本文選題：硫化氫 + 心功能�。� 參考：《寧夏醫(yī)科大學(xué)》2010年碩士論文

【摘要】：H_2S是一種內(nèi)源性氣體信號(hào)分子,在心血管系統(tǒng)中發(fā)揮著重要的生理和病理作用。已有報(bào)道H_2S可抑制心肌細(xì)胞L型鈣通道,還可濃度依賴性地降低離體灌流大鼠心臟的心功能。但灌流實(shí)驗(yàn)并未鉗制離體大鼠心臟的心率,而心率的變化對心功能也會(huì)產(chǎn)生影響,此外我們在預(yù)實(shí)驗(yàn)中發(fā)現(xiàn)低于生理濃度的H_2S有改善心臟的舒張功能。因此研究低濃度H_2S對心臟的保護(hù)作用和機(jī)制具有重要意義。 目的本工作在離體灌流大鼠心臟觀察在固定心率情況下,硫化氫對大鼠心臟冠脈流量及心功能的影響;以急性分離的大鼠心肌細(xì)胞為模型,應(yīng)用膜片鉗技術(shù),觀察硫化氫對心肌細(xì)胞L型鈣通道電流(ICa,L)、鈉鈣交換電流(INaCa)、ATP敏感鉀通道(I_(KATP))等電流的影響,以進(jìn)一步明確硫化氫對心肌舒縮功能的影響及其離子電流機(jī)制。 方法離體SD大鼠心臟采用Langendorff系統(tǒng)用KH液進(jìn)行逆行恒壓(80cmH2O)灌流,并固定離體心臟心率。觀察不同劑量NaHS對左室收縮壓(LVSP)、左室舒張末壓(LVEDP)、左心室內(nèi)壓最大上升/下降速率(±dp/dtmax),冠脈流量(CF)的影響。以LVSP和LVEDP差值作為左室發(fā)展壓(LVDP)。應(yīng)用酶解法分離心肌細(xì)胞,用膜片鉗技術(shù)記錄單個(gè)心肌細(xì)胞ICa,L、INaCa、I_(KATP),給藥組以不同濃度NaHS的細(xì)胞外液灌流心肌細(xì)胞5 min;對照組直接以細(xì)胞外液灌流5 min,記錄并觀察電流曲線的變化。 結(jié)果1、硫化氫對離體大鼠心臟功能的影響:給藥前平衡灌流期間,各實(shí)驗(yàn)組冠脈流量和各項(xiàng)心功能觀察指標(biāo)基本保持穩(wěn)定,各組間所測指標(biāo)無顯著差異。對照組灌流KH液15min,LVEDP為7.06±0.42mmHg,12.5、25μmol/L(低于生理濃度)NaHS組,灌流15min,LVEDP分別為5.88±0.97mmHg和5.42±0.88 mmHg,顯著性低于對照組(P0.01,n=8); 50μmol/L(生理濃度)NaHS組,LVEDP無明顯變化,與對照組相比無顯著差異(P0.05,n=6),100和200μmol/L NaHS組, LVEDP顯著升高,分別達(dá)15.22±3.34 mmHg、24.81±3.61 mmHg。NaHS 12.5、25μmol/L組冠脈流量增加,NaHS 50、100和200μmol/L組冠脈流量呈時(shí)間依賴性下降。以上提示,在心率固定的條件下,低濃度的NaHS可能對冠脈有擴(kuò)張作用,因而冠脈流量增加。NaHS 25μmol/L(n=8),50、100和200μmol/L(n=6)灌流15 min,LVDP均降低,分別下降10±6%、19±2%、51±5%和76±6%;+dp/dtmax的抑制百分?jǐn)?shù)分別為16±4%、23±10%、52±10%和65±9%;-dp/dtmax的抑制百分?jǐn)?shù)分別為17±4%、23±11%、61±6%、74±5%,分別與空白對照組相比較,有顯著性差異(P0.05)。以上結(jié)果表明,NaHS能夠濃度依賴性地抑制離體大鼠心臟的收縮功能。2、硫化氫對大鼠心肌細(xì)胞L型鈣電流的影響:硫化氫呈濃度依賴性抑制大鼠心肌細(xì)胞ICa,L,6.25μmol/LNaHS灌流1 min和5min后,ICa,L分別下降4±2%、8±3%,12.5μmol/LNaHS灌流1 min和5min后,ICa,L分別下降8±3%、14±5%,與空白對照組相比雖無統(tǒng)計(jì)學(xué)意義,但此濃度對ICa,L已經(jīng)有一定的抑制作用。25、50、100和200μmol/L NaHS灌流1 min后,ICa,L分別下降13±2%、19±2%、23±4%和27±5%,與對照組的3±1%相比均有統(tǒng)計(jì)學(xué)差異(P 0.05, n=8);灌流5 min后,ICa,L分別下降22±4%、25±3%、29±7%和52±9%,均顯著高于對照組的6±2%(P 0.01, n=8)。200μmol/L NaHS灌流1 min后IV曲線明顯上移,但不改變ICa,L的電壓依賴關(guān)系和反轉(zhuǎn)電位。對照組ICa,L激活曲線的半激活電位為-19.52±0.54 mV,斜率為4.85±0.52 mV;200μmol/L NaHS灌流灌流1 min后半激活電位為-19.03±0.59 mV,斜率為5.43±0.55 mV,二者比較尚無統(tǒng)計(jì)學(xué)意義(P 0.05, n=8)。對照組ICa,L失活曲線半失活電位為-23.61±0.15 mV,斜率為5.32±0.13 mV;200μmol/L NaHS灌流灌流1 min后半失活電位為-24.57±0.15 mV,斜率為5.44±0.13,二者無明顯區(qū)別(P0.05, n=8)。上述結(jié)果表明上述濃度硫化氫不改變L型鈣通道激活和失活的門控特點(diǎn)。3、硫化氫對大鼠心肌細(xì)胞鈉鈣交換電流的影響:硫化氫對正、反向鈉鈣交換電流均有增大作用。給予濃度為6.25、12.5、25、50、100、200μmol/L的NaHS 5min后,正向INaCa比給藥前分別增大15±6%、20±5%、33±8%、16±4%、25±3%、29±7%,均顯著高于對照組((P0.05,n=10),6.25、12.5、25、50、100、200μmol/L組反向INaCa分別增大2±1%、6±3%、10±2%、11±2%、21±4%、25±3%,后五組與對照組比較顯著增大(P0.05,n=10),對照組單純給予細(xì)胞外液5 min后正、反向INaCa分別減小5±2%和3±2%。4、硫化氫對大鼠心肌細(xì)胞ATP敏感性鉀通道電流的影響:在由50 mV到-100 mV持續(xù)125 ms的斜坡刺 激下記錄心肌細(xì)胞KATP通道電流。