玻璃离子水门汀氟离子的释放和再吸收及重量减少

观察缺氧及再给氧对体外培养星形胶质细胞存活能力及谷氨酸摄取功能的影响。方法：取生后１～２ｄ新生昆明小鼠大脑皮层进行星形胶质细胞原代培养,培养 １周左右予以缺氧。缺氧时间分别为 １２、２４、４８ ｈ,并取缺氧２４ ｈ后再给氧０、１２、２４、４８ ｈ的细胞观察其形态、死亡细胞数目、乳酸脱氢酶活性及谷氨酸摄取能力的变化。结果：缺氧组及再给氧组星形胶质细胞死亡数较对照组无明显变化,缺氧前、后乳酸脱氢酶活性亦无明显改变,缺氧组谷氨酸摄取功能较对照组下降３０％～６０％,并随缺氧时间延长而明显下降（与对照组相比,Ｐ＜０．０１）,再给氧２４ ｈ内谷氨酸摄取能力有所恢复,但未达正常水平。结论：星形胶质细胞与神经元相比,对缺氧培养液较为耐受,缺氧对星形胶质细胞存活能力无明显影响,但细胞摄取谷氨酸能力有所改变。     

体外培养星形胶质细胞缺氧／再给氧后谷氨酸摄取功能的变化

观察缺氧及再给氧体外培养星形胶质细胞存活能力及谷氨酸摄取功能的影响。方法：取生后１－２ｄ新生昆明小鼠大脑皮层进行星形胶质细胞原代培养,培养１周左右予以缺氧。缺氧时间分别为１２,２４,４８ｈ,。并取缺氧２４ｈ后再给氧０,１２,２４,４８ｈ的细胞观察其形态,死亡细胞数目,乳酸脱氢酶活性及谷氨酸摄取能力的变化。     

体外培养星形胶质细胞缺氧/再给氧后谷氨酸摄取功能的变化

目的:观察缺氧及再给氧对体外培养星形胶质细胞存活能力及谷氨酸摄取功能的影响.方法:取生后1～2 d新生昆明小鼠大脑皮层进行星形胶质细胞原代培养,培养1周左右予以缺氧.缺氧时间分别为12、24、48 h,并取缺氧24 h后再给氧0、12、24、48 h的细胞观察其形态、死亡细胞数目、乳酸脱氢酶活性及谷氨酸摄取能力的变化.结果:缺氧组及再给氧组星形胶质细胞死亡数较对照组无明显变化,缺氧前、后乳酸脱氢酶活性亦无明显改变,缺氧组谷氨酸摄取功能较对照组下降30%～60%,并随缺氧时间延长而明显下降(与对照组相比,P＜0.01),再给氧24 h内谷氨酸摄取能力有所恢复,但未达正常水平.结论:星形胶质细胞与神经元相比,对缺氧培养液较为耐受,缺氧对星形胶质细胞存活能力无明显影响,但细胞摄取谷氨酸能力有所改变.     

吗啡对大鼠海马星形胶质细胞[Ca2+]i的影响

目的 研究吗啡对大鼠海马星形胶持细胞[Ca^2 ]i的影响，探索吗啡对星形胶质细胞的作用效应。方法 运用荧光探针Fluo-4，利用激光共聚焦显微镜研究培养纯化大鼠海马星形胶质细胞[Ca^2 ]i的变化。结果 经0.5,1,2,10μmol/L浓的吗啡短期处理3d后，细胞内[Ca^2 ]i浓度与正常细胞比较差异无显著性（P＞0．05）。吗啡急性作用于正常星形胶质细胞后，[Ca^2 ]i呈单峰样升高；而作用于吗啡处理后的细胞，作用浓度需高于处理浓度出现[Ca^2 ]i呈单峰样升高的现象，纳络酮可阻断上述效应，NMDA受体阻断剂MK－801则不能。结论 星形胶质细胞[Ca^2 ]i对吗啡的反应性变化可能参与吗啡耐受的形成。     

