热休克蛋白90对大鼠缺氧心肌细胞丝氨酸苏氨酸蛋白激酶表达的影响

目的探讨内源性热休克蛋白90（HSP90）在缺氧心肌细胞丝氨酸苏氨酸蛋白激酶（AKT）相关信号通路中的作用。方法建立新生Wistar大鼠心肌细胞缺氧模型，将细胞分为正常组、缺氧组、加入HSP90特异性阻断剂格尔德霉素后再缺氧组（格尔德霉素＋缺氧组）。于缺氧后1、3、6、12、24、48h用噻唑蓝法检测心肌细胞的活力；缺氧24h，原位缺口末端标记法检测心肌细胞凋亡指数（AI）；缺氧1、3、6、12、24h，蛋白质印迹法检测大鼠心肌细胞中内源性HSP90及AKT表达水平。结果（1）缺氧24、48h，缺氧组、格尔德霉素＋缺氧组细胞活力均较正常组明显下降（P〈0．05）；格尔德霉素＋缺氧组细胞活力缺氧12h即开始明显下降，缺氧48h时明显低于缺氧组（P〈0．05）。（2）缺氧24h，缺氧组细胞AI为（10．7±1．2）％，明显高于正常组[（1．9±0．3）％．P〈0．05]；格尔德霉素＋缺氧组细胞AI为（26、3±5．3）％，明显高于缺氧组（P〈0．01）。（3）缺氧12h，缺氧组心肌细胞内源性HSP90及AKT表达水平高于正常组与格尔德霉素＋缺氧组；缺氧24h，缺氧组有所下降．格尔德霉素＋缺氧组则下降更明显。结论内源性HSP90对维持心肌细胞的活力有重要作用．缺氧心肌细胞AKT表达水平可受内源性HSP90表达水平的影响。     

Anti-inflammatory effect of erythropoietin pretreatment on cardiomyocytes with hypoxia/reoxygenation injury and the possible mechanism

Objective： To investigate the anti-inflammatory effect of erythropoietin （EPO） pretreatment on cardiomyocytes exposed to hypoxialreoxygenation injury （H/R） and explore the possible mechanism.
Methods： The cultured neonatal rats＇ ventricular cardiomyocytes were divided randomly into 4 groups, control group （C group）, EPO pretreatment group （E group）, EPO and pyrrolidine dithiocarbamate （PDTC） pretreatment group （EP group） and PDTC pretreatment group （P group）. After 24 hours＇ pretreatment, the cardiomyocytes were exposed to H/R. After pretreatment and H/R, the expression of tumor necrosis factor- α （TNF- α ） gene in all the groups was detected by RT-PCR and Western blot. The nuclear factor- κ B （NF- κB） activity was detected by electrophoretic mobility shift assay （EMSA） and the inhibitor- κB α （Ⅰ- κB α） protein level was detected by Western blot.
Results： The decrement of Ⅰ- κB a protein and the increasing NF- KB activity were found in cardiomyocytes pretreated with EPO before H/R compared to other groups （t=3.321, 4.183, P〈0.01）. However, after H/R, NF- κB activity and expression of TNF- α gene were significantly reduced, Ⅰ- κB a protein expression was increased in cardiomyocytes of E group compared to other groups （t=-3.425, 3.687, 3.454, P〈0.01）. All theses changes caused by EPO pretreatment were eliminated by the intervention of PDTC （an antagonist to NF- κB） during pretreatment.
Conclusions： EPO pretreatment can inhibit the activation of NF- κB and upregulation of TNF- α gene in cardiomyocytes exposed to H/R through a negative feedback of NF- κB signaling pathway, and thus produces the anti-inflammatory effect. This might be one of the ways EPO produces the anti-inflammatory effect.     

