不同浓度瑞芬太尼对新生大鼠海马区神经干细胞内钙浓度及细胞凋亡的影响

目的：探讨不同浓度瑞芬太尼对新生大鼠海马区神经干细胞凋亡及细胞内钙浓度的影响。方法：采用已建立的新生SD大鼠海马区神经干细胞单细胞克隆系细胞株,将5×105个/ml活细胞的密度均匀地接种到的12孔培养板中,每孔加入1ml含有10%胎牛血清的DMEM/F12的培养基,放入温度37℃、5%浓度CO2培养箱中孵育24小时,镜下观察细胞球贴壁后,每孔分别加入生理盐水或不同浓度的瑞芬太尼（R）,分为以下4组：（1）对照组,每孔加入生理盐水;（2）低浓度组,每孔加入瑞芬太尼5ng/ml,即R5组;（3）中浓度组,每孔加入瑞芬太尼10ng/ml,即R10组;（4）高浓度组,每孔加入瑞芬太尼20ng/ml,即R20组。每组重复4次。加药后培养300s、600s、900s用激光扫描共聚焦显微镜（LSCM）检测各组神经干细胞内游离钙浓度的动态变化,24h后流式细胞仪检测各组神经干细胞的凋亡情况。结果：(1)各组细胞内钙浓度变化情况：与对照组比较,R5组细胞内钙浓度无明显差异(P>0.05);R10、R20组细胞内钙浓度明显升高(其中R10组P<0.05 ,R20组P<0.01)。(2)各组神经干细胞凋亡变化情况：与对照组比较,R5组神经干细胞凋亡无明显差异(P>0.05);R10、R20组神经干细胞凋亡明显升高(其中R10组P<0.05 ,R20组P<0.01)。结论：低浓度的临床有效血药浓度的瑞芬太尼对新生大鼠海马区神经干细胞内钙浓度、细胞凋亡没有影响,中高浓度的临床有效血药浓度的瑞芬太尼应用使细胞内钙浓度显著升高,从而诱发细胞大量凋亡。     

新生大鼠海马和皮层神经元的原代培养及鉴定

目的建立一种条件简单易存活,适用于生理、药理学研究的原代神经元培养方法。方法应用新生(48 h以内)大鼠采用急性分离、胰酶消化的方法培养海马和皮层神经细胞,并结合膜片钳及RT-PCR技术鉴定所培养的神经元电压依赖性离子通道的表达情况。结果体外培养的神经细胞生长状态良好,免疫荧光染色鉴定结果表明该方法培养的神经元纯度可达90%以上;全细胞电压依赖性离子通道表达情况良好。结论应用该方法培养神经细胞操作简单并保持了良好的生理特性,可用于神经元生理特性考察及药理实验研究,是体外培养神经元较理想的方法。     

大川芎方提取物对神经胶质细胞内一氧化氮浓度的影响

目的：探讨大川芎方提取物对神经胶质细胞内一氧化氮浓度的影响的。方法：体外培养的人神经胶质细胞中给予1－5号被试大川芎配方的提取物（10％浓度)，剂量分别为0．06、0．12、0．24ml，作者0．5h后加入染色剂2，7－二氯荧光素，避光反应15min后用荧光分光光度计测其细胞产生的荧光强度，以反映细胞内一氧化氮的浓度。结果：即使细胞内一氧化氮浓度增高，不同比例配伍药物的提取物均可使荧光强度增强。结论：大川芎方不同配伍提取物引起神经胶质细胞内5－羟色胺（5－HT）的变化在于细胞内一氧化氮浓度的改变。     

左归丸对体外培养新生大鼠海马神经干细胞增殖分化的影响

左归丸具有滋阴补肾功效。笔者的在体实验表明左旋单钠谷氨酸模拟肾阴虚证大鼠神经干细胞(Nerve Stemceu，NSC)增殖分化和凋亡与正常组有明显差异。为探讨左归丸以及左旋单钠谷氨酸所致肾阴虚模型中有关NSC增殖分化的作用及机制，本实验从神经干细胞增殖分化方面作以下探讨。     

