【作者】 来利红；

【导师】 蒋文平；

【作者基本信息】 苏州大学 ， 心内科， 2009， 博士

【摘要】 目的:二十二碳六烯酸（DHA）在预防、减少心血管疾病发生,尤其是在抗心律失常、扩张血管、降低血压、增加冠状动脉血流量及预防心源性猝死等方面的有益作用受到广泛重视。但DHA引起的对心血管有益作用的机制尚不完全清楚。本研究拟采用膜片技术探讨DHA对大鼠心室肌细胞静息电位（RP）、动作电位（AP）、离子通道动力学以及对冠状动脉平滑肌细胞离子通道动力学的影响,旨在从细胞离子通道电生理水平上阐述DHA起到的抗心律失常、扩张血管等心血管有益作用的可能机制。为临床使用DHA提供理论依据。方法:（1）采用“四步”酶消化法,即无钙台氏液灌流、50μmol/L低钙酶液灌流、200μmol/L低钙台氏液灌流和室温下KB液中孵育。分离大鼠心室肌细胞后,记录正常大鼠心室肌细胞RP、AP、钠离子电流(I_Na)、L-型钙离子电流(I_Ca-L)、瞬时外向钾离子电流(I_to)、延迟整流性钾离子电流（I_k）和内向整流性钾离子电流(I_kl)。分别加入20、40、60、80、100和120μmol/L DHA,观察不同浓度DHA对RP、AP、I_Na、I_Ca-L、I_to、I_k和I_kl影响。（2）采用“三步”酶消化法,即含0.125%牛血清白蛋白（Bovine serum albumin,BSA）缓冲液室温孵育10分钟,酶液Ⅰ消化20分钟,酶液Ⅱ消化15分钟。分离大鼠冠状动脉平滑肌细胞后,记录正常大鼠冠状动脉平滑肌细胞大电导钙激活性钾(BK_Ca)电流、电压依赖性钾（K_v）电流。分别加入10、20、40、60和80μmol/L DHA,观察不同浓度DHA对IBK_Ca、IK_v影响结果:（1）通过“四步”酶消化法,可获得70%～90%的耐钙细胞。（2）正常大鼠心室肌细胞RP、AP和离子流:①心室肌细胞RP分别为-75.96±4.52mV（n=100）。②心室肌细胞AP最大上升速率（Vmax）、动作电位幅度（APA）和超射（OS）分别为226.37±15.36V/s、116.59±11.63mV、31.79±6.35mV（n=50）。③复极25%、50%和90%动作电位时程(APD₂₅、APD₅₀和APD₉₀)分别为4.85±1.36ms、11.76±2.23ms和52.81±5.33ms（n=50）。④I_Na:指令电压-30mV时,峰值电流为-1183.71±315.62pA,相应电流密度为-7.86±2.11pA/pF（n=50）;稳态激活的半激活电压V_1/2=-44.12±2.68mV;稳态失活的半失活电压V_1/2=-81.52±2.07mV;失活后恢复的快、慢时间常数分别为τ₁=0.66±0.32ms和τ₂=10.56±2.51ms。⑤I_Ca-L:指令电压0mV时,峰值电流为-949.52±213.51pA,相应电流密度为-6.30±1.42 pA/pF（n=60）;稳态激活的半激活电压V_1/2=-16.67±2.46mV;稳态失活的半失活电压V_1/2=-34.03±0.57mV;失活后恢复的时间常数为91.41±10.26 ms。⑥I_to指令电压+70mV时,峰值电流为4936.52±653.61pA,相应电流密度为32.91±4.36 pA/pF（n=50）;稳态激活的半激活电压V_1/2=36.39±4.17mV;稳态失活的半失活电压V_1/2=-25.68±1.01mV;失活后恢复时间常数为83.07±10.25ms。⑦I_k:指令电压+60mV时,峰值电流大小为1530.15±512.2pA,相应电流密度为10.20±3.41pA/pF（n=30）;稳态激活的半激活电压V_1/2=35.71±3.81mV;稳态失活的半失活电压V_1/2=-33.36±1.07mV;失活后恢复时间常数为168.18±16.67ms。⑧I_kl峰值电流和电流密度:指令电压-120mV时,I_kl为-4735.5±721.6pA,相应电流密度为-31.57±4.81pA/pF（n=28）。（3）DHA对心室肌细胞基本电生理影响:加入0、20、40、60、80、100和120μmol/LDHA,RP、Vmax、APA和OS差异无显著性（P＞0.05,n=10）,但加入上述浓度DHA后,APD₂₅分别为4.85±1.36ms、5.76±1.92ms、7.28±2.21ms、10.97±3.25ms、13.26±4.19ms、14.35±4.96ms和15.83±5.26ms（P＜0.05,n=10）;APD₅₀分别为11.76±2.23ms、13.21±3.02ms、14.59±3.37ms、18.92±4.16ms、20.53±4.77ms、22.19±5.34ms和23.87±5.66ms（P＜0.05,n=12）;APD₉₀分别为52.81±5.33ms、56.64±5.73ms、59.16±6.11ms、88.45±8.92ms、103.37±10.25ms、121.57±11.81ms和133.65±12.34ms（P＜0.05,n=10）。（4）DHA对心室肌细胞离子流影响:加入20、40、60、80、100和120μmol/L DHA后:①I_Na呈浓度依赖性阻滞、I-V曲线上移、稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响。在指令电压-30mV时,上述浓度DHA对I_Na阻滞分别为1.51±1.32%、21.13±4.62%、51.61±5.73%、67.62±6.52%、73.49±7.59%和79.95±7.62%（P＜0.05,n=10）,DHA对I_Na阻滞半效抑制浓度(IC₅₀)为47.91±1.57μmol/L。②I_Ca-L呈浓度依赖性阻滞、I-V曲线上移、稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响。在指令电压0mV时,上述浓度DHA对I_Ca-L阻滞分别为2.72±1.63%、21.97±3.35%、44.16±4.59%、67.89±4.87%、70.51±5.39%和72.32±5.57%（P＜0.05,n=10）,DHA对I_Ca-L抑制的IC₅₀为52.01±3.24μmol/L。