給予pinacidil后該電流明顯增加,并且能被glibenclamide阻斷(說明該電流是I_(KATP))。50、100和200μmol/L NaHS不能使KATP通道開放。400、800和1600μmol/L NaHS能使KATP通道開放,ATP敏感鉀通道開放后的電流密度分別為17.25±8.45 pA/pF、20.33±8.56 pA/pF和22.87±6.23 pA/pF。各組間比較無顯著差異(P0.05, n=8)。表明較高濃度的NaHS才能開放KATP通道,但此濃度已經(jīng)達(dá)到顯著抑制心肌細(xì)胞膜L型鈣通道。 結(jié)論高于正常生理濃度的NaHS可以濃度依賴性地抑制離體灌流大鼠心臟心功能,低濃度的NaHS能改善心肌舒張功能。NaHS可以濃度依賴性地抑制急性分離的大鼠心肌細(xì)胞膜上的L型鈣通道電流,在沒有改變鈣離子通道動(dòng)力學(xué)特征的情況下減少了通道開放時(shí)的外鈣內(nèi)流,從而減弱了心肌細(xì)胞的收縮功能。低濃度的NaHS可以增大大鼠心肌細(xì)胞的正向鈉鈣交換電流,提示低濃度的NaHS對心肌舒張功能的改善可能部分是通過增加正向鈉鈣交換電流實(shí)現(xiàn)的。高濃度的NaHS可以使急性分離的大鼠心肌細(xì)胞ATP敏感性鉀通道開放,而低濃度的NaHS能抑制心肌細(xì)胞L型鈣通道從而抑制心肌細(xì)胞收縮可能和其是ATP敏感性鉀通道開放劑的作用沒有關(guān)系。
[Abstract]:H_2S is an endogenous gas signal molecule, which plays an important physiological and pathological role in the cardiovascular system. It has been reported that H_2S can inhibit the L calcium channel in cardiac myocytes and reduce the cardiac function of isolated rat heart in a concentration dependent manner. However, perfusion test did not clamp the heart rate of the isolated rat heart, and the heart rate changes to the heart. Functions also have an impact. In addition, we have found that H_2S, which is lower than the physiological concentration, can improve the heart diastolic function in the pre experiment. Therefore, it is important to study the protective effect and mechanism of low concentration of H_2S on the heart.
Objective To observe the effect of hydrogen sulfide on cardiac coronary flow and cardiac function in rat heart under the condition of fixed heart rate, with acute isolated rat cardiac muscle cells as model, and using patch clamp technique to observe L type calcium channel (ICa, L), sodium calcium exchange current (INaCa) and ATP sensitive potassium in cardiac myocytes. The effect of current (I_ (KATP)) on the effect of hydrogen sulfide on myocardial contractile function and its ionic current mechanism were further clarified.
Methods the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (+ dp/dtmax) and coronary flow rate (CF) were observed at different doses of NaHS. The difference between LVSP and LVEDP was taken as the effect of the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and left ventricular pressure (LVEDP). Left ventricular development pressure (LVDP). Myocardial cells were separated by enzyme hydrolysis, and single myocardial cells ICa, L, INaCa, I_ (KATP) were recorded by patch clamp technique. The 5 min in the administration group was administered with different concentrations of NaHS, and the control group was injected with 5 min directly from the extracellular fluid and recorded and observed the changes of the current curve.
