4000/人胚胎干细胞源TH 阳性细胞电压门控性通道的初步研究

目的：检测人胚胎干细胞源TH阳性细胞的神经元性电生理特性。方法：采用我们实验室改良后的“无血清四步法经拟胚体培养体系”的方法,体外诱导人胚胎干细胞源性TH阳性细胞,在对其细胞核型及特异性标志物进行检测的基础上,运用全细胞膜片钳记录的方法,检测其细胞膜上电压门控性离子通道的电生理特性。结果：分化前后细胞核型保持正常;诱导得到的细胞形态一致,大多数细胞（>90%）表达多巴胺能神经元的标志β-tubulion和TH,并仍表达神经前体细胞的标志nestin;膜片钳检测显示诱导分化的TH阳性细胞具有神经元性电压门控钠、钾离子通道。结论：人胚胎干细胞经体外定向诱导分化为TH阳性细胞后具有一定的DA能神经元特性,特别是神经元性电生理特性。     

Membrane currents in identified lactotrophs of rat anterior pituitary

Qualitative features of the primary inward and outward current components of identified lactotrophs of the rat anterior pituitary were examined. Identification of lactotrophs in heterogeneous dissociated anterior pituitary cultures was accomplished by application of the reverse hemolytic plaque assay. Currents in lactotrophs were subsequently examined using whole-cell or patch recording techniques. Two components of inward calcium current were observed: a transient component and a sustained component. The transient component activated at voltages as negative as -50 mV and was the major contributor to total lactotroph calcium current. The sustained component activated at voltages above about -10 mV. The 2 currents could be qualitatively separated by differences in inactivation properties and in sensitivity to cadmium. At least 3 components of outward current were distinguished. Either 30 mM TEA or 0 calcium eliminated a major portion of sustained outward current. This is likely to represent primarily calcium- and voltage-activated potassium current. The remaining current could be further differentiated into a transient current component that could be inactivated with conditioning potentials above -60 mV. A slowly activating and deactivating potassium current remained following inactivation of the transient current. Although the time course of the transient current is reminiscent of "A" current, activation of this current required potentials above -30 mV. Candidates for the single-channel currents that underlie the whole-cell outward currents were observed in cell-attached recordings. When combined with patch-clamp electrophysiological methods, the reverse hemolytic plaque assay promises to be a powerful technique for the electrophysiological characterization of specific cell subtypes in heterogeneous dissociated cell populations.     

Electrophysiological and morphological properties of light and dark cells isolated from mudpuppy taste buds

Isolated Necturus taste receptor cells were studied by giga-seal whole-cell recording and electron microscopy to correlate electrophysiological properties with taste cell structural features. Dark (type I) cells were identified by the presence of dense granular packets in the supranuclear and apical regions of the cytoplasm. In response to a series of depolarizing voltage commands from a holding potential of ?80 mV, these cells exhibited a transient, TTX-sensitive inward Na⁺ current, a sustained outward K⁺ current, and a slowly inactivating inward Ca⁺⁺ current. Light (type II) cells were identified by a lack of granular packets and by an abundance of smooth endoplasmic reticulum distributed throughout the cell. In addition, isolated light cells had clear vesicular inclusions in the cytoplasm and blebs on the plasma membrane. Light cells were divided into two functional populations based upon electrophysiological criteria: cells with inward and outward currents, and cells with outward currents only. Light cells with inward and outward currents had voltage-activated Na⁺, K⁺, and Ca⁺⁺ currents with properties similar to those of dark cells. In contrast, the second group of light cells had only voltageactivated outward K⁺ currents in response to depolarizing voltage commands. These data suggest that dark cells and light cells with inward and outward currents are capable of generating action potentials and releasing neurotransmitters onto gustatory afferent neurons in response to taste stimulation. In contrast, light cells with outward currents only likely serve a different function in the taste bud. © 1994 Wiley-Liss, Inc.     

Acetylcholine-activated currents in mouse neuroblastoma cells

The nicotinic and muscarinic responses of differentiated mouse neuroblastoma cells from the clonal line N1E 115 to applied cholinergic agents were recorded using single channel and whole cell patch clamp techniques. An inward macroscopic current induced by acetylcholine (ACh) at the resting potential was blocked by curare; cell-attached recordings revealed a single channel conductance of 18 pS and a lifetime of 36 ms at 30°C, with 200 nM ACh. The zero current potential was close to 0 mV. The kinetics of these nicotinic currents were described by multiexponential functions for both the open and closed time distributions. An outward single channel current, present at resting and slightly depolarized potentials, was also observed and has been tentatively described as being dependent on muscarinic receptor activation, as it was usually blocked by atropine. Under our conditions of whole cell clamp, no macroscopic outward current sensitive to ACh was observed.     