吗啡活化离体培养星形胶质细胞效应研究

目的 研究吗啡活化离体培养星形胶质细胞的效应.方法 用吗啡、纳络酮处理离体培养纯化的小鼠星形胶质细胞,在第1、2、4、7天采用CCK-8检测细胞增殖,激光共聚焦扫描显微镜(CLSM)检测细胞内钙浓度[(Ca2+)i],采用ELISA检测细胞分泌TNF-α,用Griess法测定NO浓度,以期观察吗啡对星形胶质细胞的效应.结果 2μmol/mL吗啡刺激星形胶质细胞,第1天和第2天短期作用效应不明显;延长作用时间,于4、7d细胞增殖下降,(Ca2+)i升高,NO和TNF-α分泌显著升高,阿片受体阻断剂纳络酮可以翻转吗啡作用效应.结论 吗啡长期作用经阿片受体活化星形胶质细胞,表现为(Ca2+)i升高,TNF-α和NO分泌上调.     

P物质对培养脊髓星形胶质细胞谷氨酸代谢的影响

目的 观察P物质（SP）对脊髓星形胶质细胞兴奋性递质谷氨酸代谢的影响。方法 采用体外培养脊髓星形胶质细胞,施加SP刺激．分别采用^3H—Glutamate掺入和RT—PCR观察脊髓星形胶质细胞对谷氨酸摄取和转运体GLT-1表达的变化。结果 脊髓星形胶质细胞在SP的刺激下^,3H—Glu的摄取逐渐下降,10^-9mol／L与正常对照组差异有统计学意义（P〈0．05）．10^-8～10^-6mol／L组与正常对照组差异有统计学意义（P〈0．01）;脊髓星形胶质细胞GLT-1的表达在10^9-10^-8mol／L SP作用组和正常组差异无统计学意义（P〉0．05）,10^-2～10^-6 mol／L SP刺激组GLT-1表达值低于正常对照（P〈0．05、P〈0．01）。结论 高浓度的SP作用下脊髓星形胶质细胞GLT-1表达和谷氨酸掺入下降,表明周围慢性疼痛可能通过致病递质SP使脊髓星形胶质细胞对谷氨酸的摄入减少,神经间隙谷氨酸堆积而导致脊髓中枢继发的神经损害。     

吗啡对C6胶质瘤细胞嘌吟核苷酸代谢相关酶基因表达的影响

目的：研究吗啡对C6胶质瘤细胞嘌呤核苷酸合成代谢与分解代谢的影响。方法：吗啡（10μg/ml培养基）作用于C6胶质瘤细胞6h、12h、24h、48h、72h;纳络酮（1μmol/L）作用于C6胶质瘤细胞1h后,加吗啡（10μg/ml）作用24h。提取细胞总RNA,采用反转录-聚合酶链反应（RT-PCR）方法检测次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT）及腺苷激酶（AK）的基因转录产物;采用反转录-聚合酶链反应及Southern(RT-PCR-Southern)杂交方法检测黄嘌呤脱氧酶（XD）/黄嘌呤氧化酶（XO）基因转录产物。结果：C6胶质瘤细胞暴露于吗啡μ,12,24,48,HGPRT基因表达均明显降低;而C6胶质细胞AK基因表达在暴露于吗啡24h明显降低;HGPRT与AK基因表达又分别于吗啡作用胶质瘤细胞72h和48h回升;纳络酮不能阻断吗啡对HGPRT与AK基因表达降低的作用。吗啡作用于C6胶质瘤细胞与相应时段对照组相比,XD/XO基因转录产物均明显增多;纳络酮能够阻断吗啡对XD/XO基因表达增强的作用。结论：吗啡通过其他非μ受体途径介导对C6胶质瘤细胞嘌呤核苷酸补救合成关键酶HGPRT与AK基因表达降低的作用;吗啡通过μ受体途径介导对C6胶质瘤细胞嘌呤核苷酸分解代谢关键酶XD/XO基因表达增强的作用。     

