低硒心肌损伤与钾离子通道蛋白的关系研究

目的：心肌上的离子通道蛋白与心肌损伤有很大的关系,本研究通过低硒喂养对C57BL／6小鼠心肌组织损伤的影响及其对钾通道蛋白的改变。方法：将实验小鼠分为4组：对照组,低硒30天组,低硒90天组和低硒180天组。采用低硒饲料（硒含量0．0045μg／g）喂养的方法建立低硒小鼠模型,对照组给予正常饲料（硒含量0．256μg/g）,与低硒组同时喂养;硒含量的测定和HE染色方法观察心肌损伤情况,WesternBlotting方法检测其钾通道蛋白的表达。结果：低硒饲料喂养小鼠的心脏硒含量与正常饲料喂养的硒含量相比明显降低（P〈0．01）;并出现轻微的心肌损伤,钾通道蛋白的表达量在低硒30天组,低硒90天组和低硒180天组下调（P〈0．01）。结论：成功建立低硒小鼠模型,低硒能引起小鼠心肌损伤,这种改变可能有心脏的钾通道蛋白的表达水平有关。     

低硒小鼠模型的建立及低硒对心肌的影响

目的建立低硒实验动物模型,观察低硒对心肌的影响。方法利用黑龙江地产酵母配制低硒小鼠饲料,使用配制的小鼠饲料喂养BALB/c幼鼠,经过4个月的喂养,测定血清、肝脏、心肌细胞的硒含量,观察心肌超微结构的变化,测定血清心肌酶的变化。结果利用黑龙江地产酵母配制的低硒饲料,硒含量为0.016 mg/kg,符合低硒标准。BALB/c鼠用该饲料喂养4个月,心肌、肝脏、血清硒含量分别为0.187 mg/kg、0.219 mg/kg、0.241mg/kg,符合低硒诊断标准。观察低硒鼠心肌超微结构,可见心肌细胞线粒体肿胀,细胞核出现了异型性,血清心肌酶较常硒鼠升高。结论利用黑龙江地产酵母成功配制了低硒饲料。经过低硒饲料饲养可建立低硒鼠模型。低硒可以引起BALB/c鼠心肌细胞损伤。     

烟酰胺核糖抑制db/db小鼠糖尿病心肌病的作用及机制研究

目的:探讨烟酰胺核糖(NR)对2型糖尿病小鼠心肌病的治疗作用及其机制。方法:2型糖尿病模型db/db鼠和及其严格对照小鼠db/+小鼠,将小鼠分为Con (db/+)组,DM (db/db)组,DM+NR组。采用超声测小鼠心脏功能,western-blot及免疫组化测SIRT1表达含量,DHE染色、MDA含量和MnSOD活性检测反映氧化应激水平。结果:与对照组相比,db/db小鼠心脏功能显著下降(LVEF:42.3±7.2vs 73.7±10.2, P0.01;LVFS:22.1±4.2vs 42.7±6.9, P0.01),SIRT1表达量显著下调(P0.01)。NR喂养提高SIRT1表达量(P0.01),并有效改善db/db小鼠心脏功能(LVEF:53.1±8.1vs 42.3±7.2, P0.01;LVFS:33.4±6.9vs 22.1±4.2, P0.01)。同时,NR喂养显著降低了db/db小鼠心肌组织的凋亡水平和氧化应激水平(P0.05)。结论:NR有效改善了db/db小鼠的心功能障碍,降低了db/db小鼠的心肌凋亡水平和氧化应激水平,这些作用的发挥可能与NR增加SIRT1的表达量有关。     

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白 (MT) ,发现对照组小鼠肝脏MT含量为 (2 .47± 0 .90 )μg/g湿重组织 ,硫酸锌组小鼠肝脏MT含量为 (8.15± 2 .2 0 ) μg/ g湿重组织 ,硒麦芽组小鼠肝脏MT含量为(12 .80± 1.44 ) μg/ g湿重组织。锌组和硒组的MT含量与对照组相比有显著性差异 (P <0 .0 5 ,P <0 .0 5 )。硒组MT含量要显著高于锌组的MT含量 (P <0 .0 5 )。     

