H+对非洲爪蟾卵母细胞ATP-激活电流的调制作用

研究H^＋对非洲爪蟾卵母细胞内源性ATP受体的调制作用。采用双极电压钳技术记录卵母细胞膜ATP受体介导的电流。实验检测的细胞全部对ATP敏感，应用pH为7．4的ATP（10^-3mol／L）后引起一特征性电流，呈浓度依赖性。改用pH为6．5，6．0，5．5的ATP（10^-3mol／L）后，电流幅值明显减弱。结果表明，pH值降低，可抑制ATP诱导的卵母细胞膜电流。     

大黄素对辣椒素诱导的TRPV1通道电流的抑制作用

本研究通过显微注射技术将TRPV1 cRNA(1ng/nL)注射至非洲爪蟾卵母细胞(20nL/个)体内,放置于G-ORi培养液中,并在(19±1)℃的恒温培养箱内表达.同时利用灌流技术将不同浓度梯度的大黄素、辣椒素按照设计顺序依次灌流进入流动腔,同时控制流速保证非洲爪蟾卵母细胞完全浸没于流动腔内.使用双电极电压钳技术记录0.1,1.0,10.0,50.0μmol/L浓度梯度的大黄素对500nmol/L浓度的辣椒素激活TRPV1电流的影响.得到大黄素作用辣椒素激活TRPV1通道的抑制作用表现出浓度依耐性和电压非依赖性,IC50=38μmol/L,Hill系数n=0.5.结果表明了大黄素对TRPV1位点的相互作用是负协同的,在天然药物里面是一类比较弱的拮抗剂,且大黄素在ORi溶液中开始析出沉淀的浓度在50~60μmol/L之间.我们首次发现了大黄素能够抑制TRPV1通道电流,这可能为开发新的镇痛药物提供理论基础.     

ω-芋螺毒素MVIIA和MVIIC对α9α10乙酰胆碱受体的活性研究

分析和检测了ω-芋螺毒素MVIIA和MVIIC对α9α10乙酰胆碱受体(nAChR)的结合活性,并用重组表达在非洲爪蟾卵母细胞膜上的大鼠α9α10 nAChR受体,分别评价了2个ω-芋螺毒素MVIIA和MVIIC在常规的ND96灌流液和不含钙离子的钡离子ND96灌流液(Ba2+ND96)中对该受体的阻断活性.研究表明,2个ω-芋螺毒素MVIIA和MVIIC在Ba2+ND96灌流液中对α9α10 nAChR没有抑制作用,而它们在常规的ND96灌流液中却表现出微弱的抑制活性,这应是受钙离子激活产生的氯离子电流影响的结果.因此,2个ω-芋螺毒素MVIIA和MVIIC并不是α9α10 nAChR的有效阻断剂,其作用机理与α-芋螺毒素Vc1.1和Rg IA完全不同.本研究阐明了芋螺毒素抑制α9α10 nAChR和钙离子通道的机理,这对研发新型的芋螺毒素镇痛药和更好地治疗顽固性疼痛具有很重要的科学意义.     

基于USB的卵母细胞电压钳同步数据采集和分析软件设计

介绍了基于USB接口的卵母细胞电压钳系统的数据采集分析系统,应用Delphi语言开发了系统的人机操作实验界面,探讨了电压钳的USB同步传输、数据采集和和显示的实现方法.在细胞模型测试中,电压钳放大器的跨膜电流分辨达到了0.01nA,噪声测试低于10pA.在非洲爪蟾卵母细胞表达Kv4.2钾通道实验中,记录跨膜电流波形清晰,数据分析可靠.与国外同类仪器相比:该系统操作简单,价格低,数据采集和分析方便,适用于卵母细胞表达实验.     

