膜片钳技术原理及在中药研究中的应用

通过探讨记录细胞离子通道中的离子电流来反映细胞膜单一或多个离子通道分子活动的膜片钳技术和工作原理,以及其在中药单一提取成分、中药单味药以及中药复方研究中的应用,为中药在不同应用情况下的药效分析和研究提供依据。     

膜片钳芯片技术及其在细胞电生理分析中的研究进展

膜片钳芯片技术是继细胞芯片之后的又一种崭新的分析细胞电生理参数的芯片技术.由于该芯片除了具有传统膜片钳的高分辨和高准确性特点外,还具有高通量、自动化以及细胞多通道参数和细胞网络参数在线和实时检测等优点.因此,该芯片技术将大大促进细胞离子通道、细胞网络传导以及药物筛选的研究和应用.文中具体介绍了膜片钳芯片技术的发展及其应用,结合作者的研究工作,着重介绍了膜片钳芯片技术在味觉细胞研究的最新进展,并结合神经芯片研究细胞网络的方法,对采用膜片钳芯片技术在细胞和分子水平上研究味觉的敏感机理和传导机制的应用前景进行了展望.     

小鼠海马神经元急性分离和膜片钳技术的应用

为了获得适用于膜片钳实验技术的单个海马神经元,应用酶消化及机械分离法,急性分离出生10～13d的昆明小鼠得到单个海马神经元通过倒置显微镜直接观察和全细胞膜片钳技术分别对分离得到的海马细胞的形态学和电生理学特征进行研究．通过实验可得,急性分离所得活性较好的海马神经元胞体具有锥形胞体和顸树突特征,立体感强．在全细胞膜片钳记录模式下可记录到全细胞电流及钠、钾通道电流．由实验结果可知,该方法可以得到形态和生理特征良好的单个海马神经元,证实该方法适用于膜片钳技术的研究,对深入探讨药物、物理因子等对海马离子通道及信号转导机制的作用有重要的应用价值．     

用膜片钳技术筛选光学活性杀虫剂

膜片钳技术是一种以记录通过离子通道的电流来反映细胞膜上离子通道分子活动的技术。离子通道存在于几平所有细胞的质膜里，是细胞膜上的特殊蛋白质大分子形成的对适当大小和带有适当电荷的离子具有高度选择性的孔道，是多种药物的作用靶标。药物作用于离子通道后，通道蛋白的构型或开与关的状态会发生变化，从而引起细胞乃至机体的一系列反应。     

大鼠肾上腺嗜铬细胞原代培养的一种实用方法

以胶原酶Ⅰ、透明质酸酶和ＤＮａｓｅ联用消化肾上腺髓质，再经两次离心收集嗜铬细胞，建立了大鼠肾上腺嗜铬细胞的分离制备及原代培养的实用方法．经反复实验（ｎ＝１２）证明，所获得的细胞形态良好，功能正常．细胞表面光洁、完整，呈球形，直径在８～１２μｍ范围．用细胞贴附式和全细胞方式膜片钳技术在培养２～７ｄ的嗜铬细胞上记录到多种电压敏感性跨膜离子电流，说明培养细胞的膜离子通道完整，适于细胞纯度要求不很高（＜９５％）的单个细胞实验．     

一种跨膜受体偶联离子通道调控网络模型研究

以一种受体偶联钙离子通道为例,讨论了普遍存在的细胞信号跨膜传递中的离子通道启闭的控制功能,对Ｂｒａｙ和Ｌａｙ的跨膜信号传递模型进行了改进．给出了一种跨膜受体偶联离子通道调控的网络模型．运用单细胞显微荧光测量［Ｃａ２＋］ｉ技术,测量了单个ＳＨ－ＳＹ５Ｙ细胞内游离钙离子浓度的变化,说明了此模型能够较好地完成离子通道的控制功能,并给出了关于此模型的一些讨论．     

白鲜碱对气管平滑肌的舒张作用及机理研究

目的:探讨白鲜碱对异常收缩的气管平滑肌的舒张作用.方法:以80 mmol·L~(-1) KCl或100μmol·L~(-1) ACh预刺激气管,用生物机能系统检测气管的张力,用膜片钳技术检测离子通道电流,观察白鲜碱对气管平滑肌的影响.结果:白鲜碱能呈剂量依赖性地抑制高钾和ACh诱导的预收缩,而对静息状态下的气管无影响.且白鲜碱能阻断VDCCs电流,抑制细胞外Ca~(2+)内流.除VDCCs通道外,它还抑制ACh激活的其他离子通道,并抑制肌质网中的内Ca~(2+)释放.结论:白鲜碱具有舒张气管平滑肌异常收缩的作用,有望作为一种钙离子拮抗剂抑制气道高反应,缓解哮喘.     