缺氧早期大鼠心肌细胞微管损害的观察

目的 了解缺氧早期心肌细胞微管损害程度。方法将分离培养的Wistar大鼠心肌细胞分为正常组、缺氧组（建立缺氧细胞模型并设缺氧10、20、30、60min为观察时相点）。用激光共聚焦显微镜及扫描电镜观察2组细胞微管分布、形态变化，对微管蛋白荧光强度进行半定量分析．用蛋白质印迹法检测2组细胞游离d微管蛋白的表达。结果 与正常组比较，缺氧10min后，缺氧组细胞微管念珠状结构消失，但排列尚有规律、数量无明显减少；缺氧20min不仅念珠状结构消失，而且微管排列散乱，远离胞核区出现微管缺失；缺氧30、60min时微管发生扭曲、断裂，纹理紊乱，完全丧失规律性。缺氧组心肌细胞微管蛋白荧光强度较正常组下降，且随缺氧时间延长愈加明显；缺氧组心肌细胞内游离的α微管蛋白表达（缺氧10min为46644±145）高于正常组（13357±98），两组比较，差异有统计学意义（P〈0．01），随缺氧时间的延长此升高趋势愈加明显。结论在缺氧状态下，心肌细胞微管发生解聚时间较早，其结构和分布规律被破坏。微管解聚在缺氧所致心肌细胞早期病理损害中的作用值得深入研究。     

瑞芬太尼对人肠系膜小动脉平滑肌细胞L-型钙通道电流的影响

目的观察瑞芬太尼对人肠系膜小动脉平滑肌细胞(MASMCs)L-型钙通道电流的影响,探讨其扩张血管的机制。方法酶解法分离人肠系膜小动脉单个平滑肌细胞,采用全细胞膜片钳技术,以钡电流作为载流子,观察19.4nmol／L瑞芬太尼在不同钳制电压下以及不同浓度瑞芬太尼在最大激活电压下对MASMCs L-型钡电流(TBa-L)的影响。结果19．4 nmol／L瑞芬太尼可抑制TBa-L,使电流密度-电压曲线上移,最大激活电压及翻转电压均向膜的负电位方向移动(P<0．01)。瑞芬太尼浓度依赖性地抑制TBa-L,其半数最大抑制效应的浓度为(39±4)nmol／L。结论瑞芬太尼浓度依赖性地抑制人肠系膜小动脉平滑肌细胞L-型钙通道,从而产生扩张血管的作用。     

Expression of cell cycle regulators during smooth muscle cell proliferation after balloon catheter injury of rat artery

Intimal hyperplasia is defined as the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) with deposition of extracellular matrix. However, the cell cycle regulatory mechanisms of injury-induced VSMC proliferation are largely unknown. To examine the expression kinetics of cell cycle regulatory factors which is known to be worked positively or negatively, we used rat balloon injury model. Marked induction of proliferating cell nuclear antigen (PCNA), G1/S cyclin-dependent kinase (cdk2), and its regulatory subunit (cyclin E) occurred between 1 and 3 days after balloon arterial injury, and this was sustained for up to 7 days and then declined. However, the induction of the negative regulators, p21 and p27, occurred between 3 and 5 days of injury, peaked after 7 and 14 days and was then sustained. VSMC proliferation after balloon catheter injury of the rat iliac artery is associated with coordinated expression of positive (cdk2, cyclin E and PCNA) and negative (p21, p27) regulators. Cell cycle regulators such as cdk2, cyclin E, p21, p27 may be suitable targets for the control of intimal hyperplasia.     