电压依赖性钙通道 Cav3.1 的 cDNA 在爪蟾卵母细胞中的表达

Cav3.1是一种电压依赖性钙离子通道,cDNA全长7 625 bp.非洲爪蟾卵母细胞有表达外源基因的功能,被用于离子通道异源表达的研究中.pGEM-HE是卵母细胞的高效表达载体,但由于pGEM-HE多克隆位点处的酶切点较少,外源片段常不能直接插入.为了将Cav3.1的cDNA表达于卵母细胞中,首先对pGEM-HE的多克隆位点和β-Globin 3'端下游的切点进行了改造,引入需要的酶切点,删除了妨碍重组和mRNA转录的位点.并将Cav3.1的cDNA克隆到改造后的pGEM-HE载体中.将重组的质粒在爪蟾卵母细胞中表达,记录到了Cav3.1的通道电流,为以后的工作提供了基础.     

α6/α3β2β3乙酰胆碱受体在非洲爪蟾卵母细胞中的表达及其条件优化

为建立有效的α6/α3β2β3 nAChRs(α6/α3β2β3,或简写为α6β2^*,*代表其他亚基)体外重组表达实验模型,利用非洲爪蟾卵母细胞表达体系,采用显微注射RNA的方法,利用双电极电压钳技术检测电流,对α6/α3β2β3nAChRs亚型进行体外重组表达研究,并对其表达条件进行优化,获得了α6/α3β2β3 nAChRs优化的重组表达体系.烟碱型乙酰胆碱受体(nAChRs)是一类重要的配体门控离子通道,其亚型众多且结构相似,但各亚型的生理学和药理学功能截然不同.天然的α6β2^*nAChRs很难体外表达,体外表达模型的表达电流非常小.因此,本实验拟研究优化该亚型的表达,进行优化后的体外表达模型,产生的激发电流显著提高,为以该亚型为靶点的新药筛选提供实验模型和基础.     

猪卵母细胞体外成熟及电激活后发育能力的研究

探讨了不同的成熟培养液及外源激素对猪卵母细胞体外成熟培养及电激活后孤雌胚胎发育能力的影响. 结果表明：（1）改良M199（mM199）组猪卵母细胞成熟率显著高于M199组，且这两组又较NCSU-23组能极显著提高卵母细胞的成熟率. （2）孕马血清（PMSG）+人绒毛膜促性腺激素（hCG）+促卵泡素（FSH）组卵母细胞成熟率略高于FSH+促黄体素（LH）组，两者卵母细胞成熟率极显著高于尿促性腺激素（hMG）组. （3）含胎牛血清（FBS），猪卵泡液（PFF），表皮生长因子（EGF）的培养液较对照组在卵母细胞成熟率上无显著差异，但均能显著提高电激活后的卵裂率.添加EGF组桑囊率明显高于不添加组.但分别添加FBS和PFF组较对照组在桑囊率上均无显著差异. （4）卵丘细胞扩散与卵母细胞第一极体排出之间无直接相关性，但扩散程度好能提高电激活后的卵裂率.     

新型tacrine双联体对培养大鼠海马神经元NMDA受体作用位点的研究

目的：研究新型tacrine双联体bis（7）-tacrine对NMDA受体的作用位点.方法：大鼠海马神经元原代培养,全细胞膜片钳记录培养大鼠海马神经元上NMDA激活电流变化.结果：细胞外液pH值从8.1改变到6.7,在细胞外液中加入二硫苏糖醇（2 mmol/L）、精胺（10 mol/L）、镁离子（50～500 mol...     

运用热带爪蟾胚胎检测黑臭河道底泥的生态毒性

2010年9月~12月,在温州山下河（S）和九山外河（J）先后采集底泥样品5次.将热带爪蟾胚胎（NF10—NF11）暴露于1〖DK〗∶4底泥提取液（第1—4次）和间隙水（第5次）中48 h,观测其发育状况.结果表明,山下河S1样点连续5次的底泥样品均导致胚胎全部死亡,表现出很强的毒性.暴露于山下河底泥提取液的胚胎体长均显著短于空白对照组,而暴露于九山外河底泥提取液的胚胎体长仅第3次显著短于空白对照组.暴露于山下河底泥提取液的胚胎畸形率均高于暴露于九山外河底泥提取液的,尤其在第4次检测中,暴露于S2-S4底泥提取液的胚胎畸形率均高达100%,而九山外河最高仅为70%（J2）和40%（J1）.热带爪蟾胚胎直接暴露于所有采样点的山下河底泥间隙水均全部死亡.由此可见,山下河的整体毒性高于九山外河,底泥间隙水毒性高于底泥提取液.本研究表明,温州市典型的黑臭河道底泥对爪蟾胚胎的发育均有不同程度的影响,具有明显的生态毒性.同时,运用爪蟾胚胎可以有效地检测底泥的毒性效应,而且对不同河流和采样点的毒性评价具有较好的区分度.     

