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Background and Objectives@#Manipulating different signaling pathways via small molecules could efficiently inducecardiomyocytes from human induced pluripotent stem cells (hiPSC). However, the effect of transcription factors on the hiPSC-directed cardiomyocytes differentiation remains unclear. Transcription factor, p53 has been demonstrated indispensable for the early embryonic development and mesendodermal differentiation of embryonic stem cells (ESC).We tested the hypothesis that p53 promotes cardiomyocytes differentiation from human hiPSC. @*Methods@#and Results: Using the well-characterized GiWi protocol that cardiomyocytes are generated from hiPSC via temporal modulation of Wnt signaling pathway by small molecules, we demonstrated that forced expression of p53 in hiPSC remarkably improved the differentiation efficiency of cardiomyocytes from hiPSC, whereas knockdown endogenous p53 decreased the yield of cardiomyocytes. This p53-mediated increased cardiomyocyte differentiation was mediated through WNT3, as evidenced by that overexpression of p53 upregulated the expression of WNT3, and knockdown of p53 decreased the WNT3 expression. Mechanistic analysis showed that the increased cardiomyocyte differentiation partially depended on the amplified mesendodermal specification resulted from p53-mediated activation of WNT3-mediated Wnt signaling. Consistently, endogenous WNT3 knockdown significantly ameliorated mesendodermal specification and subsequent cardiomyocyte differentiation. @*Conclusions@#These results provide a novel insight into the potential effect of p53 on the development and differentiation of cardiomyocyte during embryogenesis.
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Objective To establish a rat model of cardiac hypertrophy induced by isoproterenol(ISO),and to study its basic characteristics . Methods Cardiac hypertrophy was induced in rats with ISO. The model rats received subcutaneous injections of 5 mg/kg ISO every day for 14 days. Results The heart weight/body weight and left ventricular weight/body weight ratios in model rats were significantly increased. The serum hydroxyproline level was significantly increased ,the superoxide dismutase level was significantly decreased ,and the malondialdehyde level was sig?nificantly increased in model rats. Conclusion The rat model of cardiac hypertrophy is successfully created by subcutaneous injection of ISO for 14 days. This model can be used in study of the mechanism of cardiac hypertrophy.
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Objective To explore the effect of dronedaronel on hyperpolarization-activated cyclic-nucleotide-gated(HCN) channel expression by detecting the change of HCN channel mRNA and protein level before and after giving dronedarone in neonatal rat ventricular myocytes.Methods Neonatal rat ventricular myocytes were separated and digested by type Ⅱ collagenase,and then single ventricular myocytes were collected through differential sticking wall separation method.According to the concentrations (0.1,0.5,1.0,5.0,10.0,20.0 μmol/L of dronedaronel for treating myocytes for 48 h) and time(10 μmol/L of dronedaronel for treating myocytes for 1,6,12,24,48 h)the gradient grouping was conducted.The levels of HCN2 and HCN4 channel mRNA and protein level were determined by real-time PCR and Western blot.Results The HCN2 mRNA and HCN4 mRNA expression levels in concentration gradient group and time gradient group were lower than those in the control group(P＜0.05);compared with the control group,the protein level in the 10 umol/L dronedaronel treatment for 12 h group was significantly down-regulated(P＜ 0.01).Conclusion Dronedaronel could inhibit the expression of HCN2/HCN4 channel mRNA and protein,moreover its action shows the concentration dependency and reaches the maximum at 12 h after medication.
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Objective To develop a new method to expose the stellate ganglion to increase the success rate of establishing a dog model of atrial fibrillation indinced by sympathetic stimulation .Methods A total of 28 adult dogs were randomly divided into traditional group and improvement group , 14 dogs in each group .The stellate ganglions were separated by the two different methods , respectively , to establish a sympathetic stimulation induced atrial fibrillation model in all the dogs .Changes of vital signs , survival rate of the dogs and the voltage required to stimulate the stellate ganglion were recorded intraoperatively .Changes of cardiac electrophysiology were recorded before and after electric stimulation . The levels of released neurotransmitters were detected by immunohistochemistry . Results The survival rate of the improvement group was 100%(14/14), significantly higher than the 64.3%(9/14) of the traditional group (P<0.05). The operation time of the improvement group was 122.71 ±3.62 min, significantly shorter than the 269.44 ±8.79 min of the traditional group (P<0.05).The threshold voltage of the improvement group was significantly lower than that of the traditional group ( P<0.05) .Conclusions Our modified surgical procedure can effectively reduce the mortality of dogs , significantly shorten the operation time , and reduce the intraoperative blood loss , keeping a more intact stellate ganglion , and maintains a more stable voltage of electric stimulation , Therefore, it is a new method more suitable for establishment of a sympathetic stimulation induced atrial fibrillation model in dogs .