③I_to呈浓度依赖性阻滞、I-V曲线下移、稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响。在指令电压+70mV时,上述浓度DHA对I_to阻滞分别为2.61±0.26%、21.79±4.85%、63.11±6.57%、75.52±7.26%、81.82±7.63%和84.33±8.25%（P＜0.05,n=10）,DHA对I_to抑制的IC₅₀为49.11±2.68μmol/L。④I_K呈浓度依赖性阻滞、I-V曲线下移、稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响。在指令电压+60mV时,上述浓度DHA对I_K阻滞分别为2.78±0.26%、27.23±3.97%、64.18±6.73%、77.59±7.36%、83.26±8.31%和87.93±9.35%（P＜0.05,n=10）,DHA对I_K抑制的IC₅₀为47.52±2.32μmol/L。⑤I_Kl峰值电流、I-V曲线无影响（P＞0.05,n=10）。（5）正常大鼠冠状动脉平滑肌细胞IBK_Ca和IK_V:①单通道BK_Ca:在钳制电压分别为+20、+40、+60、+80和+100mV,通道开放概率（Po）分别为:0.009±0.001、0.017±0.002、0.072±0.003、0.362±0.043和0.637±0.071（P＜0.05,n=10）,半激活电压及斜率分别为79.47±4.68mV和8.53±0.14。IBK_Ca单通道电导值为230.42±20.13pS（n=10）。②全细胞BK_Ca:指令电压+150mV时,BK_Ca峰值电流大小为784.81±261.63pA,相应电流密度为68.24±22.75pA/pF（n=20）。在指令电压+90mV,BK_Ca尾电流大小为1046.73±113.67pA,相应电流密度为91.02±13.52pA/pF（n=20）。③全细胞K_V:指令电压+50mV时,K_V峰值电流大小为553.85±106.77pA,相应电流密度为48.16±9.28pA/pF（n=20）。在指令电压+40mV,K_V尾电流大小为388.24±81.26pA,相应电流密度为33.76±7.68pA/pF（n=20）。（6） DHA对大鼠冠状动脉平滑肌细胞IBK_Ca和IK_V影响:①加入0、10、20、40、60和80μmol/L的DHA后,在钳制电压+60mV时,单通道BK₍Ca的Po分别为:0.072±0.003、0.077±0.004、O.136±0.070、0.436±0.083、0.592±0.109和0.676±0.115（P＜0.05,n=10）。DHA对单通道BK_Ca开放激活的IC₅₀为36.30±2.15μmol/L。在钳制电压分别为+20、+40、+60、+80和+100mV时,35μmol/LDHA作用前后的半激活电压及斜率分别为79.47±4.68mV、8.53±0.14和69.16±3.57mV、10.16±0.20,DHA使通道开放相对概率与钳制电压关系曲线左移。②加入10、20、40、60和80μmol/L的DHA后,IBK_Ca和BK_Ca尾电流均呈浓度依赖性增加,IBK_Ca电流密度分别为72.40题部量.49、120.44±37.96、237.48±53.22、323.60±74.83和370.61±88.16pA/pF。BK_Ca尾电流密度分别为100.23±17.34、224.02±38.76、369.19±65.39、511.39±82.77和700.14±96.64A/pF。IBK_CaI-V曲线和BK_Ca尾电流I-V曲线均上移,对稳态激活曲线无影响。在指令电压+150mV时,上述浓度DHA对IBK_Ca增加分别为6.1±0.3%、76.5±3.8%、248.0±12.3%、374.2±18.7%和443.1±22.1%（P＜0.05,n=10）。DHA对IBK_Ca激活的IC₅₀为36.22±2.17μmol/L。在测试电压+90mV时,上述浓度DHA对BK_Ca尾电流增加分别为10.12±0.33%、146.12±4.97%、305.62±9.89%、461.85±14.92%和669.22±20.37%（P＜0.05,n=10）。③加入10、20、40、60和80μmol/L的DHA后,IK_V和K_V尾电流均呈浓度依赖性抑制,IK_V电流密度分别为45.96±7.63、42.97±5.01、31.15±3.87、14.33±2.94和10.35±2.17 pA/pF。K_V尾电流密度分别为31.51±6.72、28.62±5.13、19.04±3.43、8.85±1.76和5.73±1.02 pA/pF。IK_V I-V曲线和K_V尾电流I-V曲线均下移,稳态激活曲线右移、稳态失活曲线左移。在指令电压+50mV时,上述浓度的DHA对IK_V的抑制分别为4.57±0.23%、10.77±0.54%、35.32±1.76%、70.25±3.51%和78.51±3.93%（P＜0.05,n=10）,DHA对IK_V抑制的IC₅₀为42.19±1.59μmol/L。在指令电压+40mV时,上述浓度的DHA对IK_V尾电流的抑制分别为6.65±0.27%、15.22±2.76%、43.59±7.82%、73.80±11.79%和83.02±13.65%（P＜0.05,n=10）。结论:（1）对大鼠心室肌细胞电生理影响:①DHA使APD₂₅、APD₅₀、APD₉₀延长,对Vmax、APA、OS无影响。②DHA对I_Na、I_Ca-L、I_to、I_K均有阻滞作用,使这些通道电流稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响。③DHA对I_KI电流大小和通道动力学参数均无影响。④DHA具备多通道阻滞,从而对预防心律失常和心源性猝死起有益作用。（2）对大鼠冠状动脉平滑肌细胞电生理影响:①DHA对单通道BK_Ca有激活作用,使单通道BK_Ca开放相对概率与钳制电压关系曲线左移。②DHA对全细胞BK_Ca有激活作用,对稳态激活曲线无影响。③DHA对全细胞K_V有抑制作用,稳态激活曲线右移、稳态失活曲线左移。④DHA激活BK_Ca通道作用大,抑制K_V通道作用小,综合电生理影响增加冠脉复极电流,从而起舒血管作用。更多还原

