胞内钙振荡的同步性对电磁场的响应

电磁场通常通过细胞膜将物理信号转化为生物信号,进而产生生物学效应.本文基于肝细胞钙振荡动力学模型,采用数值分析的方法,研究电磁场对细胞钙振荡同步性的影响,数值结果表明:细胞差异性导致钙振荡的不同,胞间耦合影响钙振荡的同步性;电磁场的频率、强度(调制因子)引起钙振荡的频率和幅值发生变化.     

卡维地洛对培养心肌细胞氧化损伤的保护作用

目的:探讨卡维地洛对培养心肌细胞氧化损伤的保护作用和机制。方法:H2O2作用于体外培养的新生SD大鼠心肌细胞,建立心肌细胞氧化损伤模型。用卡维地洛预处理后,镜下观察心肌细胞测定心肌细胞存活率(MTT比色法),丙二醛(MDA)、还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)等指标水平。结果:卡维地洛干预后提高心肌细胞氧化损伤的细胞存活率(P0.01),减少MDA、GSG生成以及提高GSH的生成(P0.01)。结论:卡维地洛通过抗氧化作用对心肌细胞氧化损伤起保护作用。     

克隆的BKCa通道介导AngⅡ诱导的HEK293细胞增殖

目的:探讨BKCa通道在AngⅡ诱导细胞增殖过程中的调控作用.方法:以脂质体转染法将人源性BKCa通道α亚基(hSloα)转染到HEK293细胞中(HEK293-hSloα), 用MTT法测定AngⅡ诱导细胞增殖的量效曲线, 用免疫细胞化学方法、流式细胞术分别观察AngⅡ、 IBTX及AngⅡ+IBTX对HEK293-hSloα细胞增殖细胞核抗原(PCNA)表达和细胞周期的影响.结果:AngⅡ可剂量依赖性诱导HEK293-hSloα细胞增殖, AngⅡ促进HEK293-hSloα细胞PCNA表达, 并使S期细胞比率增加.但此作用能被BKCa通道特异性抑制剂IbTX所阻断.结论:BKCa通道可介导调节AngⅡ诱导的HEK293-hSloα细胞增殖过程.     

胞间耦合和电磁场对多细胞钙振荡的影响

目的:研究电磁场对多细胞系统(三个细胞)钙振荡同步性的影响.方法:基于肝细胞动力学模型,以胞间耦合和电磁场引起的细胞三磷酸肌醇(IP3)浓度的变化作为影响因子,采用数值分析的方法,研究电磁场的频率、强度对钙振荡的影响.结果:细胞差异性导致钙振荡的不同,在相同的耦合因子下,细胞数不同导致钙振荡形式存在差异;胞间耦合影响多细胞钙振荡的同步性.结论:电磁场的频率、强度(调制因子)引起钙振荡的频率和幅值发生变化.     

极低频磁场对胞内钙振荡影响的机理分析

基于双钙库模型,本文以离子跨过细胞膜、细胞器膜迁移的几率作为细胞对电磁场的响应因子,来调制流过膜通道的离子的速率,进而影响细胞内钙离子的浓度。数值分析表明:在极低频磁场的作用下,一定能量因子下的频率条件或是一定频率因子下的能量条件可引起钙振荡形式的变化;窗效应是频率因子和能量因子共同作用的结果。     

人趋化因子受体CCR6在HEK293细胞内的稳定表达及其功能分析

目的:构建稳定表达人趋化因子受体6(CCR6)的HEK293细胞株。方法:将CCR6基因和Gα16质粒共转到HEK293细胞中,并挑取稳定表达CCR6基因的HEK293细胞克隆。采用体外趋化实验、钙流实验、RT-PCR、Western blot及免疫荧光染色法,检测CCR6在HEK293细胞表面的表达。结果:经上述实验证实,CCR6基因和Gα16质粒共转染的HEK293细胞上,可稳定表达CCR6,且具有生物学活性。结论:成功地在HEK293细胞表面稳定表达具有生物学活性的CCR6,为研究CCR6的生物学功能及筛选CCR6的拮抗剂奠定了基础。     