Membrane current possessing the properties of a mechano-electric transducer current in inner hair cells of guinea-pig cochlea

We measured the membrane current possessing the properties of a mechano-electric transducer current in isolated inner hair cells of guinea-pig cochlea. In a free-standing hair bundle, depolarization to +80 mV evoked a stable outward current attributable to the opening of transducer channels, and repolarization to -80 mV evoked a transient inward current indicating adaptation. The time constant of adaptation increased as the membrane potential depolarized. Dihydrostreptomycin diminished both the outward and inward currents.     

骨髓间充质干细胞分化为神经元样细胞后电生理特性的改变

背景：目前体外实验对骨髓间充质干细胞来源的神经元样细胞的研究多集中于形态学层面和神经标志物方面,对分化后的电生理功能研究较少。 目的：观察脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导Wistar大鼠骨髓间充质干细胞分化为神经元样细胞后电生理特性的变化。 设计、时间及地点：细胞学体外培养,对比观察,于2005-06/2007-10在天津市环湖医院细胞室和南开大学生命科学院完成。 材料：6周龄雄性Wistar大鼠3只,体质量160 g左右。 方法：贴壁培养法体外分离纯化间充质干细胞,用脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸联合诱导间充质干细胞向神经元样细胞分化。诱导前和诱导3 d后分别用膜片钳技术检测细胞膜电流。 主要观察指标：流式细胞仪检测间充质干细胞表型;倒置显微镜观察诱导分化前后细胞形态变化;免疫细胞化学鉴定神经元特异性烯醇化酶的表达,以及全细胞电流测定结果。 结果：①流式细胞仪检测结果显示,CD90阳性率(99±3)%,CD31阳性率(3.4±0.8)%,CD34阳性率(0.3±0.1)%。说明这一细胞群大部分处于未分化的干细胞状态,其纯度可达95%。②光镜下可见未经诱导的间充质干细胞多为扁平形带突起的细胞,似纤维样细胞,诱导3 d后出现神经元样细胞。③免疫细胞化学结果显示,诱导前间充质干细胞的神经元特异性烯醇化酶呈弱阳性,诱导后呈强阳性。诱导72 h时分化率为(24.01±3.76)%。④诱导组神经元样细胞外向电流峰值及最大外向电流密度高于对照组(P < 0.05),但未发现内向钠电流。 结论：脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导方法可以诱导间充质干细胞向神经元方向分化,虽未发现具有成熟神经元电生理功能,但有向成熟神经元分化的趋势。     

Calcium action on the membrane currents possessing the properties of mechano-electric transducer currents in inner hair cells of the Guinea-pig cochlea

In free-standing hair bundle, depolarization to +80 mV evoked a stable outward current and repolarization to -80 mV evoked a transient inward current attributable to the opening of mechano-electric transducer channels. The study investigated the Ca2+ dependence of this transducer-like membrane current in isolated inner hair cells of guinea-pig cochlea. The amplitude of outward currents increased and the rate of inward current decay, corresponding to adaptation kinetics, decreasing as the extracellular Ca2+ concentrations lessened, whereas the amplitude of outward current decreased and an adaptation accelerated as the extracellular Ca2+ elevated. Treatment with the cAMP agonist, 8-bromo-cAMP, induced an effect similar to that caused by elevating the extracellular Ca2+.     