【Abstract】 Objective Recently,more attentions have been paid on the beneficial effects of docosahexaenoic acid（DHA） on prevention and reduction of cardiovascular diseases, especially in anti-arrhythmia,vasodilatation,lowering blood pressure,improvement of coronary artery blood flow,prevention of sudden death,and so on.However,the molecular mechanisms underlying thatω-3 PUFAs exert their cardioprotective effects are not fully understood.The study was to investigate DHA effects on resting potentials（RP）,action potentials （AP）,ionic currents of rat ventricular myocytes and effects on ionic currents of rat in rat coronary artery smooth muscle cells（CASMCs） by patch clamp technique.Investigating DHA mechanisms from the level of cell,molecule,and ion can provide theoretical evidences for applying rationally in clinical practice.Methods（1） RP,AP,sodium current(I_Na),L-type calcium current(I_Ca-L),transient outward potassium current(I_to),delayed rectification potassium current（I_k）,and inwardly rectified potassium current(I_kl) of normal rat ventricular myocytes were respectively recorded by patch clamp after "four-step" enzyme digestion method,i.e.,perfusion with Tyrode’s solution without Ca²⁺,perfusion with 50μmol/L Ca²⁺,perfusion with 200μmol/L Ca²⁺,incubation with KB solution in room temperature.Effects on RP,AP,I_Na,I_Ca-L,I_to,I_k and I_kl were observed by addition of 20,40,60,80,100 and 120μmol/L DHA respectively.（2） BK_Ca and K_V currents in individual CASMC were recorded by patch-clamp technique after CASMCs were isolated by "three-step" enzyme digestion,i.e.,incubated in buffer soluition containing 0.125%BSA for about 10min at room temperature,digested in enzymeⅠfor about 20min and then for about 15 min in enzymeⅡ.Effects on BK_Ca and K_V currents were studied by addition of 10,20,40,60 and 80μmol/L DHA respectively.Results（1） 70～90%calcium-tolerant ventricular myocytes from rat were obtained by "four-step" enzyme digestion method.（2） RP,AP and ionic currents of normal rat ventricular myocytes:a.RP of ventricular myocytes was -75.96±4.52mV（n=100）.b.AP maximal velocity（Vmax）,AP amplitude （APA） and AP overshoot（OS） were 226.37±15.36V/s,116.59±11.63mV,31.79±6.35mV （n=50） respectively,c.25%,50%and 90%of action potential durations(APD₂₅,APD₅₀, and APD₉₀) of ventricular myocytes were 4.85±1.36ms,11.76±2.23ms and 52.81±5.33ms（n=50）,respectively,d.I_Na:I_Na currents at -30mV were-1183.71±315.62pA, and