细胞外钾对HERG基因无义突变L539fs/47表达的调控

 目的: 研究持续细胞外钾对野生型HERG与其突变体L539fs/47蛋白表达的影响。方法: 用脂质体转染法将野生型HERG(WT)及其突变体HERG-L539fs/47(MT)分别转染HEK293细胞36 h后,0.8、4.3及10 mmol/L的钾干预6 h,流式细胞术检测HERG蛋白表达量;干预12 h用激光共聚焦成像和免疫印迹法进行HERG蛋白定位及表达量检测。结果: 用激光共聚焦成像检测发现野生型HERG蛋白主要分布在细胞膜上;而HERG突变体L539fs/47蛋白大部分滞留胞浆;并且HERG蛋白的表达均随细胞外钾浓度升高而增多。流式细胞术结果显示细胞外高钾组荧光增多(P < 0.01),WT组荧光阳性细胞百分比和荧光强度均高于MT组(P < 0.05)。免疫印迹显示,不同于野生型 HERG 135 kD及155 kD 2个条带,突变体仅60 kD 1个条带,3个条带均受细胞外钾影响(P < 0.05)。结论: 细胞外高钾增强细胞膜上野生型和突变型HERG通道蛋白的稳定性,持续低钾干预时间依赖性地减少HERG通道蛋白的表达。     

稳定表达水通道蛋白-4 HEK293细胞株的建立及应用

目的:建立稳定表达水通道蛋白-4(AQP4)的HEK293细胞株,并以其检测12例视神经脊髓炎患者血清中是否存在AQP4抗体。方法:从颞叶癫痫患者脑组织提取总RNA,RT-PCR扩增AQP4的cDNA,构建AQP4-绿色荧光蛋白(GFP)融合表达重组体pEGFP-N1-AQP4,转染HEK293细胞,抗生素筛选联合流式细胞仪分选筛选稳定表达细胞株,RT-PCR和间接免疫荧光法鉴定AQP4的表达,激光共聚焦显微镜观察AQP4在细胞中的定位,并以已建立的细胞为底物间接免疫荧光法检测12例视神经脊髓炎患者血清中是否存在AQP4抗体。结果:经PCR、双酶切、测序鉴定证实成功获取AQP4的cDNA,AQP4-GFP融合表达重组质粒pEGFP-N1-AQP4构建成功,抗生素筛选联合流式细胞仪分选筛选稳定表达细胞株,稳定表达率达90%以上,RT-PCR和间接免疫荧光法鉴定存在AQP4的表达,激光共聚焦显微镜进一步确认AQP4-GFP融合蛋白主要表达于细胞膜上,11例典型视神经脊髓炎患者血清中检出AQP4抗体,对视神经脊髓炎诊断敏感性为91.7%,特异性为94.7%。结论:成功建立稳定表达AQP4的HEK293细胞株,以其作为检测底物检出视神经脊髓炎患者血清中存在AQP4自身抗体。     

小鼠FasL基因真核表达载体的构建及在HEK293细胞中的表达

目的构建含有小鼠Fas Ligand(FasL)基因的重组真核表达载体,并检测FasL蛋白在其稳定转染的HEK293细胞中的表达情况,为构建表达小鼠Fas L的树突状细胞(DC)模型,深入研究DC联合T细胞防治移植物抗宿主病(GVHD)的新方法奠定基础。方法从小鼠脾脏中提取RNA并逆转录为cDNA,以该cDNA为模板扩增FasL基因,插入pcDNA3.1(+)载体中构建pcDNA3.1(+)-FasL重组质粒,利用脂质体将其转染HEK293细胞,用G418筛选稳定表达细胞系,Western blot确定重组蛋白的表达。结果通过酶切及测序证实FasL序列正确插入表达载体pcDNA3.1(+),Western blot检测证实转染重组质粒的细胞能正确表达FasL蛋白,G418筛选后能够得到稳定表达FasL的抗性细胞株即FasL-HEK293。结论重组质粒pcDNA3.1(+)-FasL和稳定表达FasL的HEK 293细胞成功构建,为后续FasL-DC细胞的研究打下了坚实基础。     