Results 1, the effect of hydrogen sulfide on the cardiac function of rats in vitro: the observation indexes of coronary flow and heart function in each experimental group remained stable during the period of balance perfusion, and there was no significant difference between the groups. The control group was 15min, LVEDP was 7.06 + 0.42mmHg, 12.5,25 mu mol/L (below the physiological concentration) NaHS group, perfusion of 15min, LVED P was 5.88 + 0.97mmHg and 5.42 + 0.88 mmHg respectively, significantly lower than the control group (P0.01, n=8), and 50 mu mol/L (physiological concentration) NaHS group, there was no significant change in LVEDP (P0.05, n=6), 100 and 200 mu mol/L NaHS group, which was significantly higher than 15.22 + 3.34, 24.81 + 3.61 In addition, the coronary flow of NaHS 50100 and 200 mu group was time dependent. The above suggested that under the condition of heart rate fixation, the low concentration of NaHS might have dilation effect on the coronary artery, so the coronary flow rate increased by.NaHS 25 mu mol/L (n=8), 50100 and 200 micron mol/L (n=6) perfusion 15 min, LVDP decreased, respectively, 10 + 6%, 19 + 2%, 51 + 5% and 76 + 6%; +d The inhibitory percentages of p/dtmax were 16 + 4%, 23 + 10%, 52 + 10% and 65 + 9% respectively. The inhibitory percentages of -dp/dtmax were 17 + 4%, 23 + 11% and 61 + 23, respectively. Compared with the blank control group, there were significant differences (P0.05). The results showed that NaHS could inhibit the contractile function.2 of the isolated rat heart in a concentration dependent manner, and the hydrogen sulfide could be used in rats The effect of L type calcium current in cardiac myocytes: hydrogen sulfide was concentration dependent inhibition of rat myocardial cells ICa, L, 6.25 mu mol/LNaHS perfusion 1 min and 5min, ICa, L decreased 4 + 2%, 8 + 3%, 12.5 micron 1 min and 5min, ICa, respectively, 8 + 3%, 14 + 5%, although no statistical significance compared with the empty white control group, but this concentration was already After.25,50100 and 200 mol/L NaHS perfusion 1 min, ICa and L were decreased by 13 + 2%, 19 + 2%, 23 + 4% and 27 + 5%, respectively, compared with the control group's 3 + 1% (P 0.05, n=8). After 1 min perfusion, the IV curve obviously moved up, but did not change the voltage dependence and reverse potential of ICa, L. The semi activated potential of ICa, L activation curve was -19.52 + 0.54 mV, and the slope was 4.85 + 0.52 mV in the control group. 