新生大鼠海马神经干细胞的分离培养及鉴定

目的：从新生大鼠海马分离培养并鉴定神经干细胞。方法：应用含有碱性成纤维细胞生长因子（bFGF)和表皮生长因子（EGF）的无血清条件培养基，采用无底物的悬浮培养法培养神经干细胞，并用免疫细胞化学技术鉴定传代后形成的细胞团。结果：EGF bFGF可能明显地促进神经干细胞分裂增殖，免疫细胞化学检测发现传代后形成的细胞团中的细胞均表达神经上皮干细胞蛋白（Nestin)。结论：我们成功地建立了新生大鼠海马神经干细胞培养模型。     

布比卡因抑制谷氨酸诱导大鼠海马星形胶质细胞内钙升高的机制

本文以原代培养的大鼠海马星形胶质细胞为研究对象,探讨布比卡因(bupivacaine)抑制谷氨酸诱导的海马星形胶质细胞内钙升高的机制。运用免疫荧光技术确定代谢型谷氨酸受体(mGluR5受体)在神经元和星形胶质细胞上的表达情况;利用钙离子成像技术,观察布比卡因和mGluR5受体拮抗剂2-甲基-6-(苯乙炔)吡啶盐酸盐[2-methyl-6-(2-phenylethynyl)-pyridine, MPEP]对谷氨酸诱导的大鼠海马神经元和星形胶质细胞胞内游离钙(intracellular free Ca²⁺concentration,[Ca²⁺]_i)变化的影响;观察布比卡因对mGluRs (mGluR1/5)受体激动剂(RS)-3,5-二羟基苯基甘氨酸[(RS)-3,5-dihydroxyphenylglycine, DHPG]及mGluR5受体特异性激动剂2-氯-5羟苯基甘氨酸钠盐[(RS)-2-chloro-5-hydroxyphenylglycine sodium salt, CHPG]诱导的大鼠海马神经元和星形胶质细胞...     

mTOR-自噬通路对脓毒症小鼠海马小胶质细胞表型的影响

目的 探讨哺乳动物雷帕霉素靶蛋白（mTOR）-自噬通路对脓毒症小鼠海马小胶质细胞表型的影响。方法 54 只雄性 ICR 小鼠,按照随机数字表法分为 3 组（n=18）：假手术组（Sham 组）、脓毒症组（CLP 组）和脓毒症+mTOR抑制剂雷帕霉素组（CLP+Ra组）。Sham组只进行开腹手术操作;CLP组采用盲肠结扎穿孔术制备脓毒症小鼠模型;CLP+Ra 组于造模前 6 h腹腔注射雷帕霉素（1.5 mg/kg）。于造模后 24 h各组取 6只,采用酶联免疫吸附试验（ELISA）法检测海马组织肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-10和转化生长因子（TGF）-β水平;采用免疫荧光染色法检测海马组织切片离子钙接头蛋白分子（Iba）-1/CD86和 Iba-1/CD206阳性细胞数量;采用蛋白免疫印迹法测定海马组织 mTOR磷酸化（p-mTOR）水平、自噬相关蛋白微管相关蛋白 1轻链 3Ⅱ（LC3Ⅱ）和 Beclin-1表达情况。结果 与 Sham组比较,CLP组 TNF-α、IL-1β、IL-10和 TGF-β水平升高,Iba-1/CD86和 Iba-1/CD206阳性细胞数量增加,p-mTOR水平上调,LC3Ⅱ和 Beclin-1表达下调（均 P＜0.05）。与 CLP组比较,CLP+Ra组 TNF-α和 IL-1β水平降低,IL-10和 TGF-β水平升高,Iba-1/CD86阳性细胞数量减少,Iba-1/CD206阳性细胞数量增加,p-mTOR水平下调,LC3Ⅱ和 Beclin-1表达上调（均 P＜0.05）。结论 mTOR-自噬通路通过调节小胶质细胞表型转化,影响脓毒症小鼠海马炎症反应。     