TRIM22稳定过表达细胞系的构建及其抗流感病毒的功能

目的构建稳定表达TRIM22的HEK293T细胞系HEK293T-TRIM22,观察TRIM22蛋白在该细胞内的表达及对流感病毒复制的影响并初步探究其抑制机制。方法扩增TRIM22基因并与反转录病毒载体进行体外重组连接,经菌液PCR、测序确认后与反转录病毒骨架载体共转染进HEK293T细胞进行包装病毒。用包装的反转录病毒感染HEK293T细胞,感染24 h后用嘌呤霉素筛选稳定过表达TRIM22的细胞,筛选48 h后用Western blot检测TRIM22表达水平;用IAV-LUC病毒检测稳定过表达TRIM22的HEK293T对流感病毒的抑制作用;再用双分子荧光互补实验(Bi LC)检测与TRIM22相互作用的流感蛋白。结果经测序确认目的基因TRIM22成功克隆到载体p QC-XIP中。包装反转录病毒并感染HEK293T细胞,经过嘌呤霉素筛选后,稳定过表达TRIM22的HEK293T细胞中TRIM22的蛋白表达水平升高(P0.05)。在稳定过表达TRIM22的细胞内流感病毒复制减弱(P0.05)。Bi LC检测TRIM22与流感蛋白NP有相互作用。结论稳定过表达TRIM22的HEK293T细胞系构建成功,TRIM22与NP有相互作用并且抑制流感病毒复制。     

卡维地洛抑制大鼠起搏心肌细胞钙库超载诱导的钙释放*

 目的：探讨非选择性β受体阻滞剂卡维地洛对起搏心肌细胞钙库超载诱导钙释放（SOICR）的作用及其机制。方法：电刺激起搏大鼠单个心肌细胞并灌注异丙肾上腺素及咖啡因诱导钙库钙超载,从而触发肌浆网钙释放通道（兰尼碱受体2,RyR2）舒张期开放引起SOICR。应用钙离子荧光成像技术记录胞内钙浓度的实时变化。实验分为对照组、卡维地洛组、美托洛尔组、酚妥拉明组和硝苯地平组。结果：(1) 与基线刺激时比较,对照组细胞灌注异丙肾上腺素和咖啡因后,起搏细胞钙瞬变振幅显著增高（P<0.01）,SOICR的发生率显著增加（P<0.01）。(2) 在1~4 Hz起搏频率下卡维地洛组细胞SOICR发生率分别为2.00%、6.00%、10.00%和16.00%,均显著低于对照组（分别为43.59%、74.36%、87.18%和89.74%,均P<0.01）;卡维地洛对心肌细胞SOICR的抑制在不同起搏频率下无明显差异（P>0.05）。酚妥拉明、美托洛尔和硝苯地平组细胞与对照组细胞比较,SOICR发生率无差异（均P>0.05）。(3) 起搏细胞钙瞬变振幅的比较,各组间相比未见明显差异（P>0.05）;咖啡因峰值估测钙库钙总量的比较,各组间也无明显差异（P>0.05）。结论：卡维地洛可明显抑制起搏心肌细胞SOICR的发生,其作用机制可能是直接抑制RyR2的自发性开放而非源于对α1、β1受体和L型钙通道的阻滞作用。     

Effects of pH on spontaneous tension oscillation in skinned bovine cardiac muscle

