糖尿病大鼠心肌磷酸受纳蛋白基因表达和肌浆网 Ca2+-ATPase活性的变化

目的：观察糖尿病(DM)大鼠心肌磷酸受纳蛋白（PLB）基因表达和肌浆网 Ca2+-ATPase活性的变化及其与心功能的关系。方法:复制糖尿病大鼠模型，分别于4、6、8周后对糖尿病组和对照组进行左心室血流动力学检测，测定心肌PLB mRNA转录水平以及蛋白表达水平变化，检测心肌肌浆网 Ca2+-ATPase活性。结果:糖尿病大鼠心肌PLB mRNA转录和蛋白表达水平4周时与正常大鼠无明显差异，6周时和8周时明显高于正常大鼠；肌浆网 Ca2+-ATPase活性4周时无明显改变，6周时和8周时明显低于正常大鼠；糖尿病大鼠4周时LVSP、LVEDP、±dp/dtmax与正常大鼠无明显差异，6周、8周时LVSP、±dp/dtmax显著降低，LVEDP显著升高。结论：糖尿病大鼠心肌PLB表达水平升高，肌浆网 Ca2+-ATPase活性降低，引起心功能下降。     

腺相关病毒介导的PLB基因反义RNA对糖尿病大鼠心肌肌浆网

目的: 构建磷酸受纳蛋白(PLB)反义RNA腺相关病毒载体(rAAV-asPLB),建立糖尿病(DM)大鼠模型。直接心肌注射rAAV-asPLB,观察其对DM大鼠心肌PLB基因转录和蛋白表达的影响,以及对心肌肌浆网Ca²⁺-ATPase活性的作用。方法: 利用质粒辅助重组腺相关病毒系统试剂盒构建rAAV-asPLB。腹腔注射链脲佐菌素(STZ)诱导DM大鼠模型,将实验动物分为4组:正常组、DM组、盐水组和rAAV-asPLB组。盐水或rAAV-asPLB注射后6周,RT-PCR检测心肌PLB mRNA转录;Western blotting检测PLB蛋白表达水平;检测心肌肌浆网Ca²⁺-ATPase活性。结果: (1)成功构建rAAV-asPLB,诱导出DM大鼠模型。(2)DM组和盐水组PLB mRNA水平均高于正常组;rAAV-asPLB组PLB mRNA水平较DM组和盐水组明显降低。 (3)DM组和盐水组PLB 蛋白水平均高于正常组;rAAV-asPLB组PLB 蛋白水平较DM组和盐水组明显降低。(4)肌浆网Ca²⁺-ATPase活性在DM组和盐水组中较正常组明显降低,而rAAV-asPLB组较DM组和盐水组升高。结论: rAAV-asPLB抑制DM大鼠心肌PLB表达,增强Ca²⁺-ATPase活性。     

糖尿病大鼠心肌肌浆网钙调蛋白基因表达的改变

目的:探讨糖尿病大鼠心肌细胞肌浆网钙调蛋白基因表达的改变。方法: 雄性SD大鼠经尾静脉注射四氧嘧啶(40 mg/kg)复制糖尿病大鼠模型,对照组注射生理盐水,分别于4,6周处死,取心室肌组织,应用逆转录-聚合酶反应技术以肌动蛋白为内参照,测定肌浆网钙调蛋白钙泵(SERCA )、磷酸受纳蛋白(phospholamban)、ryanodine受体2型(RyR₂)、1,4,5三磷酸肌醇受体2型(IP₃R₂)mRNA的变化。结果: 4,6周后糖尿病组大鼠的体重和心脏重量均低于正常对照组,但心脏/体重比显著大于正常对照组。4周糖尿病组大鼠心肌肌浆网SERCA 、磷酸受纳蛋白、RyR₂、IP₃R₂ mRNA的表达与对照组相比无显著差异;6周的糖尿病组大鼠心肌肌浆网Ca²⁺-ATP酶 的 mRNA表达无明显变化,但磷酸受纳蛋白的mRNA表达显著高于而RyR₂、IP₃R₂的mRNA表达显著低于对照组。结论: 糖尿病大鼠心肌细胞的肌浆网磷酸受纳蛋白的mRNA表达增加,RyR₂、IP₃R₂的mRNA表达下降。     

