人参皂苷Rg1对心肌细胞氧化应激损伤的抑制作用

目的 通过构建H₂O₂诱导的H9c2细胞氧化应激模型,观察人参皂苷Rg₁对氧化应激的抑制作用,并探讨mi R-499c是否参与人参皂苷Rg₁抑制氧化应激的作用机制。方法 采用600μmol/L的H₂O₂诱导H9c2细胞氧化应激模型,给予人参皂苷Rg₁预处理24 h,检测乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)活性及丙二醛(malondialdehyde,MDA)、活性氧(reactive oxygen species,ROS)水平和线粒体膜电位(mitochondrial membrane potential,MMP)变化;采用Western blotting检测凋亡相关蛋白表达。采用Lipofiter 3.0将miR-499c转染至H9c2细胞再制备H₂O₂诱导的氧化应激模型,给予人参皂苷Rg     

人诱导多能干细胞分化来源心肌细胞特征及促成熟方法研究进展

人诱导多能干细胞分化来源的心肌细胞(Human induced pluripotent stem cells-derived cardiomyocytes,hiPSC-CMs)为心脏修复、心脏药物测试、体外心脏建模等提供了理想细胞来源。但hiPSC-CMs与心肌细胞相比并不成熟,表现在缺乏某些特定基因表达,结构、代谢、电生理不成熟,Ca 2+处理水平低等方面。许多研究都聚焦于促进hiPSC-CMs成熟的方法,包括生化刺激、物理刺激、共培养、3D培养、miRNA等。本文对hiPSC-CMs特征及其促成熟方法进行综述,并展望该领域的发展方向。     

αB-晶状体蛋白在保护过氧化氢所致心肌细胞损伤中的作用

采用热休克对新生大鼠心肌细胞进行预处理 ,以诱导αB 晶状体蛋白的表达 ,并观察其对 1mmol·L- 1 的过氧化氢 (H2 O2 )所致心肌细胞损伤的保护作用。发现 :热休克预处理组心肌细胞中αB 晶状体蛋白表达明显增加 ,H2 O2 所致的细胞死亡率及LDH释放率降低 ,总抗氧化能力增强 ;H2 O2 可引起αB 晶状体蛋白从胞浆向胞核及微丝移位。提示热休克预处理的心肌细胞保护作用与其诱导αB 晶状体蛋白的表达增加有关。αB 晶状体蛋白可能通过细胞内移位 ,保护某些核结构 ,稳定细胞骨架 ,并增强心肌细胞抗氧化能力 ,从而发挥其保护作用     

Trophic effect of angiotensin II in neonatal rat cardiomyocytes: role of endothelin-1 and non-myocyte cells

1. Angiotensin II (AII) and the endothelins (ET) are known to be potent trophic stimuli in various cells including cardiomyocytes. In order to characterize further these effects we studied, in neonatal rat ventricular cardiomyocytes, the effects of several endothelin-receptor antagonists and the AT₁-receptor antagonist losartan on AII- and endothelin-induced inositol phosphate (IP)-formation (assessed as accumulation of total [³H]-IPs in myo-[³H]-inositol prelabelled cells) and increase in rate of protein synthesis (assessed as [³H]-phenylalanine incorporation).
2. Endothelin (10 pM–1 μM) concentration-dependently increased IP-formation (max. increase at 100 nM ET-1: 130±14% above basal, n=25) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 52±4% above basal, n=16) with an order of potency: ET-1>>ET-3. Both effects were antagonized by the ET_A/ET_B-receptor antagonist bosentan and the ET_A-receptor antagonist BQ-123, but not affected by the ET_B-receptor antagonist IRL 1038 and the AT₁-receptor antagonist losartan.
3. Pretreatment of the cells with 500 ng ml⁻¹ pertussis toxin (PTX) overnight that completely inactivated PTX-sensitive G-proteins did not attenuate but rather enhance ET-1-induced IP-formation. On the other hand, in PTX-pretreated cardiomyocytes ET-1-induced [³H]-phenylalanine incorporation was decreased by 39±5% (n=5).
4. AII (1 nM–1 μM) concentration-dependently increased IP-formation (max. increase at 1 μM: 42±7% above basal, n=16) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 29±2%, n=9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ-123.
5. In well-defined cultures of cardiomyocytes (not contaminated with non-myocyte cells) AII failed to increase [³H]-phenylalanine incorporation; addition of non-myocyte cells to the cardiomyocytes restored AII-induced increase in [³H]-phenylalanine incorporation.
6. We conclude that, in rat neonatal ventricular cardiomyocytes, (a) the ET-1-induced increase in rate of protein synthesis (through ET_A-receptor stimulation) involves at least two signalling pathways: one via a PTX-insensitive G-protein coupled to IP-formation, and the other one via a PTX-sensitive G-protein, and (b) the trophic effects of AII are brought about via local ET-1 secretion upon AT₁-receptor stimulation in neonatal rat ventricular non-myocyte cells.

