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Objective To establish a method to isolate single sinoatrial node cells of adult rats and observe the cells'morphological structure and the expression of sodium channel subtype so as to provide experimental basis for further research on the role of sodium channel in sinoatrial node function.Methods We isolated adult rat sinoatrial node tissue and cut it into slices about 2 mm in width,digested the slices with type Ⅱ collagenase combinate protease, and observed the cells' morphological structure. We then performed immunofluorescence staining with HCN4 and laser confocal imaging to identify cells and observe the expressions of Nav1 .1 ,Nav1 .2 , Nav1.3,Nav1.5,Nav1.6,Nav1.7,Nav1.8 and Nav1.9.Results The isolated cells had spindle-like,arc-like, and slender and curved shapes,but were mainly spindle-shaped.The stripes of the cells were clear,had good bioactivity and could survive 6-8 hours.Sodium channels Nav1.1,Nav1.5,Nav1.6,Nav1.7,Nav1.8,and Nav1.9 were positively expressed in adult rat sinoatrial node cells,while Nav1 .2 and Nav1 .3 were negatively expressed. Conclusion The method of digesting and isolating adult rat sinoatrial node cells with type Ⅱ collagenase combinate protease is reliable,and sodium channel subtypes are differently expressed in sinoatrial node cells.
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Objective To establish a method to isolate single sinoatrial node cells of adult rats and observe the cells'morphological structure and the expression of sodium channel subtype so as to provide experimental basis for further research on the role of sodium channel in sinoatrial node function.Methods We isolated adult rat sinoatrial node tissue and cut it into slices about 2 mm in width,digested the slices with type Ⅱ collagenase combinate protease, and observed the cells' morphological structure. We then performed immunofluorescence staining with HCN4 and laser confocal imaging to identify cells and observe the expressions of Nav1 .1 ,Nav1 .2 , Nav1.3,Nav1.5,Nav1.6,Nav1.7,Nav1.8 and Nav1.9.Results The isolated cells had spindle-like,arc-like, and slender and curved shapes,but were mainly spindle-shaped.The stripes of the cells were clear,had good bioactivity and could survive 6-8 hours.Sodium channels Nav1.1,Nav1.5,Nav1.6,Nav1.7,Nav1.8,and Nav1.9 were positively expressed in adult rat sinoatrial node cells,while Nav1 .2 and Nav1 .3 were negatively expressed. Conclusion The method of digesting and isolating adult rat sinoatrial node cells with type Ⅱ collagenase combinate protease is reliable,and sodium channel subtypes are differently expressed in sinoatrial node cells.
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Objective To observe the effects of ferulic acid on cell apoptosis andβ-tubulinin vitro of rabbit sinoatrial node injured by hypoxia and low glucose, and to explore its mechanism.Methods Model cells were established on sinoatrial node cells from newborn rabbit by performing deprivation of oxygen and glucose. Then the model cells were divided into 5 groups: a normal control group, a model group, ferulic acid high, medium and low dose group. The normal control group and the model group were treated with equal volume of culture medium, and the three volume ferulic acid groups were treated with ferulic acid of 100, 20, and 10μg/ml respectively. Flow cytometry, laser scanning confocal microscopy were used to observe the sinoatrial node apoptosis rate and cytoskeleton proteinβ-tubulin in each group.Results Apoptosis rate of the model group was obviously higher than the normal group (56.95% ± 11.99%vs. 31.45% ± 6.32%,P<0.01), whileβ-tubulin cleavage was significantly lower than the normal group (5.141 ± 0.218vs. 8.035 ± 0.762,P<0.01). Apoptosis rate of ferulic acid high, medium dose group were significantly lower than the model group (24.85% ± 6.34%, 26.70% ± 9.84%vs. 56.95% ± 11.99%,P<0.01),β-tubulin structure were significantly more complete compared with the model group (7.927 ± 0.357, 5.961 ± 0.351vs. 5.141 ± 0.218,P<0.01).Conclusions Ferulic acid can suppress apoptosis of sinoatrial node cells caused by low glucose and oxygen. Protecting the sinoatrial node cell skeleton protein ofβ-tubulin may be one of the mechanisms.
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Objective To observe the effects of Tongyang Huoxue Recipe on cytoskeleton proteinβ-tubulin of ischemia reperfusion injured sinoatrial node cell in rabbits;To discuss its mechanisms in the treatment of sick sinus syndrome. Methods Sinoatrial node cells were obtained from newborn rabbit. Oxygen and glucose were deprived to simulate ischemia and were restored to simulate reperfusion. Cells were divided into 5 groups. Tongyang Huoxue Recipe high-, medium-, low dose groups were given corresponding medicine (final concentrations of 100, 20, 10 μg/mL). The normal group and model group were given equal volume of culture medium. Enzyme mark instrument and laser scanning confocal microscopy were used to observe the sinoatrial node cell activity and cytoskeleton protein β-tubulin of each group. Results Living cells of model group decreased significantly compared with normal group (P<0.01), and cytoskeleton proteinβ-tubulin cleavage significantly. Living cells of Tongyang Huoxue Recipe high, medium and low dose group were significantly higher than those in the model group (P<0.05), and β-tubulin structure remained more complete than the model group. Conclusion Tongyang Huoxue Recipe can inhibit the injury induced by ischemia reperfusion of sinoatrial node cells;the possible mechanism of Tongyang Huoxue Recipe in treating sick sinus syndrome is protecting activity and cytoskeleton proteinβ-tubulin of sinoatrial node cells.