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Objective To discuss a method combining serum‐pharmacology and electrophysiology technology ,and to research the mechanism of dilating porcine coronary artery of sodium tanshinone Ⅱ‐A sulfonate (DS‐201) .Methods To give mice intragastric administration solution and measure DS‐201 concentration in mice serum ,and apply the serum to single channel patch to research its effect on big conductance calcium‐activated potassium channels(BKca ) in porcine coronary artery smooth muscle cells (PCASMCs) . Results The linear range of concentration containing DS‐201 serum was 0 .73 to 1 .91 μg/mL (r=0 .997 7) ,the recycle rate was 99 .85% -101 .47% ,and the concentration was(7 .32 ± 4 .25)μg/mL ;the result indicates that serum containing DS‐201 has activa‐tion effects on BKCa in PCASMCs ,while there was no statistical significance (P>0 .05) .Conclusion The establishment method of the alcohol extraction of Danshen is useful and reliable .The HPLC method of measuring DS‐201 concentration is precise .Choosing higher quality drugs or raising bioavailability can improve combination of the serum pharmacology and electrophysiological tech‐nique .
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Objective To compare the differences of atria and atrial myocyte structure in patients with atrial fibrillation (AF) and sinus rhythm(SR) .To evaluate the influence of AF on cardiac function .Methods 79 patients without heart failure undergoing car-diopulmonary bypass surgery were divided into the AF group (n=39) and the SR group(n=40) .Echocardiography was performed for analysis of left ventricular end diastolic dimension(LVDd) ,left ventricular end-systolic dimension(LVDs) ,left ventricular poste-rior wall(LVPW) ,interventricular septum(IVS) and left atrial diameter(LAD) .Part of left atrial appendages was taken freshly for HE staining in order to observe atrial tissue structure .Results LVDd ,LVDs ,LVPW and IVS of the AF group were lower than that of the SR group .But LAD of the AF group was higher than the SR group .There was statistical significance in IVS and LAD between the two groups(P0 .05) .Compared with the SR group ,the AF group had thinner myocardial atrophy ,more obvious fibrosis ,smaller nucleus and darker HE staining .Conclusion The incidence of AF was mainly in rheumatic heart disease .The development of AF was mostly accompanied with the enlargement of LAD and the change of atrial tissue structure which showed that AF can reduce cardiac function .
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Objective To investigate the effects of propofol on the spontaneous transient outward K+ currents in mouse cerebral arterial smooth muscle cells.Methods Kunming mice of both sexes,weighing 18-22 g,were used in this study.Vascular smooth muscle cells were freshly isolated from cerebral arteries in two steps.Five cells were chosen and studied.When holding potential was - 30 mV,spontaneous transient outward K+ currents were recorded before and after the application of 56 μmol/L propofol by perforated whole-cell patch-clamp technique.The amplitude,frequency,area under the curve and half time width of spontaneous transient outward K+ currents were analyzed.Results Propofol 56 μmol/L significantly increased the amplitude,frequency and area under the curve of spontaneous transient outward K+ currents.There was no significant change in the half time width of spontaneous transient outward K+ currents after administration,Conclusion Propofol can activate spontaneous transient outward K+ currents in mouse cerebral arterial smooth muscle cells,and thus induces vascular smooth muscle relaxation.