醒脑静注射液组分促进缺氧复氧胶质细胞清除胞外谷氨酸提高缺氧神经元活力的研究

[目的]研究醒脑静注射液（XNJI）8种成分促进缺氧复氧（Hypo/Reox）星形胶质细胞对胞外高浓度谷氨酸的清除，及其条件培养液对缺氧后神经元活力的影响。[方法]体外培养大鼠星形胶质细胞，胶质纤维酸性蛋白（GFAP）免疫荧光鉴定。采用环磷酸腺苷（dBcAMP）和Hypo/Reox诱导胶质细胞活化并进行造模，Cell Counting Kit-8（CCK-8）测定细胞活力；谷氨酸试剂盒检测细胞培养液中谷氨酸浓度，并利用此条件培养液培养缺氧神经元，CCK-8法测神经元活力。[结果]胶质细胞体外培养成功，GFAP染色阳性。dBcAMP 0.062 5 mmol/L协同缺氧4 h复氧3 h（Hypo 4 h/Reox 3 h）处理后胶质细胞对胞外高浓度谷氨酸的清除能力显著降低。XNJI组分β-榄香烯、吉马酮、龙脑、冰片能显著提高受损胶质细胞对胞外谷氨酸的清除能力；XNJI组分吉马酮、龙脑、冰片处理的胶质细胞条件培养液能显著提高缺氧神经元的活力。[结论]XNJI组分能促进缺氧复氧胶质细胞对谷氨酸的清除并提高神经元活力。     

星形胶质细胞对兴奋性氨基酸神经递质的调控及与癫痫的关系

星形胶质细胞是中枢神经系统中最为丰富的细胞类型,其主要生理功能之一是调控中枢神经递质的稳态,从而在维持中枢神经兴奋性和抑制性平衡中扮演着关键性的角色。星形胶质细胞这种功能对调控胞外兴奋性神经递质谷氨酸和抑制性神经递质γ-氨基丁酸（γ-aminobutyric acid,GABA）尤为突出,即星形胶质细胞可通过谷氨酸-谷氨酰胺-GABA循环对正常状态下抑制性和兴奋性神经递质的循环利用具有重要意义,此外对避免因胞外谷氨酸蓄积造成的兴奋性毒性同样不可或缺。星形胶质细胞功能紊乱如谷氨酸转运体、谷氨酰胺合成酶、谷氨酸脱氢酶的减少可损伤或削弱星形胶质细胞调控神经递质的功能,可表现为胞外谷氨酸水平升高同时伴有GABA水平降低,这种病理改变与癫痫的发生发展密切相关。本文就星形胶质细胞对神经递质的调控及与癫痫的关系进行综述。     

大黄酚对原代培养小鼠星形胶质细胞谷氨酸代谢的影响?

目的探讨不同浓度大黄酚对原代培养小鼠星形胶质细胞活性及谷氨酸代谢功能的影响。方法原代培养小鼠星形胶质细胞,大黄酚10、20、40、60、80、100、200、250 mg/L处理细胞1、2、3d,MTT法检测大黄酚对星形胶质细胞活性的影响;RT-PCR检测谷氨酸/天门冬氨酸转运体(GLAST)及谷氨酸转运体1(GLT-1)mRNA表达水平。相关试剂盒检测谷氨酰胺合成酶(GS)和谷胱甘肽过氧化物酶(GSH-Px)活性。结果大黄酚可抑制星形胶质细胞活性,可诱导星形胶质细胞GLAST和GLT-1 mRNA表达上调,GSH-Px和GS活性增加,且均具有时间和浓度依赖性。但相似浓度且作用时间相同的大黄酚并不增加细胞的活性,即大黄酚对细胞外谷氨酸浓度的影响并不依赖细胞数量或活性的增加。结论大黄酚可抑制小鼠星形胶质细胞活性,对谷氨酸代谢的调节具有时间和浓度依赖性。     

