糖尿病大鼠心力衰竭时心肌细胞凋亡的研究

目的探讨糖尿病大鼠心力衰竭时是否存在心肌细胞凋亡.方法建立STZ糖尿病大鼠模型,饲养12周,经心功能检测后确认为糖尿病心力衰竭的大鼠,采用TUNEL法及TEM法,检测糖尿病大鼠左室心肌的凋亡细胞.结果 糖尿病大鼠出现心功能异常并可见凋亡的心肌细胞,而对照组左室心肌组织中未见心肌细胞凋亡.结论心肌细胞凋亡与糖尿病大鼠心力衰竭密切相关.     

三七皂甙诱导骨髓基质细胞分化为心肌样细胞的实验研究

目的探讨三七皂甙对体外定向诱导猪骨髓基质细胞(MSCs)分化为心肌样细胞的影响。方法用穿刺方法抽取猪骨髓,分离并培养骨髓基质细胞,中药三七皂甙定向诱导分化为心肌样细胞。倒置显微镜下观察细胞形态,免疫细胞化学法鉴定心肌细胞特异性抗原标志物肌钙蛋白T,用RT-PCR对心肌肌球蛋白重链(β-MHC)进行鉴定。取培养的猪心肌细胞作为阳性对照,未经三七皂甙诱导正常培养的骨髓基质细胞作为阴性对照。结果猪MSCs经三七皂甙诱导2h后,大部分MSCs可分化为心肌样细胞,免疫细胞化学检测细胞中的肌钙蛋白T呈阳性,RT-PCR显示能表达β-MHC。结论MSCs是骨髓来源的具有多向分化潜能的干细胞,在三七皂甙的定向诱导下,可以向心肌样细胞分化,有望成为心衰及心肌梗死等心肌损伤时干细胞移植治疗的理想细胞材料。     

心肌细胞凋亡的信号传导机制研究

细胞凋亡在心血管疾病发病机制中扮演了重要角色 ,细胞凋亡信号的异常表达使心肌细胞数量不断减少 ,其中含有 BH3( Bcl-2 Homology3 )结构域的凋亡促进分子 (如 Bax,Bid,BNIP3等 )在凋亡信号传导途径中发挥了重要作用。     

Decreased intercellular coupling improves the function of cardiac pacemakers derived from mouse embryonic stem cells

The aim of this study was to determine if embryonic stem cell derived cardiomyocyte aggregates (ESdCs) can act as pacemakers in spontaneously active cardiomyocyte preparations when their connexin isoform expression is tuned toward a more sinus nodal phenotype. Using microelectrode array recordings (MEAs), we demonstrate that mouse ESdCs establish electrical coupling with spontaneously active cardiomyocyte preparations (HL-1 monolayer) and obtain pacemaker dominance. WT- and Cx43(−/−)-ESdCs comparably established intercellular coupling with cardiac host tissue (Cx43(−/−): 86% vs. WT: 91%). Although both aggregates had a 100% success rate in pacing quiescent cardiac preparations, Cx43(−/−)-ESdCs had an increased likelihood of gaining pacemaker dominance (Cx43(−/−): 40% vs. WT: 13%) in spontaneously active preparations. No differences in size, beating frequency, V_m, or differentiation were detected between WT- and Cx43(−/−)-ESdCs but the intercellular coupling resistance in Cx43(−/−)-ESdCs was significantly increased (Cx43(−/−): 1.2nS vs. WT: 14.8nS). Lack of Cx43 prolonged the time until Cx43(−/−)-ESdCs established frequency synchronization with the host tissue. It further hampered the excitation spread from the cardiomyocyte preparation into the ESdC. However rectifying excitation spread in these co-cultures could not be unequivocally identified. In summary, ESdCs can function as dominant biological pacemakers and Cx43 expression is not a prerequisite for their electrical integration. Maintenance of pacemaker dominance depends critically on the pacemaker's gap junction expression benefiting those with increased intercellular coupling resistances. Our results provide important insight into the design of biological pacemakers that will benefit the use of cardiomyocytes for cell replacement therapy.     