人胚胎干细胞在无血清mTeSR®1培养基中维持培养和向内皮细胞的诱导分化

背景：传统的人胚胎干细胞培养扩增方法中应用含动物血清培养基，并依赖饲养层细胞培养，这种培养方法显著制约了干细胞的体外培养规模；另外异源动物血清成分介入，使病原污染及免疫排斥的概率显著增加。 目的：明确应用无血清培养基mTeSR®1对人胚胎干细胞进行长期体外培养的可行性，并建立诱导人胚胎干细胞分化为血管内皮细胞的相关技术平台。 方法：采用无血清培养基mTeSR®1以非饲养层细胞依赖的方式体外培养、扩增人胚胎干细胞株H9。经过40余次体外传代后，于倒置显微镜下观察其生长形态，并利用免疫荧光染色方法评估其细胞表型。此外，应用条件培养基诱导H9细胞株向内皮细胞方向分化。利用免疫荧光染色技术，定量RT-PCR以及低密度脂蛋白摄取实验对该胚胎干细胞源内皮细胞的表型及功能进行评价、分析。 结果与结论：mTeSR®1培养基能够支持H9细胞株在体外以非饲养层依赖的方式进行长期扩增，同时维持其未分化的干细胞潜能。添加血管内皮细胞的条件培养基能够定向诱导H9细胞向内皮细胞方向分化。该胚胎干细胞源内皮细胞不但表达内皮细胞的标志基因(kdr，pecam)和标记蛋白CD31，而且还能够摄取低密度脂蛋白，形成类似微血管结构。提示实验中所提供的培养及诱导分化体系能够支持胚胎干细胞的增殖与分化行为。     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Membrane currents induced by pentylenetetrazol in identified neurons of Helix pomatia

After systemic application of pentylenetetrazol (PTZ), mammalian as well as molluscan neurons generate epileptic paroxysmal depolarization shifts. For a further analysis of these potential oscillations the membrane currents induced by local application of PTZ onto identified neurons of Helix pomatia were investigated. Different types of responses were obtained at membrane potentials negative and positive to ca. -30 mV. At holding potentials more negative than -30 mV, PTZ as a rule evoked an inward current, sometimes preceded by a brief outward current. In a few experiments only a solitary outward current was found. The amplitudes of the inward and outward currents increased towards more negative potentials. The inward current was associated with a decrease and the outward current with an increase in membrane resistance. Besides these findings pharmacological and ion substitution experiments indicate that the inward current represents an unspecific current. At holding potentials more positive than -30 mV, PTZ evoked a sequence of currents which was the same in all neurons. This stereotyped current sequence consisted of (i) an early inward current, (ii) an intermediate outward current, and (iii) a late long-lasting inward current. The amplitudes of all these components increased towards more positive potentials with the outward current being particularly enhanced. The early inward current and the following outward current were associated with a decrease and the late inward current with an increase of the membrane resistance. Besides these pharmacological and ion substitution experiments suggest that the early inward current represents a mixed sodium and calcium current, the intermediate outward current a calcium activated potassium current. The late inward current is assumed to be due to a decreased potassium conductance. On the basis of the present results, it may be concluded that the unspecific inward current in the negative potential range is involved in the initiation and the calcium dependent potassium current in the termination of spontaneously occurring paroxysmal depolarization shifts.     

Voltage-gated Currents in Rabbit Retinal Astrocytes

The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was −70 ± 4.8 mV (mean ± SEM; n = 54), and the input resistance and cell capacitance were 558 ± 3.6 MΩ and 19.5 ± 1.8 pF respectively. Depolarization to potentials positive to −50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na⁺ in the bathing solution, and reduced by 50% in the presence of 1 μM tetrodotoxin. The time-to-peak of the Na⁺ current was more than twice that for the Na⁺ current found in retinal neurons. The glial Na⁺ current was half-inactivated at −55 mV. A transient component of the outward K⁺ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between −150 and −50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.     

Changes in ion channel expression during in vitro differentiation of trout oligodendrocyte precursor cells

Voltage-gated ionic currents were studied in cultured trout oligodendrocyte precursor cells derived from larval trout brain with the whole-cell mode of the patch-clamp technique. These bipolar cells which carry the ganglioside epitope A2B5 on their surface differentiated in vitro into immature multipolar oligodendrocytes expressing the myelin glycoprotein IP2, which signifies the initial step of oligodendroglial development in fish CNS. Depolarization above -40 mV activated a fast transient sodium inward current that was eliminated by substituting Na⁺ for choline and blocked in the presence of 1 μM TTX. The kinetics and the voltage-dependence of inactivation (half-maximal inactivation at -68 mV) resembled those of sodium currents described in mammalian oligodendrocyte precursor cells and CNS neurons. The expression of Na⁺ channels was developmentally regulated, since high amplitudes were measured only in A2B5⁺ cells with a characteristic bipolar morphology of glial progenitors. Depolarizing voltage steps, additionally elicited outward potassium currents that were sensitive to external 4-AP. In a subpopulation of cells this outward current consisted of a sustained and a transient component. The amplitude of both components were dependent on the prepulse potential. © 1995 Wiley-Liss, Inc.     