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AIM: To study the effect of caffeine on the large conductance calcium activated potassium (K_(Ca)) channels by patch-clamp technique on smooth muscle cells enzymatically isolated from the porcine coronary artery (PCASMC),and to investigate the effect of ryanodine on K_(Ca) being activated by caffeine.METHODS: Using the single channel patch-clamp technique,single PCASMC was isolated by collagenase,the activity of single K_(Ca) channel was recorded in porcine coronary artery smooth muscle cells.RESULTS: Caffeine (0.1-10 mmol/L) enhanced the open probability (Po) of K_(Ca) channels in a dose-dependent manner in the intracellular side of inside-out patches and its effect was almost completely abolished by washout. Caffeine decreased the mean close time markedly,but had no effect on the amplitude of K_(Ca) channels. However,ryanodine (10-40 μmol/L) decreased Po of K_(Ca) channels activated by caffeine in a dose-dependent manner in cell-attached patches. The mean open time also decreased.CONCLUSION: Caffeine directly activates K_(Ca) channels of porcine coronary artery smooth muscle cells in inside-out patches,the activity of single K_(Ca) channel is inhibited by ryanodine indirectly in cell-attached patches.
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OBJECTIVE@#In order to meet the needs of electrophysiology outer hair cells, we investigate a method to separate outer hair cells simply and effectively.@*METHOD@#The basal membrane was dissected combined with spiral ligament and axis of cochlea, then incubated with enzyme, and then mechanically triturated by micropipette.@*RESULT@#A large number of living outer hair cells were obtained. Of 80% cells could keep good condition in 6 hours.@*CONCLUSION@#If increasing the enzyme concentration slightly and the large part of cochlea without outer bone was enzymes, we can get living outer hair cells easily.
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Animals , Cell Separation , Methods , Cochlea , Cell Biology , Guinea Pigs , Hair Cells, Auditory , Cell BiologyABSTRACT
OBJECTIVE@#To investigate the degeneration mechanics of outer hair cells in guinea pig.@*METHOD@#The mechanics of outer hair cells isolated by enzyme were observed under inverted microscope for 6-8 h continuously.@*RESULT@#Over half of living outer hair cells could keep good conditions in 6 hours. During the degeneration there was always a longitudinal fold line from tip to base. Presence or absence of calcium, as well as lossing of stereociliary bundle, couldn't change the conditions of out hair cells.@*CONCLUSION@#Neither calcium nor stereociliary bundle is the decisive cause in keeping outer hair cells alive, and its degeneration may be basically related with something surrounding the cell.
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Animals , Calcium , Cells, Cultured , Cochlea , Cell Biology , Pathology , Culture Media , Guinea Pigs , Hair Cells, Auditory , Cell BiologyABSTRACT
There are some prevailing problems in the management team,such as low competency,lack of innovative ideas,lack of pioneering spirit,and serious brain drain,etc. Therefore our college has taken measures to pay close attention to the development of our management team and has made remarkable achievements with the help of assement.
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This article analyzed the current factors affecting the clinical teaching quality,proposed some measures,and discussed some aspects such as ideological recognition,implementing rules and regulations,constructing clinical teaching quality control system in order to improve the clinical teaching quality and train the high quality talents.
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<p><b>BACKGROUND</b>With the development of patch clamp and molecular biology technique, and the application of them in the investigation of tumor cellular membrane ion channnel, the ion channel is becoming the hot spot of the tumor base research gradually. The aim of this study is to investigate the electrophysiological properties of human lung adenocarcinoma cell line A-549 and the role of K+ channel in inhibition of cell proliferation by the recombinant mutant human tumor necrosis factor (rmhTNF).