Interaction between the opposing functional effects of cyclic AMP and cyclic GMP in hypertrophic cardiac myocytes

We tested the hypothesis that in isolated cardiac myocytes, the negative functional effects of cyclic GMP would be blunted when the level of cyclic AMP was increased and that this interaction would be altered in renal hypertensive (One-Kidney-One-Clip, 1K1C) cardiac hypertrophic rabbits. Using isolated control and 1K1C ventricular myocytes, cyclic AMP and cell shortening (%) data were collected: 1) at baseline, 2) after the addition of 8-Br-cGMP 10^{−7, −6, −5} M, and 3) after forskolin (10⁻⁶ M), and adenylate cyclase activator, followed by 8-Br-cGMP 10^{−7, −6,−5} M. Basal levels of cyclic AMP were similar in control vs. 1K1C myocytes (10.2 ± 1.6 vs. 11.3 ± 2.6 pmol/10⁵ myocytes). We found that 8-Br-cGMP decreased the percent shortening in a dose related manner in both control myocytes (5.1 ± 0.6 to 3.2 ± 0.4%) and hypertrophic myocytes (5.2 ± 0.4 to 3.6 ± 0.5). The level of cyclic AMP significantly increased after the addition of 8-Br-cGMP in control myocytes (14.1 ± 2.1), but not in 1K1C myocytes. Forskolin increased the percent shortening in the control myocytes (3.8 ± 0.1 to 4.8 ± 0.4), but no significant increase was noted in the hypertrophic myocytes (3.6 ± 0.3 to 3.7 ± 0.3). The level of cyclic AMP significantly increased after the addition of forskolin in both control (13.9 ± 2.0), and 1K1C cells (14.6 ± 3.8). Forskolin attenuated the negative functional effects of 8-Br-cGMP in the control (4.8 ± 0.4 to 3.2 ± 0.1) and 1K1C myocytes (3.7 ± 0.3 to 2.7 ± 0.3). The adition of 8-Br-cGMP did not affect the level of cyclic AMP after forskolin in either control (13.9 ± 2.0 to 14.8 ± 2.5) or 1K1C myocytes (14.6 ± 3.8 to 13.8 ± 1.9). These data indicated that in hypertrophic cardiac myocytes the negative functional effects of 8-Br-cGMP were similar to control, but the positive functional effects of cyclic AMP were blunted. There was an increase in cyclic AMP levels after addition of 8-Br-cGMP in control but not 1K1C cells. We conclude that in control and hypertrophic myocytes, the effects of cyclic GMP were blunted after forskolin, but this did not seem to be related to cyclic AMP phosphodiesterase activity. Received: 12 October 1999, Returned for 1. revision: 24 November 1999, 1. Revision received: 23 March 2000, Returned for 2. revision: 26 May 2000, 2. received: 16 June 2000, Accepted: 12 July 2000     

Oxidation of palmitate and lactate by beating myocytes isolated from adult dog heart.

Myocytes were isolated from hearts of adult dogs by repeated digestion with collagenase and hyaluronidase in Ca²⁺-free phosphate buffer followed by centrifugation in the presence of Ficoll. The isolated myocytes were free of other tissue elements and were contracting autorhythmically. The oxidation of palmitate and lactate were studied by incubating cell preparation with [1-¹⁴C]palmitate or [U-¹⁴C]lactate either in the presence or absence of CaCl₂ for 30 min at 37°C. [¹⁴C]O₂ was collected by KOH-soaked filter paper. Cardiac myocytes oxidized 0.1 to 0.18 nmol/mg protein min of palmitate and 1.5 to 2.2 nmol/mg protein min of lactate. The oxidation was linear for 75 min. Maximal rate of oxidation was obtained at substrate concentrations of 0.2 mm palmitate or 5.0 mm lactate. Adenine or pyridine nucleotides had no effect on the oxidation of these substrates. Palmitate oxidation was enhanced by carnitine and also by an increase in the molar ratio of palmitate/albumin, but was depressed (15 to 50%) by CaCl₂ (0.025 to 1.0 mm). CaCl₂ (0.05 to 1.0 mm) increased the rate of lactate oxidation by 50 to 68%. Lactate oxidation was also enhanced (68 to 87%) in the presence of palmitate (0.4 to 0.8 mm). These findings demonstrate the suitability of isolated adult heart myocytes for the study of cardiac metabolism.     