Effect of Morphine and Naloxone on Release of the Excitatory Amino Acids of Spinal Astrocytes Induced by TNF-α

The effect of morphine and naloxone on release of the excitatory amino acids (EAAs) of spinal astrocytes induced by TNF-α was studied. Astrocytes were purified from 2- to 3-day old SD rats and divided into 8 groups: group 1 (without any stimulatants); group 2 (10 ng/ml TNF-α); group3 (10 ng/ml TNF-α+0.5 μmol/L morphine); group 4 (10 ng/ml TNF-α+1.0 μmol/L morphine); group 5 (10 ng/ml TNF-α+2.0 μmol/L morphine); group 6 (10 ng/ml TNF-α+0.5 μmol/L naloxone); group 7 (10 ng/ml TNF-α+1.0 μmol/L naloxone); group 8 (10 ng/ml TNF-α +2.0 μmol/L naloxone). In group 2, 3, 4 and 5, 0, 0.5, 1.0 or 2.0 μmol/L morphine was added to the cells cultured with serum-free Neurobasal/B27 medium containing 10 ng/ml TNF-α respectively, while in group 6, 7 and 8, 0.5, 1.0 or 2.0 μmol/L naloxone was added respectively to the cells cultured with serum-free Neurobasal/B27 medium containing 10 ng/ml TNF-α. After 30 min incubation, high-pressure liquid chromatography (HPLC) was used to measure the levels of EAAs in all cultured cells. The results showed the level of EAAs in group 2 was significant higher than in group 1 (P<0.01). As compared with group 2, the levels of EAAs in group 3, 4 and 5 were decreased with the difference being significant between group 5 and group 2 (P<0.01) or between group 4 and group 2 (P<0.05). The levels of EAAs in group 6, 7 and group 8 was significantly lower than in group 2 (P<0.05 orP<0.01). It was concluded that TNF-α could promote the release of glutamate and aspartate from astrocytes, and morphine and naloxone might reduce the release of EAAs in cultured spinal astrocytes induced by TNF-α.     

吗啡对C6神经胶质瘤细胞Glu和Gln含量及其代谢关键酶转录活性的影响

目的：研究吗啡对C6神经胶质瘤细胞谷氨酸(Glu)-谷氨酰胺（Gln）循环代谢产物含量及其相关酶基因转录的影响,探讨吗啡依赖与耐受的作用机制。 方法： 传代培养的C6神经胶质瘤细胞,随机分为4组：对照组( C组),吗啡组（M组）,纳洛酮加吗啡组（N+M组）和纳洛酮组（N组）,应用比色法测定C6神经胶质瘤细胞培养上清液中Glu及Gln的含量,应用半定量RT-PCR法测定C6神经胶质瘤细胞Glu-Gln 循环关键酶谷氨酰胺合成酶（GS)及谷氨酸脱氢酶（GDH) mRNA相对含量的变化。 结果： 与C组比较,M组细胞外Glu含量显著性增加(P<0.01)。与M组比较,N+M组细胞外Glu含量显著性降低(P<0.01);与C组比较,M组、N组以及N+M组细胞外Gln含量均降低,但差异无显著性(P>0.05)。与C组比较,M组、N组细胞GS mRNA转录活性均显著降低(P<0.01)。与M组比较,N+M组细胞GS mRNA转录活性显著升高(P<0.01);与C组比较,M组细胞GDH mRNA转录活性升高(P<0.05)。与M组比较,N+M组细胞GDH mRNA转录活性显著性升高（P<0.01）。 结论：吗啡可能通过对Glu-Gln 循环关键酶GS和GDH基因转录活性的影响调节细胞外Gln和Glu浓度,其机制可能与吗啡依赖等作用相关。     

