induced [Ca2+]i increase by attenuating ionotropic Ca2+ influx and that this promotes the viability of PC12 cells.     

抗抑郁剂对慢性应激小鼠海马神经元再生的影响

目的　探讨抗抑郁剂作用机制。方法　以流式细胞仪法测定细胞DNA合成期 (S期 )百分率 ;用脑冷冻切片的免疫组化实验检测海马齿状回神经元先祖细胞分裂及脑源性神经营养因子 (BDNF)水平。结果　以N 甲基 D 天冬氨酸(NMDA) 6 0 0 μmol·L-1 处理PC1 2细胞 3d后 ,细胞S期百分率明显降低 ,提示高浓度NMDA可抑制细胞分裂。如果同时给予经典抗抑郁剂去甲丙米嗪 (DIM)或氟西丁 (FLU) 1 ,5 μmol·L-1 则明显提高细胞S期百分率。慢性应激 2 4d小鼠海马齿状回颗粒细胞层下区的先祖细胞分裂减2 0 0 3 10 10收稿 ,2 0 0 3 12 17修回 国家自然科学基金资助课题 ,No 3 0 3 0 0 419;北京市自然科学基金资助课题 ,No 70 42 0 5 2作者简介 :李云峰 ,男 ,3 1岁 ,博士 ,副研究员。研究方向 :精神药理学。Tel:0 10 6687460 6 5 ,Fax:0 10 682 1165 6,E mail:lyf619@yahoo .com .cn ;罗质璞 ,男 ,66岁 ,研究员。研究方向 :精神药理学。Tel:0 10 6693 1619 1,Fax :0 10 682 1165 6少 ,同时BDNF水平低下 ,均表现为阳性棕色颗粒缺失 ,若同时给予DIM或FLU 1 0mg·kg-1 (ip)则逆转上述现象 ,明显增加先祖细胞的分裂和BDNF水平 ,二者在时程上一致。结论　促进海马齿状回神经元再生可能是抗抑郁剂共同作用机制之一 ,并且可能与     

地昔帕明对皮质酮诱导培养的PC12细胞凋亡的拮抗作用

AIM: To study possible action mechanism of a tricyclic antidepressant, desipramine (DIM). METHODS: Cultured PC12 cells were exposed to corticosterone in the absence or presence of DIM for 5 d. Agarose gel electrophoresis, flow cytometry, and electron microscopy were used to detect the apoptosis of PC12 cells. RESULTS: Corticosterone 10 micromol/L treatment for 5 d elicited typical apoptotic biochemical and morphological changes including condensed chromatin shaped like crescent moon, nuclear fragmentation, and DNA degradation. The highest percentage of apoptotic cells accumulated to 28 % +/- 9 %. Agarose gel electrophoresis showed typical DNA ladders pattern. While in the presence of DIM 1 or 5 micromol/L, apoptosis percentage was markedly decreased with lightened DNA ladder and ultrastructure of the cells was improved. CONCLUSION: DIM could antagonize the apoptosis in PC12 cells induced by corticosterone, which may be one of the cellular mechanisms of its antidepressant effect.     

粉防己碱对培养乳牛基底动脉平滑肌细胞内游离钙浓度的影响

目的：研究粉防己碱对培养乳牛基底动脉平滑肌细胞游离钙浓度（[Ca^2 ]i)的影响。方法：利用AR－CM－MIC阳离子测定系统,采用Fura 2-AM为指示剂,测量单个细胞内[Ca^2 ]i。结果：粉防己碱10－100μmol/L对培养乳牛基底动脉平滑肌细胞静息[Ca^2 ]i无明显影响。在细胞外钙为1．3mmol/L,粉防己碱可浓度依赖性地抑制KC1引起[Ca^2 ]i的升高。咖啡因10mmol/L可诱导一次[Ca^2 ]i瞬间快速升高,随后自发回复到静息水平,粉防己碱10和30μmol/L对咖啡因诱导的[Ca^2 ]i瞬间升高没有作用,但高浓度（100μmol/L）粉防己碱抑制了[Ca^2 ]i瞬间升高。在细胞外钙为1.3mmol/L,苯肾上腺素10μmol/L可引起双相[Ca^2 ]i变化,包括快速升高相和持续升高相。在细胞外钙为零,苯肾上腺素仅引起[Ca^2 ]i的快速升高相。粉防己碱可浓度依赖性地抑制苯肾上腺素引起[Ca^2 ]i快速升高相。结论：在培养乳牛基底动脉平滑肌细胞,粉防己碱可能通过影响电压依赖性和苯肾上腺素受体介导的钙通道而抑制钙内流。高浓度粉防己碱也可能影响肌浆网钙释放或钙摄取。     

Effects of econazole on Ca2+ levels in and the growth of human prostate cancer PC3 cells

