微管解聚对大鼠心肌细胞自主搏动和动作电位的影响及其机制
미관해취대대서심기세포자주박동화동작전위적영향급기궤제
Effects of microtubule depolymerization on spontaneous beating and action potential of cardiac myocytes in rats and its mechanism
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2015年 3期 192-198 ,共7页

    兰晓东%党永明%李凌霏%张琼%黄跃生

    란효동%당영명%리릉비%장경%황약생

    肌细胞,心脏%微管%动作电位%延迟整流型钾离子通道电流%L型钙离子通道电流 肌細胞,心髒%微管%動作電位%延遲整流型鉀離子通道電流%L型鈣離子通道電流
    기세포,심장%미관%동작전위%연지정류형갑리자통도전류%L형개리자통도전류
    Myocytes,cardiac%Microtubules%Action potentials%Delayed rectifier K+ current%L-type Ca2+ current
    目的 观察微管解聚对大鼠心肌细胞自主搏动频率、动作电位(AP)、耗氧量的影响并探究其机制. 方法 将180只SD大鼠乳鼠分12批处理进行实验,每批取15只大鼠乳鼠处死,剪取心脏组织分离培养心肌细胞,分别接种至1个铺有6块圆形盖玻片的12孔板、1个铺有6块方形盖玻片的l2孔板、2个细胞培养瓶、2个细胞培养皿.将培养3d的各容器中细胞按随机数字表法分为正常对照组(加入3 mL 37℃复温的DMEM/F12培养液,常规培养3h)和微管解聚组(加入3 mL37℃复温的含终浓度为8 μmol/L秋水仙碱的DMEM/F12培养液,常规培养3h),每组分别3孔、1瓶、l皿.免疫荧光染色后激光扫描共聚焦显微镜下观察细胞微管形态变化,蛋白质印迹法检测细胞游离态及聚合态α微管蛋白的含量变化.倒置显微镜下观察并计算细胞自主搏动频率.氧微电极监测系统测定含有心肌细胞的DMEM/F12培养液加入秋水仙碱前后的溶解氧浓度,另测定单纯培养液和秋水仙碱+培养液溶解氧浓度.采用全细胞膜片钳记录模式记录细胞AP、延迟整流型钾离子通道电流(IK)和L型钙离子通道电流(IC.L)变化,绘制电流密度-电压(I-V)曲线.对数据行独立或配对样本t检验. 结果 (1)正常对照组细胞微管结构完整,围绕核周呈放射状分布,线性管状结构清晰.微管解聚组细胞微管结构破坏,呈现弥散性分布,线性管状结构粗糙而不光滑.(2)微管解聚组细胞游离态α微管蛋白含量为0.61±0.03,明显高于正常对照组的0.46 ±0.03,t=-6.99,P ＜0.05;聚合态oo微管蛋白含量为0.57±0.04,明显低于正常对照组的0.88 ±0.04,t=9.09,P＜0.05.(3)微管解聚组细胞自主搏动频率为(59±8)次/min,较正常对照组的(41 ±7)次/min明显增加(t =5.62,P＜0.01).(4)含心肌细胞的培养液溶解氧浓度为(138.4±2.5) μmol/L,秋水仙碱处理后下降为(121.7±3.6) μmol/L,差异明显(t=26.31,P＜0.05).单纯培养液和秋水仙碱+培养液溶解氧浓度无明显差异(t=0.72,P＞0.05).(5)与正常对照组比较,微管解聚组细胞AP形态发生明显变化,复极化平台期不明显,动作电位时程(APD)明显缩短.微管解聚组细胞的APD20、APD50、APD90分别为(36.2±3.8)、(73.7±5.7)、(ll5.1±8.0)ms,较正常对照组的(40.2±2.3)、(121.4±7.0)、(169.4 ±5.6)ms明显缩短(t值分别为2.61、15.88、16.75,P值均小于0.05).(6)微管解聚组细胞IK的I-V曲线较正常对照组上移,激活后各测试电压(0～40 mY)下微管解聚组IK电流密度均高于正常对照组(t值为2.70～ 3.76,P值均小于0.05).(7)2组细胞I.L的I-V曲线基本重叠,激活后各测试电压(-30～ 50 mV)下ICa-L电流密度相近(t值为-1.57～1.66,P值均大于0.05). 结论 微管解聚后Ik增强,ICa-L变化不明显,使得AP复极化增快,进而缩短APD,加快大鼠心肌细胞自主搏动频率,增加其耗氧量.
    목적 관찰미관해취대대서심기세포자주박동빈솔、동작전위(AP)、모양량적영향병탐구기궤제. 방법 장180지SD대서유서분12비처리진행실험,매비취15지대서유서처사,전취심장조직분리배양심기세포,분별접충지1개포유6괴원형개파편적12공판、1개포유6괴방형개파편적l2공판、2개세포배양병、2개세포배양명.장배양3d적각용기중세포안수궤수자표법분위정상대조조(가입3 mL 37℃복온적DMEM/F12배양액,상규배양3h)화미관해취조(가입3 mL37℃복온적함종농도위8 μmol/L추수선감적DMEM/F12배양액,상규배양3h),매조분별3공、1병、l명.면역형광염색후격광소묘공취초현미경하관찰세포미관형태변화,단백질인적법검측세포유리태급취합태α미관단백적함량변화.도치현미경하관찰병계산세포자주박동빈솔.양미전겁감측계통측정함유심기세포적DMEM/F12배양액가입추수선감전후적용해양농도,령측정단순배양액화추수선감+배양액용해양농도.채용전세포막편겸기록모식기록세포AP、연지정류형갑리자통도전류(IK)화L형개리자통도전류(IC.L)변화,회제전류밀도-전압(I-V)곡선.대수거행독립혹배대양본t검험. 결과 (1)정상대조조세포미관결구완정,위요핵주정방사상분포,선성관상결구청석.미관해취조세포미관결구파배,정현미산성분포,선성관상결구조조이불광활.(2)미관해취조세포유리태α미관단백함량위0.61±0.03,명현고우정상대조조적0.46 ±0.03,t=-6.99,P ＜0.05;취합태oo미관단백함량위0.57±0.04,명현저우정상대조조적0.88 ±0.04,t=9.09,P＜0.05.(3)미관해취조세포자주박동빈솔위(59±8)차/min,교정상대조조적(41 ±7)차/min명현증가(t =5.62,P＜0.01).(4)함심기세포적배양액용해양농도위(138.4±2.5) μmol/L,추수선감처리후하강위(121.7±3.6) μmol/L,차이명현(t=26.31,P＜0.05).단순배양액화추수선감+배양액용해양농도무명현차이(t=0.72,P＞0.05).(5)여정상대조조비교,미관해취조세포AP형태발생명현변화,복겁화평태기불명현,동작전위시정(APD)명현축단.미관해취조세포적APD20、APD50、APD90분별위(36.2±3.8)、(73.7±5.7)、(ll5.1±8.0)ms,교정상대조조적(40.2±2.3)、(121.4±7.0)、(169.4 ±5.6)ms명현축단(t치분별위2.61、15.88、16.75,P치균소우0.05).(6)미관해취조세포IK적I-V곡선교정상대조조상이,격활후각측시전압(0～40 mY)하미관해취조IK전류밀도균고우정상대조조(t치위2.70～ 3.76,P치균소우0.05).(7)2조세포I.L적I-V곡선기본중첩,격활후각측시전압(-30～ 50 mV)하ICa-L전류밀도상근(t치위-1.57～1.66,P치균대우0.05). 결론 미관해취후Ik증강,ICa-L변화불명현,사득AP복겁화증쾌,진이축단APD,가쾌대서심기세포자주박동빈솔,증가기모양량.