家兔颈动脉内膜损伤后血管平滑肌细胞表型转化及p38的表达变化

目的观察动脉内膜损伤后血管平滑肌细胞(VSMC)表型转化及其与p38表达的关系。方法分别用HE染色、免疫组化和免疫印迹(W estern b lot)方法检测家兔假损伤组(S组)和损伤组损伤后不同时间点血管形态学改变及血管壁中增殖细胞核抗原(PCNA)、平滑肌α肌动蛋白(SMα-actin)和p38蛋白表达的变化。结果⑴内膜损伤后1 d血管中膜管腔侧、3 d管腔内表面可见增殖的VSMC,5~7 d新生内膜(NI)形成并逐渐增厚,14~35 d NI进行性增厚。各组中膜均有增殖的VSMC向腔面集聚。⑵S组动脉中膜VSMC及内皮细胞PCNA为阴性。中膜于损伤后1~14 d,NI于5~14 d PCNA阳性细胞率逐渐增加,14 d达高峰,28 d后开始逐渐减少,且NI阳性率略高于中膜。⑶S组动脉中膜SMα-actin表达为阳性,内皮细胞为阴性。SMα-actin阳性面积于损伤后1 d开始减少,3 d最为明显,5 d后开始逐渐增加,NI阳性表达略低于中膜。⑷S组动脉中膜p38较少或无表达,损伤后1~35 d呈持续高表达,以3~14 d最为明显,NI阳性表达略高于中膜。损伤后p38表达变化与PCNA表达变化呈正相关,且早于SMα-actin表达减少。结论内膜损伤后VSMC增殖能力与其表型转化密切相关,p38参与了损伤后VSMC表型转化的信号转导。     

A Inibição do Metabolismo da Glicose por miR-34a e miR-125b Protege contra a Morte Celular de Cardiomiócitos Causada por Hiperglicemia

Background:It is well-known that insulin resistance and hyperglycemia are important pathological causes for the development of diabetic cardiomyopathy (DCM). However, its precise molecular mechanisms in the pathogenesis of DCM remain unclear.Objectives:Recent studies reveal that microRNAs (miRNA) play essential roles in the pathogenesis of DCM. This project aimed to determine the roles of miR-34a and miR-125b in hyperglycemia-induced cardiomyocyte cell death.Methods:Rat primary cardiomyocytes were isolated and exposed to normal and high concentrations of glucose. Cell viability was measured using MTT assay. Expressions of miR-34a and miR-125b were detected by qRT-PCR. Potential targets of miR-34a and miR-125b were predicted from www.Targetscan.org and validated from human heart tissues. A statistical significance of p<0.05 was considered.Results:The present study shows that miR-34a and miR-125b are downregulated in a human diabetic heart. Moreover, in vitro data from rat primary cardiomyocytes showed that short-term high glucose treatment stimulates miR-34a and miR-125b expressions. Under high glucose, it was found that rat cardiomyocytes displayed increased intracellular glucose metabolism, and glucose uptake and lactate production were significantly increased. It was also found that the key glucose metabolic enzymes, Hexokinase 2 (HK2) and Lactate dehydrogenase-A (LDHA), were direct targets of miR-125b and miR-34a, respectively. Overexpression of miR-125b and miR-34a could prevent hyperglycemia-induced cardiomyocyte cell death. Finally, the restoration of HK2 and LDHA in miR-125b and miR-34a overexpressed cardiomyocytes recovered the cardiomyocytes’ sensitivity to hyperglycemia.Conclusion:Our results proposed a molecular mechanism for the microRNA-mediated diabetic cardiovascular protection and will contribute to developing treatment strategies for diabetes-associated cardiovascular dysfunction.     

二甲基甲酰胺致心肌细胞损伤及维生素C保护作用的实验研究

目的 探讨二甲基甲酰胺(dimethylformamide,DMF)致H9c2心肌细胞损伤及维生素C(vitamin C,VC)对此损伤是否有保护作用.方法 观察DMF染毒及VC干预后心肌细胞及核形态改变;活性氧(reactive oxygen species,ROS)、总抗氧化能力(total antioxidant capacity,T-AOC)试剂盒检测不同浓度DMF染毒不同时间、特定浓度DMF合并不同浓度VC干预后细胞氧化应激水平变化.结果 DMF染毒后细胞及核形态出现不同程度异常,低浓度VC干预后有所改善;100、140、180、220、250 mM DMF染毒24h,100、140、180、200、220 mM DMF染毒48 h,100、125、140、180、200 mM DMF染毒72 h后,同时间不同剂量染毒,细胞ROS、T-AOC水平差异有统计学意义;同剂量不同时间染毒,细胞ROS、T-AOC水平差异有统计学意义(ROS:24 h:F=5 763.00,P＜0.001;48 h:F=5 861.00,P＜0.001;72 h:F =9 188.00,P＜0.001;T-AOC:24 h:F=6.25,P=0.004;48 h:F=11.48,P＜0.001;72 h:F=14.13,P＜0.001).VC (0.025、0.05、0.10、0.25 mM)干预后ROS、T-AOC水平与对照相比差异均有统计学意义(均有P＜0.05).结论 氧化损伤可能是DMF发挥毒性的重要机制;低剂量VC可缓解DMF产生的氧化损伤,对细胞产生保护效应.     