Skinned cardiac muscle preparations exhibit spontaneous tension oscillations (spontaneous oscillatory contractions; SPOCs) in the absence of Ca²⁺, and in the presence of MgATP, MgADP and inorganic phosphate (Pi; ADP-SPOC). Similar oscillations occur in the presence of sub-micromolar concentrations of Ca²⁺ under normal activating conditions without MgADP and Pi (Ca-SPOC). In the study presented here, we investigated the effects of pH on both types of SPOC in skinned bovine cardiac ventricular muscle. First, a decrease in pH increased the MgADP concentration required to induce the half-maximal isometric tension that is obtained in the absence of Ca²⁺ and in the presence of MgATP (ADP-contraction). The inhibitory effect of Pi on ADP-contractions was not affected by pH. Second, ADP-SPOCs occurred upon the addition of Pi to the solution that resulted in ADP-contraction, and the relative amplitude and the period of the tension oscillation in the presence of 2 mM MgATP, 10 mM MgADP and 10 mM Pi were unchanged under all pH conditions examined (6.6, 7.0, 7.4). On the contrary, the relative amplitude and the period of the Ca-SPOCs were markedly diminished at pH 6.6. Finally, we constructed state diagrams showing the effects of pH on SPOC conditions. The state diagram shows that SPOCs occur less frequently under acidic conditions than at neutral pH. We suggest that the intermediate state of crossbridges that is required for SPOCs is more difficult to attain at a low pH. Received: 14 September 1998 / Received after revision: 23 February 1999 / Accepted: 10 March 1999     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

卡维地洛对病毒性心肌炎小鼠的保护作用及其机制

目的：观察卡维地洛对病毒性心肌炎小鼠的保护作用并探讨其可能的作用机制。方法：188只清洁级近交系4-6周龄雄性BALB/c小鼠随机分成4组。心肌炎对照组（C组）、美托洛尔干预组（M组）、卡维地洛干预组（K组）各60只,空白对照组（B组）8只作总体对照。观察各组小鼠的心肌组织病理学改变、cTn-I水平及心肌组织磷酸化p38MAPK含量的变化。结果：美托洛尔、卡维地洛干预后,小鼠心肌组织病理改变减轻,cTn-I水平、心肌磷酸化p38MAPK含量均下降,卡维地洛干预后上述指标下降更加显著。结论：卡维地洛可能通过β1、β2肾上腺素能受体双重阻滞作用,抑制p38MAPK通路活化,减轻病毒性心肌炎引起的心肌损伤。     

APPswe mutation increases the frequency of spontaneous Ca2+-oscillations in rat hippocampal neurons

Altered calcium homeostasis is implicated in the pathogenesis of Alzheimer's disease (AD). Much effort has been put into understanding the association between protein mutations causative of this devastating neurodegenerative disease and perturbed calcium signaling. Whereas the presenilin mutations have received most attention in the context of neuronal calcium signaling, we focused on the effects of APP with the so-called Swedish mutation (APPswe) on spontaneous neuronal activity. We observed that primary hippocampal neurons from an APPswe transgenic rat showed increased frequency and unaltered amplitude of spontaneous calcium oscillations as compared to wild-type neurons. We found that the altered calcium signaling of APPswe transgenic neurons was unlikely to be due to modulation of the NMDA or nicotinic neurotransmitter systems, and did not depend on secreted APP derivates. The implications of this effect of APP are discussed.     

Control of IsAHP in mouse hippocampus CA1 pyramidal neurons by RyR3-mediated calcium-induced calcium release

In several neuronal preparations, the ryanodine-sensitive calcium store was reported to participate in the generation of slow afterhyperpolarization currents (IsAHP) involved in spike frequency adaptation. We show that calcium release from the ryanodine-sensitive calcium store is a major determinant of the triggering of IsAHP in mouse CA1 pyramidal neurons. Whole-cell patch clamp recordings in hippocampus slices show that the intracellular calcium stores depletion using an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase (5 μM cyclopiazonic acid), as well as the specific blockade of ryanodine receptors (100 μM ryanodine) both reduced the IsAHP by about 70%. Immunohistology, using an anti-RyR3 specific antibody, indicates that RyR3 expression is particularly enriched in the CA1 apical dendrites (considered as the most important site for sAHP generation). We show that our anti-RyR3 antibody acts as a functional RyR3 antagonist and induced a reduction in IsAHP by about 70%. The additional ryanodine application (100 μ M) did not further affect IsAHP, thus excluding RyR2 in IsAHP activation. Our results argue in favor of a specialized function of RyR3 in CA1 pyramidal cells in triggering IsAHP due to their localization in the apical dendrite.     