Voltage-dependent Currents in Microvillar Receptor Cells of the Frog Vomeronasal Organ

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 μm) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was −61 ± 13 mV (mean ± SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of –60 to –86 mV, with almost no spontaneous action potential. The input resistance was in the GΩ range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2 – 10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near –45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near –75 mV. An outward K⁺ current was blocked by internal Cs⁺ ions or by external tetraethylammonium or Ba²⁺ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd²⁺ ions. A voltage-dependent Ca²⁺ current was observed in an iso-osmotic BaCl₂ solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.     

Electrophysiological and immunocytological demonstration of cell-type specific responses to hypoxia in the adult cat carotid body

We have recently shown two types of cat carotid body cells based on the oxygen sensitivity of voltage-gated potassium channels. In the present study, we attempted to determine the correlation between cell types (glomus cells, sheath cells, and subtypes of glomus cells) and oxygen sensitivity of potassium channels. Further, changes in membrane potentials in response to hypoxia were also examined. Carotid body cells harvested from adult cats were cultured, and a whole cell patch clamp method was applied to determine the oxygen sensitivity of outward current. The tested cells were identified by Lucifer Yellow in the patch pipette. Glomus cells and sheath cells were immunocytochemically identified using tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) as markers. The cells whose outward current was inhibited by hypoxia showed TH-immunoreactivity but not GFAP-immunoreactivity. The cells whose outward current was not sensitive to hypoxia were GFAP-positive or TH-negative. One TH-positive cell had oxygen-insensitive outward current. The resting membrane potentials of the cells having oxygen-sensitive outward current were significantly higher (−55±3 mV) than those of the cells having oxygen-insensitive outward current (−35±2 mV). The former type of cells was depolarized during hypoxia, but not the latter type of cells. These results suggest that most glomus cells of the adult cat carotid body possess oxygen-sensitive potassium channels and are depolarized in response to hypoxia. On the other hand, sheath cells and possibly a small fraction of glomus cells possess oxygen-insensitive potassium channels and their membrane potential is not affected by hypoxia.     

Characteristics of Na+ current in Schwann cells cultured from frog sciatic nerve

Characteristics of voltage-dependent currents in cultured frog Schwann cells were investigated by the whole-cell clamp technique. An inward current was detectable at a membrane potential level more positive than-50 mV and reached a maximum value at about-10 mV, while no rectifying channel was present. The inward current was carried by Na⁺ ions, because the extrapolated reversal potential of the current agreed with the calculated E_Na, and the current was sensitive to tetrodotoxin. The membrane potential for half-maximal inactivation was-82 mV. The inactivation curve indicated that more than 90% of the Na⁺ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (-10 mV, 13°C). The electrophysiological characteristics of the Na⁺ current in the cultured frog Schwann cells were compared with those in other tissues. This Na⁺ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half-maximal inactivation potential and in the slower inactivation time course. © 1994 Wiley-Liss, Inc.     

Effect of ethanol on subthreshold currents of Aplysia pacemaker neurons

The subthreshold currents in bursting pacemaker neurons of the Aplysia abdominal ganglion were individually studied with the voltage clamp technique for sensitivity to 4% ethanol. The most prevalent effect of ethanol on unclamped bursting neurons was a hyperpolarization. This was shown to be due to a decrease of a voltage independent inward leakage current. Direct measurement of the Na-dependent slow inward current showed that this current was eliminated by 4% ethanol. Direct measurement of the Ca-dependent slow inward current showed that this current was substantially reduced by 4% ethanol. Injection of EGTA into cell bodies did not eliminate the ethanol-induced block of the slow inward calcium current. Thus, ethanol cannot be reducing the Ca-dependent slow inward current solely by an increase of internal calcium concentration. The effect of ethanol on voltage dependent outward current was measured by blockage of all inward current. The peak outward current was increased by ethanol. The rate of inactivation of this outward current was also increased. Calcium activated potassium current (IK(Ca)) is particularly complicated in its response to ethanol because it is dependent on both Ca and voltage for its activation. The level of IK(Ca) elicited in response to constant Ca injection was increased by ethanol treatment. The level of this current as activated by voltage clamp pulses was either increased or decreased depending on the neuron type. Ca2+ activated potassium conductance increased e-fold for a 26 mV depolarization in membrane holding potential. Ethanol decreased this voltage dependence to e-fold for a 55 mV change in potential. This result was interpreted to mean that ethanol shifted an effective Ca2+ binding site of these channels from about halfway through the membrane field to one quarter of the way across. The same theoretical approach allowed the further conclusion that ethanol caused an increased internal free calcium concentration probably by decreasing calcium binding by intracellular buffers.     