</p><p><b>METHODS</b>Ionic currents were recorded using the whole-cell patch clamp recording technique. The proliferation activity of A-549 cells was measured by MTT assay. The cell cycle and apoptosis rates of the carcinoma cells were measured by flow cytometric analysis (FCM).</p><p><b>RESULTS</b>Whole-cell patch clamp recording revealed a voltage-gated K+ current in A-549 cells, which could be blocked by the K+ channel blocker, TEA and CsCl. The amplitude of K+ current was markedly diminished in all cells incubated with different concentration of rmhTNF (P < 0.01). Obvious inhibitive effect of rmhTNF on proliferation of the cells was found in vitro in a dose-dependent manner (P < 0.01), the maximal inhibitory rate was 38.68% when the concentration was 400U/mL. The rmhTNF inhibited the cell cycle shifting from G1 phase to S phase and promoted apoptosis as determined by FCM analysis. The proportion of G1 cells increased from 53.02% to 72.93%, and the apoptosis rate increased from 2.08% to 8.68%. The difference were significant between the control and the high concentration groups ( 200U/mL and 400U/mL) (P < 0.01).</p><p><b>CONCLUSIONS</b>rmhTNF exerts its cytotoxic effects on A-549 cells through inhibiting cell cycle shifting and inducing apoptosis. The K+ channels on the A-549 cell membrane can be blocked by rmhTNF partly, and the effect of inhibiting proliferation and activating apoptosis on A-549 cells is a result of depression of the K+ channel.</p>
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BACKGROUND: Electrical activity of nerve cells is based on the ion channel activity on cell membrane. Epilepsy is basically characterized by abnormal neuronal discharge. The foundation is ion channel activation on cell membrane and ion transmembrane movement, however, whether Ca2+-activated K+ channel involves in epilepsy induced by Coriaria Lactone is still unclear.OBJECTIVE: Considering rat hippocampal pyramidal neurons as target,we investigate the effect of Coriaria Lactone on neuronal Ca2+-activated K+ channels in epilepsy induced by Coriaria Lactone.DESIGN: Randomized controlled experimental trials.SETTING: Department of Neurology, West China Hospital, Sichuan University and Institute. of Myocardium Electrophysiology of Luzhou Medical College.MATERIALS: This experiment was carried out in Luzhou Medical College, Sichuan Province, from May to December 2000. Totally 100 Wistar infant rats within 24-hour ages were selected.METHODS: Wistar infant rats were anaesthetized and its hippocampus was obtained under disinfected state, pyramidal neurons were cultured for 7-10 days, neurons growing well with typical shape model were colleted normal control group, 19 dishes were added with DMEM culture medium,given different membrabe voltage and then followed by adding in te3 subgroups with 8 dishes each one. Added seperately DMEM culture medium containing f0-8, 10-7, 10~ mol/L concentration of calcium ion, and 2.0 mL/L Coriaria Lactone induced epilepsy group: added with DMEM culture medium with different dosages of Coriaria Lactone and finally tetraethylamine in each concentration of 26 dishes for totally 130 dishes.Cell-attache method and inside-out method of patch-clamp technique were used to record the neuronal single channel electricity. The open probability, average opening hour and closing hour, electric current amplitude of channel were analyzed.activated K+ channels of pyramidal neurons at normal, various membrane To observe and record the influence of Coriaria Lactone on the activation of pyramidal neuronal cell membrane, as well as the role of tetraethylamine.were only small amount of pyramidal neurons randomly opening its Ca2+-activated K+ channels and it displayed obvious voltage-dependent property.The channel electric conductance was (122.79±21.68) pS. The channels the inside-out condition, Ca2+-activated K+ channel displayed calcium iondependent property. The average opening rate was 0.022±0.006, 0.040±0.007, 0.142±0.049 when the calcium concentration was 10-8, 10-7,aria Lactone could increase the opening rate of Ca2+-activated K+ channels when the free calcium ion in bath solution was 10-8 mol/L and memLactone, 1.0 mL/L Coriaria Lactone could increase the average opening time of Ca2+-activated K+ channels (1.867±0.210, 6.900±0.120, P ＜ 0.01), and reducing the average closing time (78.505±7.192,6.233±0.854, P ＜ 0.01).CONCLUSION: In epilepsy induced by Coriaria Lactone, the activation of Ca2+-activated K+ channels might play an important role of negative modulation.