孕激素受体激活系统建立及对细胞周期的影响

建立孕激素受体（PR）转录激活系统,同时探索孕激素受体对细胞周期的影响。有助于进一步阐明其在乳腺癌的发生发展中发挥重要作用的调控机制。运用双荧光报告系统,建立内、外源孕激素受体转录激活系统,运用细胞流式技术检测孕激素受体对细胞周期的影响。发现孕激素可以激活外源构建的孕激素受体,孕激素能够通过结合内源受体促进细胞生长。我们成功建立了孕激素受体转录激活系统,并发现孕激素可以通过其受体增加细胞的S期比例,促进肿瘤细胞T47D的增殖。     

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.     

Expression of functional acetylcholine receptor from cloned cDNAs

The cloned cDNAs encoding the four subunits of the Torpedo californica acetylcholine receptor, each carried by a simian virus 40 vector, direct the synthesis of the functional receptor in a combined expression system consisting of COS monkey cells and Xenopus oocytes. Our results suggest that all four subunits are required to elicit a normal nicotinic response to acetylcholine, whereas only the alpha-subunit is indispensable for alpha-bungarotoxin binding activity.     

Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes

The Shaker gene of Drosophila melanogaster encodes a potassium-selective ion channel, the 'A' channel, or one of its subunits. A single Shaker messenger RNA species suffices to direct the synthesis of functional A channels in Xenopus oocytes. Physiological characteristics of the A currents induced by two different mRNA species are compared.     

Cloning by functional expression of a member of the glutamate receptor family

We have isolated a complementary DNA clone by screening a rat brain cDNA library for expression of kainate-gated ion channels in Xenopus oocytes. The cDNA encodes a single protein of relative molecular mass (Mr) 99,800 which on expression in oocytes forms a functional ion channel possessing the electrophysiological and pharmacological properties of the kainate subtype of the glutamate receptor family in the mammalian central nervous system.     

组织胺对大鼠DRG神经元ATP-激活电流的抑制作用

目的:探讨组织胺对大鼠DRG神经元ATP-激活电流的调制作用.方法:采用全细胞膜片钳技术,在新鲜分离的大鼠背根神经节细胞上进行.结果:实验观察到组织胺在DRG神经元可引起内向电流,并有明显的浓度依赖性.在被检测的DRG神经元中,85%(30/35)的细胞对ATP敏感,可引起一浓度依赖性的去敏感的内向电流.预加10-8、10-7、10-6、10-5、10-4mol/L组织胺后,对ATP-激活电流的抑制分别是:(12.50±3.2%(n=5)、(24.49±3.5%(n=5)、(35.18±4.5%(n=6)、(32.62±5.8%(n=8)、(23.53±4.2%(n=5),呈浓度依赖性.结论:组织胺对大鼠DRG神经元ATP-激活电流有明显的抑制作用.     

Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation

Modulation of neuronal excitability by regulation of K+ channels potentially plays a part in short-term memory but has not yet been studied at the molecular level. Regulation of K+ channels by protein phosphorylation and oxygen has been described for various tissues and cell types; regulation of fast-inactivating K+ channels mediating IK(A) currents has not yet been described. Functional expression of cloned mammalian K+ channels has provided a tool for studying their regulation at the molecular level. We report here that fast-inactivating K+ currents mediated by cloned K+ channel subunits derived from mammalian brain expressed in Xenopus oocytes are regulated by the reducing agent glutathione. This type of regulation may have a role in vivo to link metabolism to excitability and to regulate excitability in specific membrane areas of mammalian neurons.     

Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.