牛磺酸对家兔缺血/再灌注心肌细胞凋亡的影响

目的:研究牛磺酸(Tau)对家兔缺血/再灌注损伤心肌细胞凋亡的影响.方法:阻断家兔心脏左冠状动脉前降支45 min,再灌注180 min引起心肌缺血/再灌注损伤,在心肌缺血前5 min耳缘静脉注射牛磺酸(200mg/kg),应用DNA片段原位末端标记法 (TUNEL染色),DNA凝胶电泳和流式细胞仪(FCM)观测心肌细胞凋亡.结果:琼脂糖凝胶电泳显示损伤对照组(I/R) 心肌DNA呈云梯状改变,而Tau I/R组无此改变.与损伤对照组(I/R) 比较,Tau I/R组缺血心肌凋亡细胞明显减少(TUNEL染色).流式细胞仪测定I/R组及Tau I/R组缺血心肌凋亡率分别为17.66%±1.54%和4.86%±1.23%.I/R组的缺血心肌Fas和Bax蛋白表达较非缺血心肌高 (P<0.01),Bcl-2/Bax比例较非缺血心肌低(P<0.01);而在Tau I/R组,Fas和Bax蛋白表达较I/R组的低 (P<0.01),Bcl-2/Bax比例较I/R组高(P<0.01).结论:牛磺酸可减少I/R家兔心肌细胞凋亡,其机制与调控凋亡相关基因 Fas,Bax和Bcl-2的蛋白表达有关.     

黄芪注射液对缺血性心肌病大鼠心肌的保护作用*

目的: 探究黄芪注射液对缺血性心肌病大鼠心肌保护作用及其机制。方法: 将36只雄性鼠随机分成:对照组(12只)、缺血性心肌病组(12只)及黄芪注射液组(12只);缺血性心肌病组和黄芪注射液组的大鼠开胸结扎冠状动脉,建立缺血心肌病大鼠模型;建立心肌缺血模型后,黄芪注射液组术后注射黄芪注射液(每周一次,剂量:10 g/kg体重),共注射4次,其他两组腹腔均注射相同剂量的生理盐水;4周后给予3组大鼠麻醉后行心电图及心脏彩超后,处死大鼠取心肌标本行电镜检查,观察其心肌病理超微结构的变化,检测大鼠心肌细胞线粒体Ca²⁺浓度和心肌细胞线粒体融合蛋白mitofusin 1(Mfn1)及凋亡因子C/EBP 同源蛋白(chop)表达,以及黄芪注射液对大鼠心肌细胞ATP敏感钾通道电流的作用。结果: 与对照组比较,缺血性心肌病组中大鼠出现心律失常现象;心室扩大,EF值降低;心肌排列紊乱,线粒体空泡化严重;线粒体Ca²⁺浓度增加(P＜0.01);Mfn1表达减低(P＜0.05),chop表达增加(P＜0.01); 与缺血性心肌病组比较,黄芪注射液组中大鼠心律失常发生率明显减少,心肌细胞动作电位时程缩短,心脏彩超及心肌病理明显改善并存在大量线粒体融合,心肌线粒体Ca²⁺浓度和chop表达明显减少(P＜0.01),而Mfn1表达明显增加(P＜0.01),心肌细胞ATP敏感钾电流明显增加(P＜0.01),该作用可被ATP敏感钾通道特异性阻断剂格列本脲阻断。结论: 黄芪注射液明显减少缺血性心肌病大鼠心律失常的发生率,继而改善缺血性心肌病大鼠心脏功能、减轻心肌病理损伤,其作用机制可能通过心肌细胞ATP敏感钾通道所介导。     

硒对小鼠柯萨奇性病毒性心肌炎脂质过氧化反应的影响

目的：探讨硒对小鼠病毒性心肌炎发生中脂质过氧化反应的影响。方法：昆明种小鼠分为常规饲料喂养和适量补硒喂养4周后,腹腔接种柯萨奇B3病毒建立小鼠心肌炎模型,测定小鼠谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）、丙二醛（MDA）含量及心肌病变情况。结果：光镜下补硒组小鼠的心肌病理变化轻,其病理积分明显低于常规喂养组（P〈0.05）。补硒组小鼠GSH-Px、SOD明显高于正常喂养组;MDA明显低于常规喂养组（P〈0.05）。结论：补硒能提高脂质过氧化酶的活性、减少脂质过氧化产物的产生,减轻病毒感染引起的心肌细胞损伤。     