    Objective To explore the effects of microtubule depolymerization (MD) on the spontaneous beating rate,action potential (AP),and oxygen consumption of eardiac myocytes in rats and its mechanism.Methods One-hundred and eighty neonatal SD rats divided into 12 batches were used in the experiment,and 15 rats in each batch were sacrificed for the isolation and culture of cardiac myocytes after the heart tissues were harvested.The cardiac myocytes were respectively inoculated in one 12-well plate filled with 6 round cover slips,one 12-well plate filled with 6 square cover slips,two cell culture flasks,and two cell culture dishes.After routine culture for three days,the cardiac myocytes from all the containers were divided into normal control group (NC,routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 ℃ for 3 h) and group MD (routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 ℃ and containing 8 μmol/L colchicine for 3 h) according to the random number table,with 3 holes,1 flask,or 1 dish in each group.The morphological changes in microtubules were observed with confocal laser scanning microscope after immunofluorescent staining.The content of polymerized or dissociative α-tubulin was determined by Western blotting.Spontaneous beating rate of the cells was observed and calculated under inverted microscope.Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was determined by oxygen microelectrode system before and after the addition of colchicine.Additionally,dissolved oxygen concentration of DMEM/F12 solution and colchicine + DMEM/F12 solution was determined.The whole-cell patch-clamp technique was used to record AP,delayed rectifier K + current (IK),and L-type Ca2+ current (ICa-L.) in cardiac myocytes;current density-voltage (I-V) curves were drawn based on the traces.Data were processed with independent or paired samples t-test.Results (1) In group NC,microtubules of cardiac myocytes were around the nucleus in radial distribution with intact and clear linear tubiform structure.The microtubules in group MD were observed in dispersive distribution with damaged structure and rough linear tubiform structure.(2) In group MD,the content of dissociative α-tubulin of cells (0.61 ± 0.03) was obviously higher than that in group NC (0.46 ± 0.03,t =-6.99,P ＜ 0.05),while the content of polymerized α-tubulin (0.57 ± 0.04) was significantly lower than that in group NC (0.88 ± 0.04,t =9.09,P ＜0.05).(3) Spontaneous beating rate of cells was (59±8) times per min in group MD,which was distinctly higher than that in group NC [(41 ±7) times permin,t =5.62,P ＜0.01].(4) Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was (138.4 ±2.5) μmol/L,and it was reduced to (121.7±3.6) μmol/L after the addition ofcolchicine (t =26.31,P ＜0.05).There was no obvious difference in dissolved oxygen concentration between DMEM/F12 solution and colchicine + DMEM/F12 solution (t =0.72,P ＞0.05).(5) Compared with that of group NC,AP morphology of cells in group MD changed significantly,with unobvious repolarization plateau phase and shorter action potential duration (APD).The APD20,APD50,and APD90 were respectively (36.2 ± 3.8),(73.7 ± 5.7),and (115.1 ±8.0) ms in group MD,which were significantly shorter than those of group NC [(40.2 ±2.3),(12 1.4 ± 7.0),and (169.4 ± 5.6) ms,with t values respectively 2.61,15.88,and 16.75,P values below 0.05].(6) Compared with that of group NC,the I-V curve of IK of cells in group MD moved up with higher current density under each test voltage (0 to 40 mV) after activation (with t values from 2.70 to 3.76,P values below 0.05).(7) There was not much alteration in current density of ICa-L under each test voltage (-30 to 50 mV) between2 groups (withtvalues from-1.57 to 1.66,Pvalues above0.05),and their I-V curves were nearly overlapped.Conclusions After MD,the IK is enhanced without obvious change in ICa.L,making AP repolarization faster and APD shortened.Then the rapid spontaneous beating rate increases oxygen consumption of cardiac myocytes of rats.
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