     

环维黄杨星D延长大鼠心室肌细胞APD作用的分子机制研究

目的　研究环维黄杨星D对分离的大鼠心室肌细胞内向整流钾电流 (IK1 )、瞬时外向钾电流 (Ito)、L 型钙电流(ICa L)和动作电位时程 (APD)的影响。方法　采用全细胞膜片钳技术记录大鼠心室肌细胞IK1 、Ito、ICa L 和APD。结果　 1和10 μmol·L- 1 环维黄杨星D明显延长分离大鼠心室肌细胞APD50 和APD90 ,10 μmol·L- 1 可明显降低静息膜电位 (RP)。环维黄杨星D对IK1 内向电流和外向电流均有明显抑制作用 ,当指令电压为 - 10 0mV时 ,1和 10 μmol·L- 1 环维黄杨星D分别使IK1 电流密度从给药前的 ( - 8.0± 1.1)pA pF降至 ( - 4 .1± 0 .7)pA pF和 ( - 3.4± 0 .8)pA pF ;当指令电压 - 30mV时 ,分别使IK1 电流密度从 ( 1.10± 0 .2 4 )pA pF降至 ( 0 .6 1± 0 .18)pA pF和 ( 0 .36± 0 .11)pA pF ;在钳制电位从 0到 + 6 0mV之间 ,环维黄杨星D明显抑制Ito,当指令电压 4 0mV时 ,1和 10 μmol·L- 1 环维黄杨星D分别使Ito电流密度从给药前的 ( 8.9± 2 .0 )pA pF降至 ( 5 .5± 1.2 )pA pF和 ( 4 .9± 0 .9)pA pF。环维黄杨星D浓度依赖性抑制ICa L,在指令电压为 10mV时 ,1和 10 μmol·L- 1 分别使ICa L电流密度从给药前的 ( - 9.9± 1.8)pA pF降至 ( - 6 .4± 1.4 )pA pF和 ( - 4 .2± 0 .6 )pA pF。结论　环     

心气虚细胞模型的研制

目的探索心气虚细胞模型的制作方法。方法ＮＩＨ小白鼠乳鼠培养心肌细胞用缺氧（１～３小时）再给氧损伤方法研制心气虚细胞水平动物模型。结果培养心肌细胞采用缺氧再给氧损伤后，出现ＬＤＨ和ＣＫ含量增加，ＳＯＤ活性降低，ＭＤＡ含量上升，与对照组比较，差别有显著性（Ｐ＜０．０５）或高度显著性（Ｐ＜０．０１）。结论根据缺血缺氧是导致心气虚证的重要因素之一，探索采用缺氧再给氧损伤制作离体心气虚细胞模型具有一定的研究价值     

眼睛蛇毒心脏毒素对大鼠心肌细胞收缩及细胞内钙离子的作用

目的 研究眼睛蛇毒心脏毒素（Cardiotoxin，CTX）对心肌细胞的形态、收缩幅度和细胞内钙离子（[Ca^2＋]i）的作用。方法 应用荧光计量法（以Fura-2／AM为荧光染料）及光学成像系统来测定单个心肌细胞[Ca^2＋]i和收缩幅度。结果 0．001～1μmol／L的CTX使心肌细胞由杆状变成圆形，药物的作用从第1分钟时开始，到第20分钟时趋于稳定。在电刺激存在的情况下，1μmol／L的CTX最初导致电诱导的[Ca^2＋]i和收缩幅度瞬间增加，接下来[Ca^2＋]i时程延长，最终细胞对电刺激不敏感、突然收缩、[Ca^2＋]i持续增高。在缺乏电刺激的情况下，1μmol／L的CTX可诱导Ca^2＋震荡波、持续性[Ca^2＋]i增高，这种作用与40mmol／L的KCl和10mmol／L咖啡因所引起的[Ca^2＋]i瞬间增加不同。结论 CTX作用初期使[Ca^2＋]i增高，使细胞[Ca^2＋]。超载，同时伴随细胞形状的改变。     