依折麦布辛伐他汀片对兔腹主动脉粥样硬化斑块逆转作用的实验研究

目的：观察腹主动脉粥样斑块内炎性巨噬细胞和平滑肌细胞的表达情况,以探索依折麦布联合他汀类药物在逆转动脉斑块中的作用及机制。方法选取24只健康雄性新西兰大耳白兔,随机分为对照组（n＝8）和高胆固醇血症组（n＝16）。对照组给予普通饲料,喂养12周。高胆固醇血症组喂饲致动脉粥样硬化饲料（由普通颗粒饲料＋15g/L胆固醇＋100g/L猪油＋150g/L蛋黄粉组成）2周后行腹主动脉内膜球囊拉伤术,术后再随机分为模型亚组和依折麦布辛伐他汀（ES）亚组（给予5/10mg/（kg·d）每组8只,两亚组均继续喂饲致动脉粥样硬化饲料10周。喂养第12周时活杀动物,取腹主动脉进行石蜡切片。检测不同时间点脂质和脂蛋白,应用光学显微镜观察动脉粥样硬化进程,采用免疫组化方法分析巨噬细胞和平滑肌细胞在斑块处的表达。结果 ES亚组的血清总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）浓度明显低于模型亚组（P＜0.01）。病理检测显示两亚组及ES亚组斑块直径、斑块厚度和动脉内/中膜厚度经单因素方差分析,差异有统计学意义（P＜0.05）。免疫组化检测结果示ES亚组血管壁中巨噬细胞的表达量较模型亚组显著减少（P＜0.05）,而平滑肌细胞的表达量较模型亚组显著增多（P＜0.01）。结论 ES可能通过减少细胞外脂质的沉积,减少内膜和中膜巨噬细胞的数量和胆固醇的含量,增加胶原和平滑肌细胞面积,从而起到逆转斑块的作用。     

RhoA/ROCK pathway regulates hypoxia-induced myocardial cell apoptosis

Objective:To observe the regulatory effects of RhoA/ROCK pathway on the apoptosis of cardiac myocyte induced by anoxia and its mechanism.Methods:The model of cardiac myocyte anoxia was established.The beat pulsations and apoptosis rales after 1 h,3 h,6 h,9 h and 12 h of anoxia were recorded and the expressions of RhoA,ROCK1/2,p-PI3 K,p-AKT and caspae-3 were detected,too.The apoptosis and the expressions of related proteins were detected after RNAi of RhoA and the inhibition of ROCK by Y-27632.Results:The beat pulsations after 1 h,3 h,6 h.9h and 12 h decreased gradually but the apoptosis rates increased gradually,and the expressions of RhoA,ROCK1/2,p-P13 K,p-AKT and caspase-3 were increasing along with the increasing duration of anoxia.The apoptotic rales after 1 h,3 h,6 h.9 h and 12 h of anoxia were(4.36±0.98)%,(8.36±2.12)%,(15.32±3.62)%,(18.68±4.83)%and(24.56±6.22)%.respectively and decreased more significantly than control group in different time points of anoxia(P0.05).and the expressions of RhoA,ROCK1/2,p-PI3 K,p-AKT and caspase-3 decreased significantly(P0.05).The apoptosis rate and the expressions of RhoA,ROCK1/2,p-PI3 K,p-AKT and caspase-3 decreased significantly(P0.05) after the inhibition of ROCK by Y-27632(P0.05).Conclusions:RhoA/ROCK pathway plays a critical role in the regulation of the apoptosis of cardiac myocyte induced by anoxia,which may be accompanied by regulating the activity of PI3K/AKT/Caspase-3 pathway.