铅对大鼠神经胶质培养细胞ERK活力及总量的影响

目的探讨不同浓度、不同时间染铅对体外原代培养的神经胶质细胞中细胞外信号调节激酶(ERK)活力及总量的影响。方法出生4~8d Wistar大鼠脑神经胶质细胞原代培养,细胞不同浓度及不同时间染铅组及丝裂原激活蛋白激酶(mitogen activated proteinkinase,MAPK)特异抑制剂PD098059预处理组分别收集作为样品,应用Western印迹法测定样品ERK活力及总量的变化。结果0.2、1.0、10μmol/L染铅后,神经胶质细胞中ERK活力均显著增高,分别比对照增高了19.7%、69.8%和16.1%(P<0.05);PD98059抑制铅激活ERK活力,1.0μmol/L染铅30min时ERK活力最高,约为对照的2倍(P<0.05),120min时降至接近正常。染铅后ERK总量无变化。结论低浓度染铅可使原代培养的神经胶质细胞中磷酸化的ERK含量短暂升高,一段时间后又恢复正常,而对ERK的总量无影响。     

糖皮质激素对小胶质细胞内钙的影响

目的观察糖皮质激素对小胶质细胞内钙的影响并初步探讨其机制。方法体外培养神经小胶质细胞株BV-2,使用Fluo3-AM作为钙荧光染料,激光共聚焦显微镜实时扫描观察氢化可的松处理后BV-2细胞内钙浓度的动态变化情况。结果氢化可的松和阳性对照药尼古丁均能显著升高BV-2细胞内钙水平(P<0.05);实时扫描显示氢化可的松即刻引起细胞内钙升高,15 s左右达到峰值,持续约10 s后开始下降,200 s左右恢复至基态,并且这一作用显示出与尼古丁升高细胞内钙效应的一致性。糖皮质激素受体拮抗剂RU486不能取消氢化可的松升高BV-2细胞内钙的效应(P>0.05);而α7烟碱型乙酰胆碱受体(α7nAChR)阻断剂甲基牛扁亭碱(MLA)可以拮抗氢化可的松升高BV-2细胞内钙的效应(P<0.05)。结论氢化可的松通过影响α7nAChR升高小胶质细胞内钙水平,这一作用不但证实了糖皮质激素的非基因组效应,同时提示糖皮质激素可能作为α7nAChR的内源性配体。     

Effects of dexamethasone on intracellular Ca 2 in its sensitive cells from neonatal mouse hippocampus and cultured cortical neurogliocytes

目的：研究地塞米松（Ｄｅｘ）对神经元和胶质细胞内钙浓度（［Ｃａ２＋］ｉ）的影响．方法：Ｆｕｒａ２ＡＭ负载小鼠海马细胞（ＮＭＨＣ）和培养的胶质细胞（ＣＣＮ）．单细胞内［Ｃａ２＋］ｉ由ＡＲＣＭＭＩＣ检测系统测定．结果：Ｄｅｘ使多数ＮＭＨＣ［Ｃａ２＋］ｉ浓度依赖地迅速升高,９６个ＮＭＨＣ中仅１０％出现［Ｃａ２＋］ｉ降低．［Ｃａ２＋］ｉ升高被无镁细胞外液阻滞、被氯化镧逆转,但不受氯化锂影响．无钙Ｈａｎｋｓ液悬浮、米非司酮（Ｍｉｆ）或河毒素均可阻断Ｄｅｘ４０－９０μｍｏｌ·Ｌ－１的升［Ｃａ２＋］ｉ效应,而Ｄｅｘ２００μｍｏｌ·Ｌ－１的效应仍被保持．４０个ＣＣＮ中５０％对Ｄｅｘ产生浓度依赖的［Ｃａ２＋］ｉ升高,并被无钙或无镁的细胞外液和Ｍｉｆ预处理抑制．结论：Ｄｅｘ快速改变海马神经元和胶质细胞内［Ｃａ２＋］ｉ．［Ｃａ２＋］ｉ的这种改变是由Ｍｇ２＋和受体相关的外钙内流及高浓度Ｄｅｘ诱发的内钙释放介导的．     

Effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pig

The effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pigs were studied by means of a whole-cell voltage-clamp method and Ca2+-sensitive fluorescent dye. Thiopental inhibited L-type voltage-dependent Ca2+ currents in a concentration-dependent manner (IC50=2.8.10(-5) mol/l). Moreover, the mode of Ca2+ current inhibition by thiopental showed no use dependency. Electrical stimulation-induced intracellular Ca2+ transient was significantly suppressed by 10(-5) mol/l thiopental. However, the caffeine-sensitive Ca2+ releasing pathway from sarcoplasmic reticulum was not affected by thiopental. Our results indicate that thiopental inhibits L-type Ca2+ currents, but not release of Ca2+ from sarcoplasmic reticulum. These results suggest that the negative inotropic action of thiopental is mainly due to inhibition of L-type Ca2+ channels in guinea pig atrial myocytes.     

大蒜新素对分离大鼠脑细胞内游离Ca^2＋的影响

观察大蒜新素对不同刺激剂所致分离大鼠脑细胞内游离钙的影响。方法：以Ｆｕｒａ２－ＡＭ为细胞内游离钙的荧光指示剂,用ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统,直接测定了分离新生大鼠脑细胞内游离钙值,观察了大蒜新素的影响。结果;大蒜新素对脑细胞静息「Ｃａ＾２＋」ｉ无明显影响,大蒜新素１－１００μｍｏｌ．Ｌ＾－１能剂量依赖性地抑制高Ｋ＾＋和谷氨酸引起的「Ｃａ＾２＋」ｉ升高,其中ＩＣ５０分别为５９．７和６９．９μｍ     

Pneumadin-evoked intracellular free Ca2+ responses in rat aortic smooth muscle cells: effect of dexamethasone.

The direct vascular effect of pneumadin (PN) was determined by studying the changes in intracellular free calcium ([Ca2+]i) levels in cultured rat aortic smooth muscle cells maintained between the second and fifth passages. PN evoked a rapid, concentration-dependent, biphasic increase in [Ca2+]i. The [Ca2+]i level rose from a basal value of 108 nM to a maximum increase in peak value of 170 nM. Although the level of maximal [Ca2+]i response evoked by PN was less than with other vasoactive agonists, it was more potent (EC50 0.5 nM) than even endothelin-1 (EC50 3.1 nM). At concentrations > 100 nM, [Ca2+]i elevations induced by PN above basal levels were no longer observed. Pretreatment with dexamethasone (100 nM for 24 hr) resulted in a significant increase (P < 0.01) in the peak [Ca2+]i response (310 nM) to PN. However, the biphasic pattern in the peak [Ca2+]i responses encountered with increasing concentrations of PN remained unaffected. The exaggerated [Ca2+]i response to PN was abolished by preincubation of cells with either the glucocorticoid antagonist mifepristone (RU 486) or the protein synthesis inhibitor cycloheximide. Inclusion of either an AT1 antagonist (losartan), a V1 selective vasopressin antagonist (d(Ch2)5 Tyr (Me) AVP), or an alpha-adrenoceptor antagonist (phentolamine) failed to affect the increases in [Ca2+]i induced by PN. PN-evoked increases in inositol 1,4,5-trisphosphate levels paralleled the [Ca2+]i changes. These data suggest that PN increases Ca2+ mobilization in rat aortic smooth muscle cells via activation of phospholipase C coupled receptors. This effect is up-regulated by dexamethasone.     