体外诱导脂肪来源细胞向心肌细胞分化的实验研究

目的通过5-氮杂胞苷(5-AZA)体外诱导脂肪来源细胞(adipose-derived cells,ADC),来观察其向心肌细胞分化的潜能,探讨脂肪来源细胞治疗缺血性心脏病的可能性。方法应用酶消化法获得兔ADC,并进行体外培养、扩增、鉴定。然后5-AZA体外诱导3周后,应用RT-PCR检测心肌细胞特异性α肌球蛋白重链(-αMHC)基因。结果RT-PCR显示与正常心肌组织-αMHC阳性条带对照,诱导后ADC也在相同位置出现-αMHC的阳性条带,而未诱导ADC没有阳性条带出现。结论ADC可在一定诱导条件下向心肌细胞分化,是一种较有潜力的治疗缺血性心脏病的移植细胞。     

苓桂术甘汤对慢性心律失常大鼠心肌损伤的保护作用及对Nrf2/HO-1通路的影响

[目的]探讨苓桂术甘汤对慢性心律失常大鼠心肌损伤的保护作用及对核因子E2相关因子2(nuclear factor E2 related factor 2,Nrf2)/血红素氧合酶-1(heme oxygenase-1,HO-1)通路的影响。[方法]45只雄性SD大鼠中随机选择10只作为正常对照组,其余35只经尾静脉注射氯化钡溶液制备慢性心律失常模型,将30只建模成功的大鼠随机分为苓桂术甘汤组、阳性对照组、模型对照组,每组各10只。苓桂术甘汤组大鼠采用苓桂术甘汤灌胃,阳性对照组采用酒石酸美托洛尔灌胃,模型对照组和正常对照组接受等体积0.9%氯化钠注射液灌胃,2次/d,均干预4周。采用心电图检验大鼠造模成功情况,以酶联免疫吸附法检验大鼠血清谷胱甘肽(glutathione,GSH)、谷胱甘肽硫基转移酶(glutathione S-transferase,GST)含量,苏木精-伊红(hematoxylin-eosin,HE)染色观察心肌组织病理变化,DNA缺口末端标记法(TdT-mediated dUTP nick-end labeling,TUNEL)染色检测各组大鼠心肌细胞凋亡情况,实时荧光定量聚合酶链式反应(Real-time fluorescence quantitative polymerase chain reaction,Real-time q PCR)与Western blot检测心肌组织中Nrf2、HO-1基因及蛋白表达水平。[结果]与正常对照组比较,模型对照组、阳性对照组、苓桂术甘汤组GSH和GST含量较低,心肌细胞凋亡率较高,Nrf2、HO-1 mRNA与蛋白相对表达量较高(P0.05)。与模型对照组比较,阳性对照组、苓桂术甘汤组大鼠的GSH和GST含量较高,心肌细胞凋亡率较低(P0.05);与阳性对照组比较,苓桂术甘汤组大鼠的GSH和GST含量较低,心肌细胞凋亡率较高(P0.05)。模型对照组心肌组织水肿、充血严重,肌束紊乱,心肌细胞间隙增宽;阳性对照组、苓桂术甘汤组心肌组织水肿、肌束及心肌细胞间隙明显改善。与模型对照组比较,阳性对照组、苓桂术甘汤组大鼠Nrf2、HO-1 mRNA及蛋白相对表达量均较高(P0.05);与阳性对照组比较,苓桂术甘汤组大鼠Nrf2、HO-1 mRNA及蛋白相对表达量均较低(P0.05)。[结论]苓桂术甘汤可能通过激活Nrf2/HO-1通路,发挥对慢性心律失常大鼠心肌损伤的保护作用。     

Bufalin,a bufanolide steroid from the parotoid glands of the Chinese toad,inhibits L‐type Ca2+ channels and contractility in rat ventricular myocytes

Bufalin is a bufanolide steroid compound in Chan Su. Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads and has been used as an oriental drug. However, the effect of bufalin on cardiac function and its underlying cellular mechanisms remain unclear. Here, we explore the cellular mechanisms of bufalin on myocardial protection via the whole‐cell patch‐clamp recording and video‐based edge detection system. Exposure to bufalin resulted in a concentration‐dependent blockade of I_Ca‐L, with the half‐maximal inhibitory concentration (IC₅₀) of 60 μm and the maximal inhibitory effect of 71.50 ± 2.67%. Bufalin at 100 μm reduced cell shortening by 33.83 ± 4.01%. Bufalin restrained L‐type Ca²⁺ channels conductance, and contractility in rat ventricular myocytes. Thus, the protective effects of bufalin on the heart may be determined by the inhibitory effect on I_Ca‐L and the negative inotropic action caused by the decrease of intracellular Ca²⁺ in rat myocardial cells.