1. Econazole is used clinically as an antifungal drug with many different in vitro effects. However, the effects of econazole on prostate cancer cells are unknown. The effects of econazole on intracellular Ca2+ concentrations ([Ca2+]i) in and the proliferation of human PC3 prostate cancer cells was explored in the present study using fura-2 and tetrazolium as fluorescent dyes. 2. At a concentration of 0.1 micromol/L, econazole started to increase [Ca2+]i in a concentration-dependent manner. The econazole-induced increase in [Ca2+]i was reduced by 48% by removal of extracellular Ca2+, suggesting that the econazole-induced increase in [Ca2+]i was composed of extracellular Ca2+ influx and intracellular Ca2+. 3. This econazole-induced Ca2+ influx was via an L-type Ca2+ channel-like pathway. In Ca2+-free medium, 1 micromol/L thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic increase in [Ca2+]i, after which the effect of econazole to increase [Ca2+]i was substantially inhibited. Conversely, pretreatment with 5 micromol/L econazole to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. 4. The phospholipase C (PLC) inhibitor U73122 (2 micromol/L) abolished the increase in [Ca2+]i induced by 10 micromol/L ATP (a Ca2+ mobilizer that needs inositol 1,4,5-trisphosphate). 5. Overnight incubation with 1-30 micromol/L econazole inhibited proliferation of PC3 cells in a concentration-dependent manner. 6. These findings suggest that, in PC3 cells, econazole increases [Ca2+]i by stimulating Ca2+ influx into cells and Ca2+ release from the endoplasmic reticulum via a PLC-independent mechanism. Econazole is cytotoxic at submicromolar concentrations.     

Inhibition of N-methyl-D-aspartate receptor function appears to be one of the common actions for antidepressants

In order to explore the possible common action mechanisms of three kinds of classical antidepressants, inhibition of drugs on the N-methyl-D-aspartate (NMDA)-Ca(2)-nitric oxide synthase (NOS) signal pathway was observed. With 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and lactic dehydrogenase (LDH) assay, classical antidepressants, desipramine (1, 10 microM), fluoxetine (0.625-10 microM) or moclobemide (2.5, 10 microM) antagonized NMDA 300 M induced-lesion in PC12 cells. Using fura-2/AM (acetoxymethyl ester) labelling assay, desipramine or fluoxetine at doses 1, 5 microM attenuated the intracellular Ca(2) overload induced by NMDA 200 microM for 24 h in PC12 cells. Meanwhile, using confocal microscope, it was also found that desipramine 5 microM, fluoxetine 2.5 microM or moclobemide 10 microM decreased the NMDA 20 microM induced intracellular Ca(2) overload in primarily cultured rat hippocampal neurons. Furthermore, desipramine (1, 5 microM), fluoxetine (1, 5 microM) or moclobemide (2.5, 10 microM) significantly inhibited NOS activity in NMDA (300 microM) treated PC12 cells for 4h. In summary, we suggest that inhibition on the function of NMDA-Ca(2) -NOS signal pathway appears to be one of the common actions for antidepressants despite their remarkably different structures, which is expected to have great implication for the evaluation and screening in vitro of new antidepressants.     

Tonic potentiation and attenuation produced by membrane depolarization in guinea-pig trachealis

1. We studied how membrane depolarization directly affected intracellular Ca2+ signalling when voltage-operated Ca2+ channels (VOCC) were not available in guinea-pig tracheal smooth muscle. To block VOCC, we used 3 micromol/L verapamil, which completely abolished high K+ (20-60 mmol/L)-induced contraction, and elevation of fura-2 signal. 2. Muscle tone was generated by adding Ca2+ to the extracellular Ca2+-free solution containing prostaglandin (PG)E2 (100 nmol/L) after abolishing basal tone with indomethacin (1 micromol/L). 3. In the absence of verapamil, high K+ (20-60 mmol/L) solution potentiated 2.4 mmol/l Ca2+-induced sustained contractions. Even in the presence of 3 micromol/L verapamil, replacement with 20 and 40 mmol/L K+ solution induced tonic potentiation, which was changed to attenuation with a higher K+ solution (60 mmol/L), lower extracellular Ca2+ concentration ([Ca2+]o) and pretreatment with cyclopiazonic acid (10 micromol/L), a Ca2+ sequestration inhibitor. 4. These results indicate that the balance between depolarization-dependent Ca2+ release and receptor-operated cation channel inhibition may determine whether tonic potentiation or attenuation is manifested, depending on the availability of VOCC, the magnitude of the depolarization, [Ca2+]o and Ca2+ content in the sarcoplasmic reticulum.     

Effect of the antidepressant maprotiline on Ca2+ movement and proliferation in human prostate cancer cells

1. The effect of maprotiline, an antidepressant, on human prostate cells is unclear. In the present study, the effect of maprotiline on [Ca2+]i and growth in PC3 human prostate cancer cells was measured using the fluorescent dyes fura-2 and tetrazolium, respectively. 2. Maprotiline caused a rapid, concentration-dependent increase in [Ca2+]i (EC50 = 200 micromol/L). The maprotiline-induced [Ca2+]i increase was reduced by removal of extracellular Ca2+ or pretreatment with nicardipine. 3. The maprotiline-induced Mn2+ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca2+ influx. 4. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i increase, after which the effects of maprotiline of increasing [Ca2+]i were abolished. In addition, pretreatment with maprotiline reduced a major portion of the thapsigargin-induced increase in [Ca2+]i. 5. U73122, an inhibitor of phospholipase C, abolished the ATP (but not maprotiline)-induced increase in [Ca2+]i. 6. Overnight incubation with 1-10 micromol/L maprotiline did not alter cell proliferation, although incubation with 30-50 micromol/L maprotiline decreased cell proliferation. 7, These findings suggest that maprotiline rapidly increases [Ca2+]i in human prostate cancer cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release and that it may modulate cell proliferation in a concentration-dependent manner.