低浓度凝血酶预处理对星形胶质细胞氧糖剥夺损伤的影响

目的:研究低浓度凝血酶(TB)预处理对培养的鼠脑星形胶质细胞(As)在氧糖剥夺(OGD)诱导损伤后的影响.方法:原代培养的大鼠大脑皮层AS,预先用不同低浓度(0.005～5.000 kU/L)的TB处理(TB预处理,TPC),观察在缺糖和缺氧(氧糖剥夺)培养状态下细胞的功能状态和损伤情况.以四甲基偶氮唑盐(MTT)还原试验检测细胞活力,乳酸脱氢酶(LDH)漏出率作为细胞损伤指标,流式细胞术检测细胞凋亡发生率,[3H]-谷氨酸摄取法测定As对谷氨酸(Glu)摄取能力的变化.结果:OGD引起细胞活力下降,LDH漏出率增高,OGD诱发细胞凋亡,降低As对Glu的摄取,上述指标与正常对照组相比均具有统计学意义(P＜0.01);而经低浓度TB预处理后,OGD损伤造成的LDH漏出率较低,细胞凋亡发生率降低,同时MTT值提高,As对Glu的摄取也增高,与OGD组比较有统计学意义(P＜0.05),其中最佳TB效应剂量为0.10 kU/L.结论:低浓度TB预处理的As,可增强在OGD环境下的损伤耐受能力,具有细胞保护作用.     

足底注射吗啡可阻止谷氨酸和天然辣椒碱引起的动物对放射热的逃避行为

目的探讨足底注射吗啡对由谷氨酸和天然辣椒碱引起的动物对放射热的逃避行为的影响,从而了解阿片和谷氨酸受体在末梢神经痛觉传递中的相互作用。方法120只健康SD雄性大鼠随机分为12组,每组10只。在大鼠后肢左侧足底分别注射（正常对照组不用药）、生理盐水组（saline组）、谷氨酸组（Glu组）、天然辣椒碱组（Cap组）、吗啡组（Mor组）、盐酸纳洛酮组（NLX组）、谷氨酸牟吗啡组（Glu＋Mor组）、谷氨酸＋盐酸纳洛酮组（Glu＋NLX组）、谷氨酸＋盐酸纳洛酮＋吗啡组（Glu＋NLX＋Mor组）、天然辣椒碱＋吗啡组（Cap＋Mor组）、天然辣椒碱＋盐酸纳洛酮组（Cap＋NLX组）、天然辣椒碱＋盐酸纳洛酮＋吗啡组（Cap＋NLX＋Mor组）;注射药物15min,1、2、3、4、5、6h后分别观察SD雄性大鼠双足温度逃避域值的变化。结果大鼠双足逃避域值的变化,正常对照组左足的温度逃避域值为（11．7±0．29）s,右足的温度逃避域值为（11．5±0．4）s,两者的比为100％;saline组、Mor组、NLX组左右足的温度逃避域值比均为100％,3组比较差异无统计学意义（P〉0．05）;Cap组最大减少幅度发生在15～180min之间[150nmol为（66．4±3．9）％;300nmol为（70±4．3）％],与saline组比较差异有统计学意义（P〈0．05）;Glu组最大减少幅度发生在15～120min之间[1．5μmol为（85．9±1.3）％,5±mol为（78．7±2．7）％],与saline组比较差异有统计学意义（P〈0．05）;Glu＋Mor组逃避域值比最大值为（130±4）％,显著高于G1u组（P〈0．05）;Glu＋NLX＋Mor组最大值为（81±6）％,低于Glu＋Mor组（P〈0．05）;Cap＋Mot最大值为（203±10）％,显著高于Cap组（P〈0．05）;Cap＋NLX＋Mor组最大值为（89±9）％,低于Cap＋Mor组（P〈0．05）。结论谷氨酸受体参与末梢神经痛觉传递,激活阿片受体可以阻止外源性和内     