原发性甲状腺功能减退与肾功能的相关性分析

目的:探讨原发性甲状腺功能减退症(原发性甲减)患者甲状腺功能与肾功能的关系.方法:检测68例原发性甲减患者和50例正常对照组的空腹血清游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)、高敏促甲状腺激素(hs-TSH)、尿素氮(BUN)、肌酐(Cr)、尿酸(UA)、肌酐清除率(CCr)、尿蛋白定性,并进行相关性分析.结果:原发性甲减患者BUN、Cr、UA、尿蛋白阳性率均明显高于正常对照组,而CCr明显低于正常对照组,并且FT3与CCr呈正相关(r=0.682,P<0.05),与UA呈负相关(r=-0.415,P<0.05).结论:提示原发性甲减患者常伴BUN、Cr、UA、尿蛋白阳性率升高,CCr下降;血清hs-TSH、FT4与肾功能无明显相关性,而CCr水平下降与FT3密切相关.     

心肌肌浆网Ca2+-ATP酶基因转导改善慢性心力衰竭犬心功能的研究

目的： 探讨以重组腺相关病毒(rAAV)为载体的心肌肌浆网Ca2+-ATP酶(SERCA2a)基因转导对慢性心力衰竭犬心功能的影响。方法: 选取成年比格犬17只，随机分为对照组4只和心力衰竭组11只。快速右心室起搏建立慢性心力衰竭犬模型并随机分为心力衰竭组4只、心力衰竭＋绿色荧光蛋白（EGFP）组4只、心力衰竭＋SERCA2a组5只(其中1只在开胸后死亡)。接受基因导入的心力衰竭犬行开胸术，分别向心肌内注射携带EGFP和SERCA2a基因的rAAV载体。于基因转导30d时停止起搏后进行超声心动图和血流动力学检查。结果: 基因转导30 d时，心力衰竭＋SERCA2a 组犬的症状及超声心动图指标与心力衰竭＋EGFP组相比有显著好转（P<0.05）；与对照组相比无显著差别。血流动力学监测发现，转导SERCA2a的犬LVSP、+dp/dtmax和-dp/dtmax明显升高，平均值较EGFP组分别增加54.12％［(214.72±31.74)mmHg vs (139.32±36.79)mmHg］、146.81％［(6 779.43±217.58)mmHg/s vs (2 746.85±931.23)mmHg/s］和71.52％［(-4 341.42±322.02)mmHg/s vs (-2 531.14±616.15 mmHg/s)］，LVEDP则降低了63.43％［(21.86±6.95)mmHg vs (59.78±6.92)mmHg］，所有参数与对照组相比无显著差异。心力衰竭＋EGFP组犬心肌冰冻切片在激光共聚焦显微镜下可见弥漫绿色荧光。结论: 以rAAV为载体介导SERCA2a基因转导能够改善慢性心力衰竭犬心脏的收缩和舒张功能，是1种有前景的治疗慢性心力衰竭的方法。     

钙释放通道稳定蛋白过度表达对心力衰竭心室肌细胞肌浆网功能的影响

目的： 探讨钙释放通道稳定蛋白FKBP12.6过度表达对心力衰竭心室肌细胞肌浆网（SR）功能的影响。方法： 用携带FKBP12.6基因的重组腺病毒Ad.FKBP12.6-GFP感染分离的心力衰竭心室肌细胞，通过RT-PCR和Western blotting技术检测转基因的表达；通过局部场刺激诱发胞内钙瞬变，SR钙容量则由咖啡因诱发的钙释放估测；以X-rhod-1-AM作为Ca2+指示剂，采用激光共聚焦线扫描检测细胞内Ca²⁺ 浓度的变化。结果： Ad-FKBP12.6-GFP组的FKBP12.6 mRNA 和蛋白表达水平分别比对照组升高5倍和4倍；Ad-FKBP12.6-GFP感染细胞的钙瞬变幅度(F/F₀峰值，3.16±0.42 vs 1.43±0.38, P<0.01)和SR钙容量(F/F₀峰值, 4.15±0.54 vs 2.23±0.44, P<0.01)均显著高于Ad-GFP感染细胞。结论： 采用基因转移技术上调FKBP12.6的表达可能是心力衰竭治疗的一条新途径。     