单离子通道的隐马氏模型与状态的还原

对细胞膜单离子通道建立隐马氏模型，其中通道潜在的开关状态序列{xt}为马氏链，而膜片钳记录数据{xt}为依赖通道状态的正态分布。用EM算法估计模型中的转移概率以及正态分布的均数与方差等参数，并利用隐马氏模型的性质，用观察到的{Yt}来还原通道潜在的开关状态{Xt}。     

细胞膜上的离子通道

离子通道是细胞膜上控制离子进出的功能蛋白,在细胞生命活动中发挥重要作用．离子通道具有对离子的选择性、通透的饱和性和开关的可控制性等特点;膜电压的变化、机械刺激和某些信号分子都可以调控离子通道开关;离子通道担负着离子吸收、渗透压调控、电冲动的形成和信号转导等重要的生理功能．离子通道的结构或功能失常会导致一些严重的疾病,对离子通道进行研究,寻找和设计调控离子通道的有效药物是治疗相关疾病的重要手段。     

膜片钳实验中玻璃微电极的拉制技术研究

介绍了膜片钳实验中玻璃微电极的拉制技术,并测试了不同温度对电极电阻的影响,同时讨论了拉制过程中的影响因素和注意事项。结果表明:拉制过程中的温度设置等对电极电阻有不同程度的影响,为膜片钳实验中玻璃微电极的制备提供了参考依据。     

Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.

Atrial natriuretic peptide, acting through its second messenger guanosine 3',5'-cyclic monophosphate (cGMP), suppresses Na+ absorption across the renal inner-medullary collecting duct and increases urinary Na+ excretion. Patch clamp studies show that cGMP reduces Na+ absorption by inhibiting an amiloride-sensitive cation channel in the apical membrane. We have now examined, using the patch clamp technique, the molecular mechanisms of cGMP inhibition. Cyclic GMP directly and specifically reduced the probability of a single channel being open (open probability, Po) by 39% (inhibition constant, Ki = 7.6 x 10(-7) M) by a phosphorylation-independent mechanism. Cyclic GMP also inhibited the channel by activating cGMP-dependent protein kinase (cGMP-kinase). Exogenous cGMP-kinase completely inhibited the channel by a phosphorylation-dependent mechanism. Activation of a pertussis toxin-sensitive G protein by GTP-gamma-S blocked cGMP-kinase inhibition of the channel. By contrast, cGMP-kinase inhibition of Po was completely reversed by GTP-gamma-S. Taken together with the results of a previous study showing that a G protein activates the cation channel, these data indicate that cGMP-kinase and a G protein sequentially regulate the cation channel. Our results show that atrial natriuretic peptide, acting through cGMP, inhibits Na+ absorption across the inner-medullary collecting duct by a dual mechanism, and that cGMP-kinase inhibits the channel by a pathway involving a G protein.     

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.     

The Reconstructing of Low Signal-noise Ratio Single Ion Channel Signal from Patch-clamp Recordings Sampled in the Colored Background Noise

The single ion channel signal is an ionic current that can be recorded by the patch clamp technique. Hidden Markov model(HMM)algorithm has been used to convert the low signal-noise ra-tio (SNR) noisy recording into an idealized quantal one in the case of white background noise. The traditional HMM algorithm is extended and adapted to the colored background noise.A new algorithm called EHMM (Extended HMM) algorithm is proposed,and mainly validated by simulati-on.Results show that it's effective.     