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Objective To explore the regularity of dynamic morphologic changes of the sinoatrial node (SAN) cells of rabbits after the constitution was damaged by formaldehyde wet dressing. Methods Sixty rabbits were randomly divided into 6 groups (n=10 in each group): the control group and 5 experimental groups observed at 2 h, 1, 2, 3, and 4 weeks respectively after the operation. The SAN constitution of rabbits was damaged by 20% formaldehyde wet dressing. The constitutions of SAN were cut quickly at 2 h after wet dressing in the control group and at the corresponding time points in other experimental groups respectively. The specimens were made for light microscopical and electron microscopical observations. The apoptosis of SAN cells was detected by TUNEL method. Results Compared with those in the control group, SAN cells in all experimental groups were damaged at various degrees observed by light microscopy and electron microscopy. Distinct cell tumefaction, soak of grain cells, cytoclasis, hyperplasia of collagen fibers, and other pathological changes were detected. Electron microscopical observation showed distinct pathological changes of the super-micro construction of cells. Apoptosis was not detected in the control group, but various degrees of apoptosis in all experiment groups. The apoptotic rates in all experimental groups were significant as compared with that in the control group (P
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Objective To study the effects of mimic ischemic preconditioning (IP) on i and I-LCa of sinoatrial node cells and explore the protective effects of IP. Methods Cells were randomized to three groups: control, ischemia/reperfusion (I/R), IP. After labeled with fura4, fluorescence intensity of i was studied with laser confocal microscopy and I-LCa was recorded by whole-cell patch clamp technology. Results IP significantly reduced the fluorescence intensity of i and enhanced the peak of I-LCa as compared with I/R, shifted the I-V curve to more negative value. Conclusion IP can reduce the overload of i caused by I/R and increase I-LCa weakened by I/R.
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Objective To compare the effects of different ischemic preconditioning (IP) protocols on the viability of primary cultured sinoatrial node cells from neonatal rats for investigating the protective effect of IP. Methods The cells were randomized into eleven groups: control, ischemia/reperfusion (I/R), IP1 (preconditioned with ischemia/reperfusion for 5 min), IP2 (preconditioned with 2 cycles of ischemia/ reperfusion for 5 min), IP3 (preconditioned with 3 cycles of ischemia/reperfusion for 5 min), IP4 (preconditioned with ischemia/reperfusion for 10 min), IP5 (preconditioned with 2 cycles of ischemia/reperfusion for 10 min), IP6 (preconditioned with 3 cycles of ischemia/reperfusion for 10 min), IP7 (preconditioned with ischemia/reperfusion for 20 min), IP8 (preconditioned with 2 cycles of ischemia/reperfusion for 20 min), and IP9 (preconditioned with 3 cycles of ischemia/reperfusion for 20 min). PI positive staining rate and changes of D 490 after 3 h ischemia/4 h reperfusion were determined by PI staining and MTT chromatometry. Results ① D 490 value in sinoatrial node cells in each experimental group was significantly lower than that in the control group, but that in IP2, IP3, IP4, IP5, and IP7 groups was significantly higher than that in I/R group (P
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Objective To compare the effects of stimulated ischemic preconditioning (IP) and ischemia/reperfusion (I/R) on the hyperpolarization-activated current (I_ f ) of sinoatrial node cells and explore the mechanisms of electrophysiological protection of IP. Methods The sinoatrial node cells cultured for 2 d were randomized to three groups: ① Control in which the cells were cultured at 37 ℃ in the mimic reperfusion solution, meanwhile the mixture of 95% O_ 2 and 5% N_ 2 was ventilated; ② I/R in which the cells were cultured in the mimic ischemic solution and simultaneously the mixture of 95% O_ 2 and 5% N_ 2 was ventilated for 3 h, then cultured in the mimic reperfusion solution and simultaneously the mixture of 95% O_ 2 and 5% N_ 2 was ventilated for 4 h; ③ IP in which the cells underwent ischemia for 10 min and reperfusion for 10 min, repeated once. Then the cells were treated as the same as the I/R group. After 4-hour reperfusion, I_ f was recorded by whole-cell patch clamp technology. Results I/R significantly enhanced the current density of I_ f , shifted the current activation curve to more positive value by changing the half-activation voltage from (-98.9?2.4) mV to (-85.2?2.5) mV (P
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Objective To understand morphological characteristics of sinoatrial node cells(SNCs) by taking purified isolation and culture of neonatal rat's SNCs in vitro and to understand the expression characteristics of surface antigens hyperpolarization activated cyclic nucleotide gated cation channel 4(HCN4) and connexin 45(Cx45) on SNCs.Methods The SNCs were isolated from sinus node tissues removed from neonatal rats by trypsin and purified cultured with differential attachment and 5'-bromodeoxyuridine(BrdU) treatment.Results Three different types of cells were observed among the purified cultured SNCs: spindle,triangular and irregular cells.The spindle cells took up the greatest proportion,had the fastest beats,and had less developed organelle and scarce muscular fibrils.Immunofluorescence staining was positive against HCN4 and Cx45 on the spindle cells.Small amounts of HCN4 was observed on triangular cells.Conclusion ① Isolation by trypsin combined with differential attachment and BrdU treatment is a reliable approach to culturing sinoatrial node cells.② The spindle cells with the fastest beats and positive expressions of HCN4 and Cx45 may be the sinus node pacemaker cells.