corresponding current densities were -7.86±2.11pA/pF（n=50）.50%stably activated potential was -44.12±2.68mV;50%stably inactivated potential was -81.52±2.07mV;the fast and slow recovered time from inactivation wasτ₁=0.66±0.32ms,τ₂=10.56±2.51ms.e. I_Ca-L:I_Ca-L currents at 0mV were-949.52±213.51pA,and corresponding current densities were -6.30±1.42 pA/pF（n=60）.50%stably activated potential was-16.67±2.46mV;50% stably inactivated potential was -34.03±0.57mV;recovered time from inactivation was 91.41±10.26 ms.f.I_to:I_to currents at +70mV were 4936.52±653.61pA,and corresponding current densities were 32.91±4.36 pA/pF（n=50）.50%stably activated potential was36.39±4.17 mV;50%stably inactivated potential was -25.68±1.01 mV;recovered time from inactivation was 83.07±10.25ms.g.I_k:I_k currents at +60mV were 1530.15±512.2pA, and corresponding current densities were10.20±3.41pA/pF（n=30）.50%stably activated potential was35.71±3.81mV;50%stably inactivated potential was -33.36±1.07mV; recovered time from inactivation was 168.18±16.67ms.h.Peak currents and current densities of I_kl:I_kl currents at -120mV were -4735.5±721.6pA,and corresponding current densities were -31.57±4.81pA/pF（n=28）.（3） Effects on basic electrophysiology of rat ventricular myocytes by DHA:RP,Vmax, APA and OS were not significant（P＞0.05,n=10）,but APD changed after application 20, 40,60,80,100 and 120μmol/L DHA.APD₂₅ were 4.85±1.36ms,5.76±1.92ms, 7.28±2.21ms,10.97±3.25ms,13.26±4.19ms,14.35±4.96ms and 15.83±5.26ms respectively （P＜0.05,n=10）.APD₅₀ were 11.76±2.23ms,13.21±3.02 ms,14.59±3.37ms,18.92±4.16ms, 20.53±4.77ms,22.19±5.34ms and 23.87±5.66ms respectively（P＜0.05,n=12）.APD₉₀ were 52.81±5.33ms,56.64±5.73ms,59.16±6.11ms,88.45±8.92ms,103.37±10.25ms, 121.57±11.81ms and 133.65±12.34ms respectively（P＜0.05,n=10）.（4） Effects on ionic currents of ventricular myocytes by DHA:when 20,40,60,80,100 and 120μmol/L DHA were gradually applicated:a.I_Na were gradually blocked,Ⅰ-Ⅴcurves were upward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged（P＜0.05,n=10）,and stably activated curves were no remarkable significance（P＞0.05,n=10）.I_Na was blocked to 1.51±1.32%,21.13±4.62%, 51.61±5.73%,67.62±6.52%,73.49±7.59%and 79.95±7.62%under manding potential equal to -30mV（P＜0.05,n=10）,and 50%inhibition concentration(IC₅₀) of DHA was 47.91±1.57μmol/L.c.I_Ca-L were gradually blocked,Ⅰ-Ⅴcurves were upward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged with augmentation of DHA（P＜0.05,n=10）,and stably activated curves were no remarkable significance（P＞0.05,n=10）.I_Ca-L was blocked to 2.72±1.63%,21.97±3.35%, 