霉酚酸对大鼠肺动脉平滑肌细胞增殖的影响

目的 在细胞水平研究霉酚酸对大鼠肺动脉平滑肌细胞增殖的影响.方法 采用生长曲线、甲基噻唑基四唑(MTT)方法、流式细胞仪检测生长细胞数、细胞的存活值、DNA含量,计算G1,S,G2M和增殖指数.结果 除低浓度(100 nmol/L)外,各霉酚酸组(1、10、100μmol/L)生长细胞数均较对照组减少,差异有统计学意义(p<0.01).MTT法检测霉酚酸组细胞的存活值降低,呈剂量依赖性(100 nmol/L～100 μmol/L).霉酚酸组G1期比例增加,S期比例减少:G2M期比例减少,增殖指数降低,呈剂量依赖性.结论 霉酚酸可有效地抑制大鼠肺动脉平滑肌细胞的增殖,主要作用于DNA合成期,并呈剂量依赖性.其有效浓度在临床可应用的范围内.     

支气管哮喘大鼠气道重塑中气道平滑肌细胞凋亡及地塞米松的干预作用

目的 观察支气管哮喘(简称哮喘)大鼠气道重塑中气道平滑肌细胞(airway smoothmuscle cells,ASMC)凋亡及地塞米松对ASMC凋亡的影响.方法 将清洁级雄性SD大鼠按随机数字表法分为正常对照组、哮喘组和地塞米松干预组,每组12只.以卵蛋白致敏和激发的方法制备大鼠慢性哮喘模型.用脱氧核糖核苷酸末端转移酶(TdT酶)介导的dUTP切口末端标记法(TUNEL法)检测ASMC凋亡并计算凋亡指数.用免疫组织化学和原位杂交法分别检测气道平滑肌Bcl-2、Bax蛋白及其mRNA的表达情况.经SPSS 11.5软件进行统计学分析,实验数据用x±s表示.多组间比较采用方差分析,多组样本均数两两比较,两变量的相关程度采用直线相关分析.结果 (1)对照组、哮喘组和干预组大鼠ASMC的凋亡指数分别为:0.201±0.022、0.030±0.016和0.118±0.043;(2)对照组、哮喘组和干预组大鼠气道平滑肌层Bcl-2蛋白表达的吸光度值分别为0.060±0.012、0.112±0.028和0.080±0.010,Bcl-2 mRNA表达的吸光度值分别为0.065±0.019、0.157±0.019和0.099±0.029;(3)对照组、哮喘组和干预组大鼠气道平滑肌层Bax蛋白表达的吸光度值为0.120±0.020、0.062±0.012和0.093±0.010,Bax mRNA表达的吸光度值分别为0.155±0.025、0.074±0.019和0.118±0.031;(4)相关分析结果显示,ASMC的凋亡指数与气道平滑肌厚度以及Bcl-2蛋白的相对含量呈显著负相关(r值分别为-0.860、-0.783,P<0.01);ASMC的凋亡指数与气道平滑肌Bax蛋白相对含量呈正相关(r=0.837,P<0.01).结论 ASMC凋亡的减少可能参与了哮喘气道重塑过程;地塞米松可以增加促凋亡蛋白Box的表达,同时减少抑凋亡蛋白Bcl-2的表达,从而增加ASMC的凋亡.