细胞膜单通道非平衡态离子动力学研究

速率学说更合理地解释离子通过离子通道中孔道的过程。本文将速率学说建立在Markor过程理论基础上,得到非平衡定态时越过能垒的离子流和膜钳制电位之间关系的一般模型。并将系统辨识中的单纯形法引入非平衡定态模型中的参数估计。采用膜片钳制技术记录了离体培养的新生大鼠大脑皮质神经元钾离子单通道电流。在膜两侧对称高钾溶液下,通道电流和电压之间呈线性关系的范围为：细胞贴附模式下,±60mv,内膜向外时±40mv。在此范围之外,通道电流和电压之间呈非线性关系。能垒分布接近三能垒模型。细胞贴附时膜外侧能垒最高,中间能垒最低,这种分布导致通道电流和电压之间的S型关系。内膜向外模式下,从膜内到膜外,能垒高度依次递增。孔道内能垒不对称是导致通道电流整流现象的基础。     

IS4 peptide forms ion channel in rat skeletal muscle cell membrane

A 22-mer peptide, identical to the primary sequence of domain I segment 4 (IS4) of rat brain sodium channel I, has been synthesized. IS4 peptide can incorporate into cultured rat skeletal myotube membranes and form ion channels. With patch clamp cell-attached technique single channel currents through IS4 channels can be recorded. The single channel conductances of IS4 channels are distributed heterogeneously. With different holding potentials, the mean open time, the mean closed time and the mean open probability are different respectively. IS4 channels are selective for Na⁺, Li⁺ and K⁺, but not for Cl⁻.     

血竭对大鼠背根神经节细胞钠通道电流的影响

研究血竭对大鼠背根神经节细胞电压门控性钠通道电流的影响。方法：采用全细胞膜片钳记录法，在新鲜分离的大鼠背根神经节细胞上观察血竭对钠通道电流幅值的影响。结果：血竭剂量依赖地抑制单个DRG细胞电压门控性钠通道电流，高剂量的血竭抑制作用不可逆。结论：血竭对背根神经节细胞钠通道电流的这种抑制作用，可能是其产生躯体镇痛作用的药理机制之一。     

Currents through the fusion pore that forms during exocytosis of a secretory vesicle

Exocytosis, or the fusion of cytoplasmic vesicles with the cell membrane, occurs in nearly all eukaryotic cells, but its mechanism is not understood. Morphological and electrophysiological studies have suggested that membrane fusion begins with the formation of a 'fusion pore', a narrow channel across the closely adjacent membranes of vesicle and cell that forms the first connection of the vesicle lumen with the cell exterior and later dilates to allow release of vesicle contents. We used the patch clamp technique to study exocytosis of single giant secretory vesicles in mast cells of beige mice. The first opening of the fusion pore was found to generate a brief current transient, whose size and direction indicated an initial pore conductance of about 230 pS and a lumen-positive vesicle membrane potential. In time-resolved a.c. admittance measurements, the pore conductance was found to increase to much larger values within milliseconds, as if the pore dilated soon after opening. We conclude that the earliest fusion event may be the formation of a structure similar to an ion channel. Its conductance is of the same order of magnitude as that of a single gap junction channel, the only other known channel that spans two membranes.     

氯离子通道生理药理学特点及相关疾病的研究进展

离子通道（Ion channel）研究近几年发展较快,逐渐与一些临床疾病联系起来,从基础研究迅速转向临床综合应用性研究,成为现阶段研究热点之一。离子转运（ion transportation）是离子通道的首要功能属性,其具体转运形式存在于种各细胞中。氯离子跨膜转运对细胞的功能起着关键性的作用,有些转运形式的改变或离子通道的变化都会对机体产生一定的影响,从而导致部分疾病的发生。就离子跨膜转运而言,氯离子通道产生的影响更为显著。其电生理及药理学特点值得关注,通过对其深入研究,帮助我们找到相关治疗疾病药物的作用位点,以达到有效治疗疾病及靶标药物筛选的目的,由此我们对氯离子转运过程、其通道形式和其相关疾病的研究进展做以下阐述。     

Single ionic channels observed in tissue-cultured muscle

The extracellular patch clamp technique developed by Neher et al. to record the responses of single channels in skeletal muscle has provided firm evidence for the two-state nature of the conductance event in nicotinic endplate channels. We report here the use of the extracellular patch technique to record single-channel responses from tissue-cultured chick skeletal muscle cells. The temperature dependence of channel conductance and gating kinetics shows no evidence of discontinuous behaviour between 17 and 37 degrees C.