Glutamate Impairs Mitochondria Aerobic Respiration Capacity and Enhances Glycolysis in Cultured Rat Astrocytes

Objective To study the effect of glutamate on metabolism, shifts in glycolysis and lactate release in rat astrocytes.
Methods After 10 days, secondary cultured astrocytes were treated with 1 mmol/L glutamate for 1 h, and the oxygen consumption rates (OCR) and extra cellular acidification rate (ECAR) was analyzed using a Seahorse XF 24 Extracellular Flux Analyzer. Cell viability was then evaluated by MTT assay. Moreover, changes in extracellular lactate concentration induced by glutamate were tested with a lactate detection kit.
Results Compared with the control group, treatment with 1 mmol/L glutamate decreased the astrocytes’ maximal respiration and spare respiratory capacity but increased their glycolytic capacity and glycolytic reserve. Further analysis found that 1-h treatment with different concentrations of glutamate (0.1-1 mmol/L) increased lactate release from astrocytes, however the cell viability was not affected by the glutamate treatment.
Conclusion The current study provided direct evidence that exogenous glutamate treatment impaired the mitochondrial respiration capacity of astrocytes and enhanced aerobic glycolysis, which could be involved in glutamate injury or protection mechanisms in response to neurological disorders.     

K-ATP channel openers facilitate glutamate uptake by GluTs in rat primary cultured Astrocytes

Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels(K-ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K-ATP channel openers(KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that K-ATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters(GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium(MPP+). These potentiated effects were completely abolished by mitochondrial K-ATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C(PKC). These findings are the first to demonstrate that activation of K-ATP channel, especially mitochondrial K-ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.     

K-ATP channel openers facilitate glutamate uptake by GluTs in rat primary cultured Astrocytes

Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels(K-ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K-ATP channel openers (KCOs) have been pro-posed as potential neuroprotectants. Our previous studies demonstrated that K-ATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters(GluTs) of astrocytes play crucial roles in glutamate up-take and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium (MPP+). These potentiated effects were completely abolished by mitochondrial K-ATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species(ROS) and phosphoryla-tion of protein kinases C(PKC). These findings are the first to demonstrate that activation of K-ATP channel, especially mitochondri-al K-ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phospho-rylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also pro-vides a new strategy for neuroprotection.     

纳洛酮对离体人脑神经元缺氧损伤谷氨酸释放的影响

目的建立原代培养人脑神经元缺氧模型,探讨纳洛酮对神经元缺氧损伤中谷氨酸释放的影响.方法应用无血清原代培养人胚胎大脑神经细胞,经神经微丝染色证实可获得富含神经元的混合培养细胞.将神经元细胞随机分为对照组、缺氧组和给药组(纳洛酮终浓度分别为0.25、5、10 μg/ml).对照组为正常培养,缺氧组和给药组细胞进行缺氧结合无糖处理(氧糖剥夺)l h并继续复氧培养24h.于复氧24 h后观察细胞形态学变化,测定细胞外液乳酸脱氢酶(LDH)浓度,应用四甲基偶氮唑蓝法(MTT)观察细胞存活率,应用高效液相色谱法测定细胞外液谷氨酸浓度.结果缺氧组细胞外液LDH、谷氨酸浓度显著高于对照组(P＜0.01),MTT法测得光密度(OD)值则显著低于对照组(P＜0.01);应用纳洛酮后,随药物浓度增加,各组细胞外液谷氨酸浓度与缺氧组比较呈现逐渐降低趋势(P＜0.05,P＜0.01),MTT法测得OD值呈现逐渐升高趋势(P＜0.01),至纳洛酮10μg/ml时恢复至对照组水平(P＞0.05).结论纳洛酮可以减少缺氧神经元的谷氨酸释放,减轻兴奋性神经毒性,对神经元缺氧损伤具有保护作用.