ApoE基因缺陷小鼠的心肌肥厚及辛伐他汀的干预研究

目的:观察高胆固醇喂养的不同周龄ApoE基因缺陷(ApoE-/-)小鼠心肌细胞和心肌间质成分的改变,并观察辛伐他汀对其的影响.方法:36只8周龄雄性ApoE-/-小鼠饲以高胆固醉饲料喂养8周即至16周龄,随机被分为三组继续喂养至24周龄组、32周龄组和40周龄组,每一周龄组为12只,再随机分为模型组6只和辛伐他汀干预组6只(25mg/kg/d),相同周龄的C57BL/6J小鼠设为对照.分别在24周、32周、40周结束时处死小鼠.常规检测血浆胆固醇水平,留取新鲜心脏组织测定总胆固醉及一氧化氮(NO)、超氧化物歧化酶(SOD)、丙二醛(MDA):另取心脏组织固定,石蜡切片,HE染色观察各组小鼠心肌细胞的变化,Masson染色观察心肌胶原改变.结果:24、32和40周龄模型组ApoE-/-小鼠血浆、心脏组织胆固醇和MDA水平逐渐增加(p<0.05),NO和SOD水平逐渐降低(p<0.05),心肌细胞直径和心肌胶原含量逐渐增加(p<0.05).与相同周龄模型组相比,辛伐他汀干预组血浆、心脏组织胆固醇和MDA水平明显降低(p<0.05),心肌细胞直径明显减小;40周龄辛伐他汀干预组左室壁平均厚度明显降低(p<0.05),32周龄和40周龄辛伐他汀干预组心肌胶原含量明显减少(p<0.05).结论:高胆固醇喂养的ApoE基因缺陷小鼠,随着周龄增加、胆固醇水平增加,抗氧化能力降低,心肌细胞直径和心肌胶原含量显著增加,辛伐他汀可能减轻心脏重构.     

三氧化二砷对心肌细胞膜延迟整流钾通道蛋白表达的影响研究

目的:观察不同剂量的三氧化二砷(arsenic trioxide,As2O3)对心肌细胞膜上延迟整流钾电流蛋白表达的影响。方法:将豚鼠随机分为4组:正常对照组、As2O3小剂量组(0.4 mg/kg)、中剂量组(0.8 mg/kg)、大剂量组(1.6 mg/kg),给药后不同时间间隔记录心电图,测量QT间期和RR间期,计算QTc的值的变化,同时应用荧光免疫组化技术检测心肌延迟整流钾通道IKr、IKs通道蛋白的表达量。结果:1在不同剂量的As2O3作用下,0.8 mg/kg和1.6 mg/kg As2O3组的豚鼠QTc明显延长,并且这种延长作用与给药剂量和时间密切相关。在2 h的观察时间内,0.8 mg/kg和1.6 mg/kg As2O3分别使QTc从对照组的324±7 ms延长到368±11 ms(P0.01)和388±11 ms(P0.01)。2大剂量组豚鼠心肌缓慢型延迟整流钾通道Kv LQT1和GPERG蛋白表达与对照组相比显著降低(P0.01)。结论:As2O3对豚鼠心肌QT间期有明显延长效果,其机制可能与降低Kv LQT1和GPERG蛋白的表达,影响了钾通道的功能有关。     

纳米硒通过抗氧化应激调节大脑NO含量改善睡眠剥夺小鼠认知功能

研究了纳米硒对睡眠剥夺(SD)小鼠(Mus musculus)认知功能的影响,并探讨其作用机制。将120只雄性昆明小鼠随机分成两批,第一批24只分为3组:对照组(NC)、亚硒酸钠组(SE)和纳米硒组(NS),分别给予硒浓度为4μg/ml的亚硒酸钠和纳米硒溶液每只0.5ml/d,NC组给等体积蒸馏水,连续30d,第31天测定SE和NS两组小鼠的血硒及全血GSH-Px活性,评价两种硒源的生物利用性;第二批96只分为4组:对照组(N-SeC),纳米硒低、中、高剂量组(L、M、H),L、M和H组分别给予硒浓度为2μg/ml、4μg/ml、8μg/ml的纳米硒溶液每只0.5ml/d,N-SeC组给予同体积蒸馏水,连续30d。第二批小鼠每组又各自分为4小组:SD对照组(SDC)及SD18h、SD36h、SD54h组,采用单平台水环境法(SPM)制作小鼠SD模型。在SD后,N-SeC、L、M和H组利用Y-型迷宫试验测定认知能力,同时测定小鼠大脑GSH-Px、NO、MDA含量。结果表明,纳米硒对GSH-Px活性的提高优于传统硒源亚硒酸钠,但血硒无显著差异;与SDC组比较,SD降低了小鼠的认知能力及大脑GSH-Px活性,提高了NO和MDA含量;与N-SeC比较,纳米硒使SD小鼠的认知功能得到改善,大脑GSH-Px活性提高,MDA和NO含量下降。上述结果表明,纳米硒能够改善SD小鼠的认知功能,这可能与其提高大脑GSH-Px活性并降低了自由基对大脑神经的损害有关。     