卡维地洛对氧自由基诱导的内皮细胞内源性一氧化氮合酶抑制物代谢的影响

目的： 观察卡维地洛对氧自由基（OFR）培养的人脐静脉内皮细胞（HUVECs）二甲精氨酸-二甲赖氨酸水解酶 (DDAH)活性及表达的影响，以探讨卡维地洛对不对称二甲精氨酸（ADMA）代谢机制的影响。 方法： 采用改良的Jaffe法培养原代人脐静脉内皮细胞（HUVECs），取生长良好的3-6代HUVECs用于实验，分为①空白对照组：加DMEM培养液；②OFR组:加入OFR（0.01 mmol/L,0.1 mmol/L）；③OFR+卡维地洛组: 同时加入0.1 mmol/L OFR及卡维地洛（10 μmol/L）共孵24 h后, 检测上清液中一氧化氮（NO）、内皮素（ET）、ADMA含量、L-胍氨酸(L-cit)浓度及一氧化氮合酶（NOS）活性。用Western blotting法测定细胞裂解液中二甲基精氨酸-二甲基赖氨酸水解酶 (DDAH)的蛋白表达。 结果： OFR条件培养下,内皮细胞的代谢产物ADMA、ET的量均高于空白对照组,而NO的量及NOS的活性少于空白对照组;反映DDAH酶活性的L-cit浓度显著降低,且有浓度依赖性，而DDAH的表达无明显变化。卡维地洛干预组的ADMA、ET的量均低于OFR组，NOS活性及NO、L-cit浓度明显高于OFR组。 结论： OFR培养下，内皮损伤ADMA的增加与DDAH的活性减弱有关，而与DDAH的表达无关。卡维地洛通过增加DDAH活性促进ADMA代谢，使NOS活性增加，抑制OFR对内皮功能的损伤。     

阿托伐他汀钙对高血压大鼠血管内皮细胞的保护作用

目的:观察高血压状态下阿托伐他汀钙对SD大鼠胸主动脉血管内皮细胞的保护作用。方法:构建两肾一夹SD大鼠肾性高血压模型。研究动物随机分为高血压组、他汀治疗组和假手术组,每组8只,术后第4周他汀治疗组给予阿托伐他汀钙20mg·kg-1.d-1灌胃8周。术后第4、12周测血压、血脂,第12周通过扫描电镜方法观察胸主动脉内皮细胞层的完整性,检测血液中循环内皮细胞数量,并测定循环内皮祖细胞(CEPCs)的数量及增殖能力、黏附能力及凋亡情况。结果:他汀治疗组大鼠胸主动脉内皮细胞受损轻于高血压组,但重于假手术组,高血压组、他汀治疗组和假手术组大鼠循环内皮细胞数量(5.9×106、3.9×106、2.0×106)与CEPCs凋亡率(22.1%±2.1%、13.4%±1.6%、7.4%±1.3%)依次降低(均P0.01);而CEPCs数量(21.63±2.33、40.38±6.00、65.38±2.97)、CEPCs增殖能力(0.13±0.01、0.17±0.01、0.29±0.03)与CEPCs黏附能力(12.25±2.49、21.50±2.20、28.88±2.85)依次增高(均P0.01)。结论:(1)高血压状态可导致大鼠血管内皮细胞严重受损,CEPCs数量增加,而CEPCs数量与功能降低,凋亡率增高,继而引起CEPCs对血管内皮细胞的修复能力降低。(2)阿托伐他汀钙对血管内皮细胞具有直接保护作用,并可能通过提高CEPCs数量及功能、降低CEPCs凋亡、增强CEPCs对血管内皮细胞的修复能力而发挥作用。