甲状腺机能减退和亚临床甲状腺机能减退时缺血修饰蛋白的变化

目的 探讨甲状腺功能减退和亚临床甲状腺功能减退时缺血修饰白蛋白(IMA)以及丙二醛(MDA)水平的变化,同时观察甲状腺激素与氧化应激之间的关系.方法 选取35例甲状腺功能减退和35例亚临床甲状腺功能减退患者作为病例组,35例健康人作为正常对照组,分别检测病例组和对照组的促甲状腺激素(TSH)、游离三碘甲状原氨酸(FT3)、游离甲状腺素(FT4)、IMA、白蛋白(ALB)、IMA/ALB、MDA、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)水平.结果 与对照组相比,甲减组和亚临床甲减组MDA和IMA水平显著升高(P＜0.05),IMA/ALB也显著升高(P＜0.05).甲减组,TSH水平与MDA(r=0.372,P=0.043),IMA(r =0.409,P=0.025)和IMA/ALB比值(r=0.445,P=0.014)呈显著正相关;亚临床甲减组各变量之间没有显著相关性.结论 由于氧化应激的存在,甲减患者抗氧化状态降低导致IMA和MDA水平增加.     

妊娠10-16周甲状腺功能减退的流行病学研究

目的通过孕期血清检查,统计本地妊娠期临床及亚临床甲状腺功能减退的发病情况,探讨妊娠10-16周进行甲状腺功能检查的筛查意义。方法随机选取来429位我院定期产检的妊娠10-16周的孕妇,进行产科常规检查的同时进行血液甲状腺功能三项的检测:促甲状腺激素(TSH),血清游离三碘甲状腺原氨酸(FT3)与血清游离甲状腺素(FT4)。并进一步追踪其妊娠结局及新生儿结局。结果 429例妊娠10-16周孕妇中临床及亚临床甲状腺功能减退共24例,患病率为5.6%,其中临床甲减为2例,占全部孕妇的0.47%。甲状腺功能亢进20例,患病率为4.7%,20孕周时复查甲状腺功能调整亚甲减患病率为3.72%。早产发生率在甲减及亚甲减组高于正常人群组。结论在妊娠10-16周进行甲状腺功能检查,对及时发现妊娠期甲状腺功能异常有重要意义,可作为该孕期常规产检项目。     

急性心肌梗死大鼠钙调蛋白的改变及卡维地洛的干预作用

目的：研究急性心肌梗死（AMI）后心力衰竭大鼠心肌钙调蛋白肌质网Ca2+-ATP酶（SERCA）和受磷蛋白（PLB)的变化及卡维地洛对其干预作用。 方法： 选取AMI术后成活的雄性SD大鼠随机分为AMI组、卡维地洛组两组。给药6周后观察血流动力学参数、心室重构指标及钙调蛋白SERCA、PLB的蛋白和mRNA表达。另设正常对照组及假手术组。 结果： AMI组左室舒张末压（LVEDP）、各心室重量均显著大于假手术组，左室内压最大收缩和舒张速率（±dp/dt）显著低于假手术组；SERCA蛋白和mRNA表达显著低于假手术组（P<0.01），PLB蛋白和mRNA表达高于假手术组（P<0.01）。卡维地洛组的LVEDP、心室重量均显著低于AMI组，±dp/dt显著高于AMI组；卡维地洛治疗使SERCA蛋白和mRNA表达明显升高（P<0.05），但未能改变PLB蛋白和mRNA水平(P>0.05）。 结论： 急性心肌梗死后心力衰竭中钙调蛋白SERCA和PLB的变化可能是心肌收缩功能失调的重要机制；卡维地洛能有效地抑制大鼠AMI后心室重构并改善血流动力学，其分子机制可能与钙调蛋白SERCA含量正常化有关。     

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium

The enhanced diastolic Ca²⁺ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium- ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (V _max) was significantly reduced in failing heart preparations (NF crude membranes, 130±8; DCM crude membranes, 102±5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488±35; DCM crude membranes, 494±42; P=0.92), phospholamban (NF crude membranes, 497±51; DCM crude membranes, 496±45; P=0.98) and calsequestrin (NF crude membranes, 109±06; DCM crude membranes, 107±08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca²⁺ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca²⁺ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function. Received: 2 September 1997 / Accepted: 2 December 1997     