Effects of phenylalkylamines and benzothiazepines on Ca(v)1.3-mediated Ca2+ currents in neonatal mouse inner hair cells

Calcium currents (I(Ca)) in inner hair cells (IHCs) are carried by the Ca(v)1.3 subtype of L-type calcium channels. They play an important role in synaptic transmission of sound-evoked mechanical stimuli. L-type calcium channels are targets of the organic blocker classes dihydropyridines, phenylalkylamines and benzothiazepines. Previously a low sensitivity of the Ca(v)1.3 subtype towards dihydropyridines has been demonstrated. Therefore, this study evaluates the effect of two phenylalkylamines (verapamil and gallopamil) and the benzothiazepine diltiazem on I(Ca) through Ca(v)1.3 channels in mouse IHCs. Whole-cell I(Ca) was measured using the patch-clamp technique in mouse IHCs aged postnatal day 3-7 with 5 mM calcium as a charge carrier. The phenylalkylamines verapamil and gallopamil and the benzothiazepine diltiazem inhibited I(Ca) in IHCs in a concentration-dependent manner. This block was largely reversible. Dose-response curves revealed IC(50) values of 199+/-19 microM for verapamil, 466+/-151 microM for gallopamil and 326+/-67 microM for diltiazem. The inhibition of peak I(Ca) by phenylalkylamines and benzothiazepines was voltage-independent. Verapamil (300 microM) enhanced current inactivation from -20 to +20 mV while diltiazem (300 microM) did so only at very depolarised potentials (+20 mV). In conclusion, the concentrations of phenylalkylamines and benzothiazepine necessary to inhibit 50% of I(Ca) in IHCs were one order larger compared to concentrations which inhibited I(Ca) through Ca(v)1.2 channels in native cells or expression systems. However, inhibitory concentrations were in the same range as those required for block of I(Ca) in turtle hair cells.     

Hydroxylated PCB induces Ca2+ oscillations and alterations of membrane potential in cultured cortical cells

Polychlorinated biphenyls (PCBs) are known as environmental pollutants that may cause adverse health effects. Although some congeners have been shown to affect brain development or function, the molecular mechanisms mediating their toxicity are not yet fully understood. Since signal transduction via intracellular Ca²⁺ is crucial for neuronal development and plasticity, we investigated the effect of PCBs on Ca²⁺ homeostasis and membrane potential in cultured mouse cortical cells. Acute exposure to hydroxylated PCB 106 [4(OH)‐2′,3,3′,4′,5′‐pentachlorobiphenyl, OH‐PCB 106, 0.1 μm ] caused recurring Ca²⁺ oscillations that were classified into three prototypes. Although extracellular Ca²⁺ deprivation significantly reduced the oscillations, 54% of the cells still showed different patterns of oscillations or gradual increase in the intracellular Ca²⁺ concentration, indicating possible involvement of multiple Ca²⁺ channels in a cell‐specific manner. Such a possibility was further confirmed by differential responses to several channel/receptor blockers, including nifedipine, ryanodine, xestospongine and tetrodotoxin. Although all chemicals had partial inhibition action in different subsets of neurons, nifedipine blocked the OH‐PCB 106 action in the largest subpopulation of cells and with the greatest magnitude. Ryanodine also blocked the action with a similar magnitude, but in a smaller subpopulation of cells. Moreover, OH‐PCB 106 induced depolarization of the plasma membrane in all the recorded cells. Taken together, our results indicate that OH‐PCB 106 alters membrane potential as well as Ca²⁺ dynamics in part by inducing extracellular influx and/or intracellular release of Ca²⁺. These mechanisms may be responsible for their neurotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Role of intracellular Ca2+ on histamine release from rat peritoneal mast cells.

The benzoic acid derivative 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) strongly inhibited histamine release from rat peritoneal mast cells induced by various secretagogues. TMB-8 also inhibited Ca2+ mobilization from intracellar Ca2+ stores. However, histamine release induced by compound 48/80 (condensation product of N-methyl-p-methoxy-phenethylamine and formaldehyde) was not affected by TMB-8. These results indicate that Ca2+ mobilization is necessary to elicit the release of histamine from mast cells.     