超极化激活环化核苷酸门控通道基因转染猪骨髓间充质干细胞

背景：研究证实超极化激活环化核苷酸门控通道（hyperpolarization-activated cyclic nucleotide-gated,HCN）电流在调控心脏的自发搏动中起着非常重要的作用。 目的：观察HCN2基因重组腺病毒转染后猪骨髓间充质干细胞目的基因的表达及电生理特征。 设计、时间及地点：细胞-基因学体外观察,于2007-07/2008-03在解放军第二军医大学胸心外科实验室完成。 材料：Yorkshire猪由解放军第二军医大学动物所提供,HCN2质粒由意大利Dario DiFrancesco教授惠赠,重组腺病毒Ad.HCN2由本实验室采用Ad5系统构建并保存。 方法：Percoll密度梯度离心+贴壁法体外分离纯化猪骨髓间充质干细胞,以感染复数=50进行Ad.HCN2转染,同时设立未转染组和转染Ad.Null组。 主要观察指标：通过RT-PCR、免疫荧光染色检测各组细胞HCN2 mRNA和蛋白的表达,用全细胞膜片钳检测各组细胞电生理学变化。 结果：未转染组和转染Ad.Null组均未见扩增片段,而Ad.HCN2扩增后在250~500 bp可见扩增片段,与携带HCN2基因的质粒扩增出的片段位置相同。转染后细胞核染色强度明显弱于胞膜和胞浆,与HCN2蛋白的分布相符合,未转染组及转染Ad.Null组无HCN2蛋白阳性表达。全细胞膜片钳可记录到起搏电流,其激活电位约为-60 mV,完全激活电位-140 mV,呈电压依赖性,当给予4 mmol/L CsCl后,内向的起搏电流即被明显抑制;未转染组及转染Ad.Null组细胞均无起搏电流。 结论：通过起搏基因HCN2重组腺病毒载体可成功转染猪骨髓间充质干细胞,使其能够表达HCN2通道蛋白,全细胞膜片钳可检测到起搏电流。     

Voltage-gated potassium currents in cultured ovine oligodendrocytes

Cultured oligodendrocytes (OLGs) develop processes and form myelin following attachment to a substratum. We applied the whole-cell voltage-clamp technique to identify and characterize the ionic currents of OLGs in culture. Within 2 d after attachment, OLGs extended processes and began to express an outward current that represents a composite response of an inactivating/transient component and a non-inactivating component. The current had a reversal potential of -66 mV and was sensitive to potassium channel blockers. After 4-5 d in culture, the transient component was less prominent, often accompanied by an increase in noninactivating or steady-state outward current. In addition, there was an increase in inward rectifier current. Four of 7 cells that failed to develop processes exhibited only linear high-resistance membranes. We conclude that cultured OLGs express 3 voltage-gated potassium conductances: (1) a transient outward current, (2) a noninactivating outward current, and (3) an inward rectifier current. The sequential appearance of the several currents may relate, at least in part, to process formation.     

CALCIUM ACTION ON THE MEMBRANE CURRENTS POSSESSING THE PROPERTIES OF MECHANO-ELECTRIC TRANSDUCER CURRENTS IN INNER HAIR CELLS OF THE GUINEA-PIG COCHLEA

In free-standing hair bundle, depolarization to +80 mV evoked a stable outward current and repolarization to ?80 mV evoked a transient inward current attributable to the opening of mechano-electric transducer channels. The study investigated the Ca²⁺ dependence of this transducer-like membrane current in isolated inner hair cells of guinea-pig cochlea. The amplitude of outward currents increased and the rate of inward current decay, corresponding to adaptation kinetics, decreasing as the extracellular Ca²⁺ concentrations lessened, whereas the amplitude of outward current decreased and an adaptation accelerated as the extracellular Ca²⁺ elevated. Treatment with the cAMP agonist, 8-bromo-cAMP, induced an effect similar to that caused by elevating the extracellular Ca²⁺.