兔膈肌肌浆网Ca2+-ATPase活性及钙离子的转运

本文分别用定磷法测定兔膈肌SR Ca 2 -APTase 活性、Fura-2 荧光法测定SR Ca 2 释放、摄取动力学和[ 3 H] -Ryanodine 与RyR 结合实验测定SR RyR 的量,分析其功能特性。 结果显示兔隔肌、心肌和骨骼肌SR Ca 2 -APTase 活性分别为70.13 ±8.25、 41.25 ±6.25 和120.17± 17.03 m mol/L pi/mg 蛋白/h1 。膈肌的SRCa 2 -APTase 活性显著高于心肌P<0.01 。但明显低于骨骼肌P<0.01; 膈肌SR Ca 2 释放量和摄取速度显著快于心肌（P<0.01）,但明显低于骨骼肌（P<0.01）;膈肌SR RyR 同[ 3 H] Ryanodine 的最大结合值（Bmax）是0.78 ±0.05pmol/mg 蛋白,其解离常数（KD）是6.93 1.13nmol/L,分别位于心肌和骨骼肌范围内。本文认为膈肌Ca 2 释放单位、SR Ca 2 -APTase 和SR Ca 2 释放摄取动力学分别具有心肌和骨骼肌的一些特征,其Ca 2 释放可能具有变构偶联和CICR 偶联两种形式,心肌型DHPR 亚型,RyR 3 和SERCA 2 a 的存在可能是膈肌ECC 依赖于细胞外Ca 2 的主要原因。     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

苯那普利对自发性高血压大鼠心肌缺血-再灌注心功能、氧自由基和肌浆网Ca2+-ATP酶的影响

目的:探讨血管紧张素转换酶抑制剂(ACEI)对自发性高血压大鼠(SHR)心肌缺血-再灌注心功能、氧自由基和肌浆网Ca²⁺-ATP酶的影响。方法:30只10周龄雌性SHR分为2组,SHR对照组(SHR)、SHR+B组(每日10mg/kg苯那普利);另15只同周龄、同性别Wistar大鼠作为对照组(Wistar)。治疗12周后,每组大鼠结扎左冠状动脉前降支30min,再灌注30min,观察血流动力学参数,左室心肌丙二醛(MDA)含量、超氧化物岐化酶(SOD)活性及肌浆网(SR)Ca²⁺-ATP酶活性。结果:与Wistar组比较,SHR组血压、左心室重/体重较高,左心功能损害程度较重,心肌MDA含量较高,SOD活性和SRCa²⁺-ATP酶活性较低;SHR+B组血压、左心室重/体重、左心功能损害程度,SRCa²⁺-ATP酶活性无显著性差异,但心肌MDA含量较低,SOD活性较高。结论:苯那普利可逆转SHR左室肥厚,促进缺血-再灌注心肌SRCa²⁺-ATP酶活性的恢复和减少氧自由基损害,从而减轻缺血-再灌注心功能损伤。     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

RNA阵列技术检测自发性高血压大鼠肌浆网Ca2+-ATP酶和受磷蛋白基因表达

目的:探讨肌浆网Ca²⁺-ATP酶(SERCA)和受磷蛋白(PLB)在原发性高血压发病过程中的变化特点及其相互关系。方法:提取2、4、6、8、10、12不同周龄雄性自发性高血压大鼠(SHR)和正常血压大鼠(WKY)的心室肌、血管平滑肌、肝脏和肾脏组织的总RNA,共294个样品,利用高通量RNA阵列技术(RNAarray)检测SERCA和PLB基因在不同周龄SHR和WKY中mRNA表达谱改变。结果:SHR在6、8、10、12周龄血压出现显著高于同周龄WKY(均P<0.01),10、12周龄心室肌重量／体重比出现显著增加(均P<0.01),心肌和血管平滑肌SERCA的表达在4、6、8、10、12周龄出现显著高于同周龄WKY(P<0.05或P<0.01)。PLB基因表达在两组间无显著差异(P>0.05)。心肌的SERCA与PLB表达量比值在6、8、10、12周龄出现显著大于同周龄WKY(P<0.05或P<0.01),而血管平滑肌的SERCA与PLB表达量比值在4、6、8、10、12周龄出现显著大于同周龄WKY(P<0.05或P<0.01)。结论:肌浆网SERCAmRNA表达改变及SERCA与PLB比例失常是高血压发生和发展过程中重要的分子生物学机制。