Mg2+ and caffeine-induced intracellular Ca2+ release in human vascular endothelial cells.

Interaction of ionized magnesium ([Mg2+]o) and caffeine in regulation of intracellular free calcium concentration ([Ca2+]i) in human aortic endothelial cells was studied using fura-2 and digital imaging microscopy. In 1.2 mM [Mg2+]o, basal [Ca2+]i was 73.7 +/- 22.4 nM, with a heterogeneous distribution within the cells. No significant changes of basal [Ca2+]i were found either when cells were treated with 10 mM caffeine or when [Mg2+]o was lowered from 1.2 mM to 0.3 mM. However, a combined superfusion of the cells with 0.3 mM [Mg2+]o and 10 mM caffeine resulted in a significant elevation of [Ca2+]i to 382.8 +/- 57.1 nM, probably by release of Ca2+ from internal stores, which was attenuated by NiCl2 (1 mM). These results suggest that a Ca(2+)-induced Ca2+ release mechanism is involved in regulation of [Ca2+]i in endothelial cells, which may be either regulated or modulated by Mg2+.     

Failure of endothelin-1 to activate store-operated Ca2+ channels by lack of mobilization from intracellular Ca2+ stores in cultured bovine adrenal chromaffin cells

We have recently shown that in addition to L-type voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) stimulation opens two types of Ca2+-permeable nonselective cation channels [designated nonselective cation channel-1 (NSCC-1) and NSCC-2]. However, in this Ca2+ entry, the involvement of store-operated Ca2+ channel (SOCC), which is suggested to exist in chromaffin cells, was unclear. Those NSCCs as well as SOCC can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. To clarify whether SOCC should actually exist and play a role in Ca2+ entry in chromaffin cells stimulated with ET-1, we examined the effects of removal of extracellular Ca2+, thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca2+-ATPase), LOE 908 and SK&F 96365 on cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured bovine adrenal chromaffin cells. After the cells were exposed to Ca2+-free medium followed by exposure to TG to deplete Ca2+ from the intracellular Ca2+ store, restoration of extracellular Ca2+ caused a gradual increase in [Ca2+]i (to about 200% of control). The increase was unaffected by LOE 908, but completely abolished by SK&F 96365. In the Ca2+-free medium, no increase in [Ca2+]i by ET-1 was observed, but the subsequent restoration of extracellular Ca2+ induced a rapid increase in [Ca2+]i (to the same level of [Ca2+]i as that evoked by ET-1 in the normal medium (1.0 mM Ca2+)). Since SK&F 96365 is also a blocker of SOCC, these results indicate that in bovine adrenal chromaffin cells, Ca2+ entry through SOCC (Ca2+ influx through the capacitative Ca2+ entry system) occurs but is comparably weak, and that it virtually does not work on the stimulation of ET-1.     

灯盏花素对人脐静脉内皮细胞胞内Ca~(2+)水平的调控作用

目的探讨灯盏花素对培养的人脐静脉内皮细胞(HUVECs)胞内Ca2+水平([Ca2+]i)的调控作用。方法采用新一代Ca2+荧光探针Fluo-3/AM标记培养的HUVECs,激光共聚焦显微镜检测细胞胞内钙荧光信号,观察灯盏花素对培养的HUVECs胞内Ca2+水平的调控作用。结果在胞外有Ca2+或无Ca2+的情况下,灯盏花素均可引起[Ca2+]i的短暂性升高;灯盏花素的Ca2+释放作用与钙泵抑制剂CPA存在着交迭;灯盏花素能够抑制由KCl所引起的[Ca2+]i的升高;灯盏花素对胞内Ca2+池耗竭后胞外复Ca2+所引起的钙内流无明显阻断作用。结论灯盏花素可引起胞内Ca2+池的Ca2+释放,其释放的Ca2+来自CPA敏感的Ca2+池。灯盏花素也可抑制经电压依赖性Ca2+通道的Ca2+内流,对Ca2+池耗竭后引起的Ca2+内流通道无明显阻断作用。