44.16±4.59%,67.89±4.87%,70.51±5.39%and 72.32±5.57%under manding potential equal to 0mV（P＜0.05,n=10）,and IC₅₀ of DHA was 52.01±3.24μmol/L.d.I_to were gradually blocked,Ⅰ-Ⅴcurves were downward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged with augmentation of DHA （P＜0.05,n=10）,and stably activated curves were no remarkable significance（P＞0.05, n=10）.I_to was blocked to 2.61±0.26%,21.79±4.85%,63.11±6.57%,75.52±7.26%, 81.82±7.63%and 84.33±8.25%under manding potential equal to +70mV（P＜0.05,n=10）, and IC₅₀ of DHA was49.11±2.68μmol/L,e.I_K were gradually blocked,Ⅰ-Ⅴcurves were downward,stably inactivated curves were shifted to the left,and recovered time from inactivation was prolonged with augmentation of DHA（P＜0.05,n=10）,and stably activated curves were no remarkable significance（P＞0.05,n=10）.I_to was blocked to 2.78±0.26%,27.23±3.97%,64.18±6.73%,77.59±7.36%,83.26±8.31%and 87.93±9.35% under manding potential equal to +60mV（P＜0.05,n=10）,and IC₅₀ of DHA was47.52±2.32μmol/L,f.DHA at different concentrations did not have any effect on I_Kl （P＞0.05,n=10）.（5） BK_Ca and K_V currents of normal rat CASMCs:a.Open probability（Po） of BK_Ca channel in individual CASMCs were observed using a patch-clamp technique in an inside-out configuration.Under manding potential equal to +20,+40,+60,+80 and +100mV,Po of BK_Ca channel were 0.009±0.001,0.017±0.002,0.072±0.003,0.362±0.043 and 0.637±0.071 respectively（P＜0.05,n=10）,and half activated voltage and slope rate of BK_Ca channel was 79.47±4.68mV and 8.53±0.14.Conductance of BK_Ca channel was 230.42±20.13pS（n=10）.b.Peak currents and current densities of IBK_Ca under whole-cell configuration:BK_Ca currents at +150mV were 784.81±261.63pA,and corresponding current densities were 68.24±22.75pA/pF（n=20）.BK_Ca tail currents at +90mV were 1046.73±113.67pA,and corresponding current densities were 91.02±13.52pA/pF（n=20）.d. Peak currents and current densities of IK_V under whole-cell configuration:K_V currents at +50mV were 553.85±106.77pA,and corresponding current densities were 48.16±9.28pA/pF（n=20）.K_V tail currents at +40mV were 388.24±81.26pA,and corresponding current densities were 33.76±7.68pA/pF（n=20）（6） Effects on BK_Ca and K_V currents of normal rat CASMCs by DHA:a.Po of BK_Ca channel at +60mV were 0.072±0.003,0.077±0.004,0.136±0.070,0.436±0.083, 0.592±0.109 and 0.676±0.115（P＜0.05,n=10） by addition of 0,10,20,40,60 and 80μmol/L DHA respectively,and IC₅₀ of DHA on BK_Ca channel was 36.30±2.15μmol/L. Curves of relative Po and manding potential relation were shifted to the left under manding potential equal to +20,+40,+60,+80 and +100mV