Protective effects of the potassium channel opener-diazoxide against injury in neonatal rat ventricular myocytes

The mitochondrial ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. Similarly to plasma membrane K(ATP), the mitochondrial channel is inhibited by antidiabetic sulfonylureas and activated by potassium channel openers, such as diazoxide. In the present work, the cytoprotective properties of diazoxide on the H9c2 cardiac myoblast cell line and neonatal rat ventricular cardiomyocytes were analysed. It was observed that 100 micromol/l diazoxide protected neonatal rat ventricular cardiomyocytes, but not H9c2 myoblasts, against injury induced by hydrogen peroxide or simulated ischemia. Moreover, diazoxide prevented hydrogen peroxide-induced mitochondrial potential depolarisation in neonatal rat ventricular cardiomyocytes. Diazoxide, at the same time, did not affect the expression level of the anti-apoptotic protein bcl-2 in these cells. The protective effects of diazoxide were suppressed by 5-hydroxydecanoic acid, a potassium channel blocker. These observations suggest that activation of the mitochondrial ATP-regulated potassium channel plays an important role in protection of neonatal cardiomyocytes against injury.     

Long-term Selenium Deficiency Increases the Pathogenicity of a Citrobacter rodentium Infection in Mice

Citrobacter rodentium is a mouse pathogen that causes infectious colitis and shares characteristics with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, including the ability to cause attaching and effacing lesions in the colon and serves as a useful model to study the pathogenicity of these bacteria. In this study, mice were fed a selenium-deficient diet for 5 or 20?weeks and then infected with C. rodentium. Colonization of the colon by C. rodentium was similar in mice fed adequate or selenium-deficient diets, but total bacterial colonization of the spleen was elevated in mice fed selenium-deficient diet for 20?weeks. Infection-induced changes to the colon included inflammatory cell infiltration, gross changes in crypt architecture, and ulceration and denuding of the epithelial layer that were greatest in mice fed a selenium-deficient diet for 20?weeks. Expression of pro-inflammatory genes was significantly higher 12-days post-infection in mice fed the selenium-deficient diet for 20?weeks compared to mice fed a selenium-adequate diet or selenium-deficient diet for 5?weeks. Diarrhea was prevalent in mice fed the selenium-deficient diet for 20?weeks but not 5?weeks, and this was associated with decreased expression of solute carrier family 26a3 and carbonic anhydrase IV, genes involved in ion transport. These results indicated that selenium played an important role in resistance to the pathological effects of a C. rodentium infection, and therefore, selenium status may be important in the expression of human disease caused by common food-borne bacteria.     

Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family

Selenium is an essential micronutrient that function through selenoproteins. Selenium deficiency results in lower concentrations of selenium and selenoproteins. The brain maintains it's selenium better than other tissues under low-selenium conditions. Recently, the selenium-containing protein selenoprotein P (Sepp) has been identified as a possible transporter of selenium. The targeted disruption of the selenoprotein P gene (Sepp1) results in decreased brain selenium concentration and neurological dysfunction, unless selenium intake is excessive However, the effect of selenoprotein P deficiency on the processes of memory formation and synaptic plasticity is unknown. In the present studies Sepp1(-/-) mice and wild type littermate controls (Sepp1(+/+)) fed a high-selenium diet (1 mg Se/kg) were used to characterize activity, motor coordination, and anxiety as well as hippocampus-dependent learning and memory. Normal associative learning, but disrupted spatial learning was observed in Sepp1(-/-) mice. In addition, severe alterations were observed in synaptic transmission, short-term plasticity and long-term potentiation in hippocampus area CA1 synapses of Sepp1(-/-) mice on a 1 mg Se/kg diet and Sepp1(+/+) mice fed a selenium-deficient (0 mg Se/kg) diet. Taken together, these data suggest that selenoprotein P is required for normal synaptic function, either through presence of the protein or delivery of required selenium to the CNS.     