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<p><b>BACKGROUND</b>Oncogenesis, development, invasion and metastasis of lung cancer are modulated by relative genes of lung cancer, and the expression, deletion or mutation of these genes are regulated by cell membrane signal transduction system and cell membrane ionic channels. The aim of this study is to explore the electrophysiological properties of human lung adenocarcinoma cell line A549 and cell proliferation affected by tetraethylammonium chloride (TEA), one potassium channel blocker, so as to know whether the voltage-gated potassium channels are required for the proliferation of A549 cell line.</p><p><b>METHODS</b>Ionic currents were recorded by whole-cell patch-clamp recording technique. The proliferation activity of A549 cells was measured by MTT assay.</p><p><b>RESULTS</b>Membrane current was observed when cells were held at -70mV and test potentials ranged from -30 to +110mV. The current exhibited properties of voltage-dependent, outward rectification and no or little inactivation over the 500ms voltage pulse. Exposure of tumor cells to 10mmol/L TEA reduced the peak outward potassium current (evoked by depolarization to +110mV) from (1057.52±59.17)pA to (212.26±11.96)pA, the ratio of suppression was 79.92% (P < 0.01). Obvious inhibitive effect of TEA with different concentrations ranged from 20 to 60mmol/L on proliferation activity of the cells was found.</p><p><b>CONCLUSIONS</b>The voltage-gated potassium channels exist in human lung adenocarcinoma cell line A549 and play a great role in proliferation of A549 cells. TEA can inhibit proliferation of A549 cell through blocking the potassium channels and the inhibition is dose-dependent.</p>
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Objecitve:To study the effects of ketamine on the large conductance Ca~(2+)-activated potassium (BK)channels in cultured newborn rat hippocampal neurons. Method:All experiments were carried out on 3-7 days old neurons. Single channel current signals were recorded in symmetrical high K~+ solution(140mM) under cell-attached patch and inside-out patch. Result:(1)In inside-out patch, with the unitary conductance being 176.5?16.5pS,the BK channel activity was markedly activated by internal Ca~(2+) but was blocked by potassium channel blocker (TEA). (2) In cellattached patch,when the concentration of ketamine was 0.01,0.05,0.20mmol/L and membrane potential kept at?60mV,the open probability (Po) decreased from 0.042?0.011 at baseline to 0.027?0.008,0.030?0.015,0.032?0.021 respectively(P0.05). Conclusion:Ketamine has dual effects on the BK channels,the inhibition may be related to its side effects,and the activation to the mechanism in anesthesia.
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AIM To study the mechanism of puerarin against arrhythmias. METHODS Standard microelectrode intracellular recording technique was used RESULTS ①Puerarin 0 005, 0 01,0 015 mmol?L -1 prolong the action potential duration at 50% and 90% of repolarization (APD 50 ,APD 90 ) respectively APD 50 was prolonged from (176 43?51 37) ms to (192 86?60 82) ms ( n=7, P
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AIM:To study the effect of caffeine on the large conductance calcium activated potassium (KCa) channels by patch-clamp technique on smooth muscle cells enzymatically isolated from the porcine coronary artery (PCASMC),and to investigate the effect of ryanodine on KCa being activated by caffeine.METHODS:Using the single channel patch-clamp technique,single PCASMC was isolated by collagenase,the activity of single KCa channel was recorded in porcine coronary artery smooth muscle cells.RESULTS:Caffeine (0.1-10 mmol/L) enhanced the open probability (Po) of KCa channels in a dose-dependent manner in the intracellular side of inside-out patches and its effect was almost completely abolished by washout. Caffeine decreased the mean close time markedly,but had no effect on the amplitude of KCa channels. However,ryanodine (10-40 ?mol/L) decreased Po of KCa channels activated by caffeine in a dose-dependent manner in cell-attached patches. The mean open time also decreased.CONCLUSION:Caffeine directly activates KCa channels of porcine coronary artery smooth muscle cells in inside-out patches,the activity of single KCa channel is inhibited by ryanodine indirectly in cell-attached patches.
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AIM: To investigate the changes of current and gene expression of acetylcholine sensitive K~+ channel (K_ ACh ) in chronic human atrial fibrillation (AF) and to evaluate the roles of K_ ACh expression in the occurring and maintenance of AF. METHODS: Acetylcholine sensitive K~+ currents (I_ KACh ) were recorded with the whole-cell patch clamp technique in single atrial myocyte of AF group and normal sinus rhythm group (SR group). The current densities-voltage relations were analyzed. The Kir3.4 mRNA was measured by reverse transcription-polymerase chain reaction. RESULTS: (1) Compared with SR group, acetylcholine sensitive K~+ current densities in AF group were reduced under testing potential between -80 mV and -120 mV . Acetylcholine sensitive K~+ current density was ( -11.665 ?1.027) pA/pF (n=11) in AF group vs ( -19.486 ?0.766) pA/pF (n=11) in SR group at -100 mV testing potential (P