200 mu mol/L NaHS perfusion flow 1 min half activation potential was 0.59, and the slope was 5.43 + 0.55. Two there was no statistical significance. Meaning (P 0.05, n=8). The semi inactivation potential of the control group ICa, L inactivation curve is -23.61 + 0.15 mV, the slope is 5.32 + 0.13 mV, and the second half inactivation potential of 200 u mol/L NaHS perfusion flow 1 min is -24.57 + 0.15 mV, and the slope is 5.44 + 0.13. The above results show that the above concentration of hydrogen sulfide does not change the activation and inactivation of the calcium channel. .3, the effect of hydrogen sulfide on the sodium and calcium exchange current of rat cardiac myocytes: hydrogen sulfide has an increasing effect on positive and reverse sodium calcium exchange current. After NaHS 5min with a concentration of 6.25,12.5,25,50100200 mol/L, the positive INaCa is increased by 15 +, 20 +, 33 + 8%, 16 + 4%, 25 + 3%, 29 + 7%, respectively. ( P0.05, n=10), the reverse INaCa of group 6.25,12.5,25,50100200 mu mol/L increased by 2 + 1%, 6 + 3%, 10 + 2%, 11 + 2%, 21 + 4%, 25 + 3%, then significantly increased (P0.05, n=10) in the five group compared with the control group (P0.05, n=10). Effect of flow: slope stab at 125 ms from 50 mV to -100 mV
The current of KATP channel of myocardial cells was stimulated. After pinacidil, the current increased obviously and could be blocked by glibenclamide (indicating that the current was I_ (KATP)).50100 and 200 mu mol/L NaHS did not open the KATP channel open.400800 and 1600 mu mol/L NaHS. The current density of the sensitive potassium channel was 17.25 + 8, respectively. There was no significant difference in.45 pA/pF, 20.33 + 8.56 pA/pF and 22.87 + 6.23 pA/pF. (P0.05, n=8). It showed that a higher concentration of NaHS could open the KATP channel, but this concentration had significantly inhibited the L type calcium channel in the myocardial cell membrane.
Conclusion NaHS, which is higher than normal physiological concentration, can inhibit the cardiac cardiac function of rat perfusion in vitro. The low concentration of NaHS can improve the myocardial diastolic function.NaHS, which can inhibit the L type calcium channel current on the membrane of acute isolated rat cardiac myocytes, without changing the dynamic characteristics of calcium ion channel. The low concentration of NaHS can increase the positive sodium calcium exchange current in rat cardiac myocytes, suggesting that the low concentration of NaHS may be partly achieved by increasing the positive sodium calcium exchange current. The high concentration of NaHS can make it urgent. The ATP sensitive potassium channel of isolated rat cardiomyocytes is open, and the low concentration of NaHS can inhibit the L calcium channel in cardiac myocytes and inhibit the contraction of cardiac myocytes, which may not be related to the effect of the ATP sensitive potassium channel opener.

【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

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