严重烫伤延长大鼠心室肌细胞动作电位时程的机制

严重烫伤引起心肌细胞动作电位时程（action potential duration,APD）延长,通过加重烫伤心肌细胞钙紊乱和诱发室性心律失常,促进烫伤心功能障碍的发生,但APD延长的机制尚不清楚。通过制作约40％体表面积（total body surface area,TBSA）Ⅲ度烫伤大鼠模型,在伤后12h大鼠心功能明显减弱时分离其心肌细胞,采用膜片钳技术观察心肌细胞APD以及动作电位复极化相关的重要离子通道电流,包括瞬间外向钾电流（transient outward K^＋ current,Ito）,L-型钙电流（L-type Ca^2＋ current,ICa-L）和内向整流钾电流（inward rectifier K^＋ current,IK1）。结果显示,烫伤后12h单个心肌细胞APD明显延长,APD50和APD90在烫伤组分别为（46．02±3．78）ms、（123．24±12．48）ms（n=19）,明显长于对照组的（23．28±4．85）ms、（72．12±3．57）ms（n=17）（P〈0．01）。烫伤引起,Ito电流密度降低,＋60 mV下烫伤组的电流密度（20．39±1．98）pA／pF（n=25）明显低于对照组的（34．15±3．78）pA／pF（n=20,P〈0．01）;烫伤组在-120至-80mV电压刺激下所产生的IK1电流密度显著低于对照组：而两组之间ICa-L电流密度、电压依赖性的激活和失活无显著性差异。结果提示,烫伤引起心肌细胞APD延长的机制与瞬间外向钾通道和内向整流钾通道功能下调有关。     

调节性T细胞参与调控新生小鼠心肌再生

为探究调节性T（regulatory T,Treg）细胞在新生小鼠心肌损伤后再生中的作用,首先建立新生小鼠心肌再生模型。C57BL/6J（C57）新生1 d小鼠20只随机分成2组。实验组进行心尖切除（apex resection,AR）,假手术（Sham,SH）组只进行开胸。术后7 d取心脏组织,利用在细胞核表达的增殖标志物磷酸化组蛋白H3（phospho-histone H3,pH3）和Ki67分别与在心肌细胞胞质特异表达的α-辅肌动蛋白（alpha-actinin cytoskeletal isoform,α-actinin）,进行免疫共染检测心肌细胞增殖。结果显示,与SH组相比,AR组pH3+及Ki67+的心肌细胞明显增多。而且Masson三色染色结果显示,术后21 d被切除的心肌组织完全再生。为研究Treg细胞是否参与调控新生小鼠心肌损伤后的再生,Western印迹检测Treg细胞特异转录因子叉头/翼状螺旋转录因子3（forkhead box P3,Foxp3）蛋白表达水平。结果显示,术后7 d、14 d,AR组心和脾中Foxp3与SH组相比显著升高（P<0.05）。同时,免疫组化染Foxp3结果显示,术后7 d、14 d, AR组与SH组相比,心尖处有大量的Treg细胞富集。为更直观地检测AR后Treg细胞的数目变化,利用流式细胞仪检测术后7 d Treg细胞数目。结果显示,AR组心和脾中Treg细胞数目与SH组相比显著增多（P<0.01）。为研究Treg细胞对AR后心肌再生的影响,引入注射白喉毒素（diphtheria toxin,DT）的Foxp3DTR小鼠,可特异性敲除Treg细胞。实时定量PCR结果显示,AR+DT组与AR+PBS组相比,抑炎因子白介素IL（interleukin,IL）-10、IL-13与转化生长因子TGF（transforming growth factor,TGF）-β表达均降低（P<0.05,P<0.01,P<0.01）。而促炎因子IL-6、IL-1β和肿瘤坏死因子-α（tumor necrosis factor,TNF-α）表达均升高（P<0.01,P<0.001,P<0.01）。免疫荧光染色检测结果显示,AR+DT组与AR+PBS组相比,术后7 d pH3+及Ki67+的心肌细胞明显减少;并且Masson三色染色结果显示,术后21 d AR+DT组被切除的心肌组织不能再生。综上所述,敲除Treg细胞会加剧AR后的炎症反应,抑制心肌细胞增殖,最终导致新生小鼠心肌再生能力丢失。     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.