respectively,and half activated voltage and slope rate of BK_Ca channel was 69.16±3.57mV and 10.16±0.20 after addition of 35μmol/LDHA,b.IBK_Ca were gradually increased,Ⅰ-Ⅴcurves were upward,and BK_Ca tail currents were gradually increased,BK_Ca tail currentsⅠ-Ⅴcurves were upward with augmentation of DHA（P＜0.05,n=10）,and stably activated curves were no remarkable significance（P＞0.05,n=10）.IBK_Ca were increased to 6.1±0.3%,76.5±3.8%,248.0±12.3%, 374.2±18.7%and 443.1±22.1%（P＜0.05,n=10） with 20,40,60,80 and 100μmol/L of DHA under manding potential equal to +150mV,and IC₅₀ of DHA was 36.22±2.17μmol/L. BK_Ca tail currents were increased to 10.12±0.33%,146.12±4.97%,305.62±9.89%, 461.85±14.92%and 669.22±20.37%with augmentation of DHA under manding potential equal to +90mV（P＜0.05,n=10）,c.IK_V and K_V tail currents were gradually blocked,Ⅰ-Ⅴcurves were downward,stably activated curves were shift to the right,and stably inactivated curves were shifted to the left.IK_V were decreased to 4.57±0.23%, 10.77±0.54%,35.32±1.76%,70.25±3.51%and 78.51±3.93%（P＜0.05,n=10） with 20,40, 60,80 and 100μmol/L of DHA under manding potential equal to +50mV,and IC₅₀ of DHA was 42.19±1.59μmol/L.K_V tail currents were decreased to 6.65±0.27%,15.22±2.76%, 43.59±7.82%,73.80±11.79%and 83.02±13.65%with augmentation of DHA under lnanding potential equal to +40mV（P＜0.05,n=10）.Conclusion（1） Effects on Electrophysiology of rat ventricular myocytes:a.APDs are gradually prolonged with augmentation of DHA,but DHA has no effects on Vmax,APA and OS.b.DHA have blocked effects on I_Na,I_Ca-L,I_to and I_K,stably inactivated curves of these currents were shifted to the left,and recovered time from inactivation were prolonged with augmentation of DHA,and stably activated curves were no remarkable significance,c. DHA with different concentrations has no effects on I_Kl and its channel dynamics,d.The blocked effects of DHA on I_Na,I_Ca-L,I_to and I_K,are beneficial to prevent arrhythmia and sudden death.（2） Effects on Electrophysiology of rat CASMCs:a.DHA actived BK_Ca channel in an inside-out configuration.Curves of relative Po and manding potential relation were shifted to the left.b.DHA actived BK_Ca channel in an whole-cell configuration,and stably activated curves were no remarkable significance,c.DHA blocked K_V channel,tably activated curves were shift to the right,and stably inactivated curves were shifted to the left.d.The activation effects on BK_Ca channel by DHA are larger than its inhibitation effects on K_V channel.The complex action on BK_Ca channel and K_V channel induced by DHA result in vasodilatation.更多还原