改良的乳鼠原代心房肌细胞的培养及鉴定方法

目的:主要探讨原代心房肌细胞的培养及鉴定方法,为进一步研究心房颤动的重构机制及治疗方法奠定基础。方法:选取1-3 d的SD乳鼠40只,雌雄不限,分离心房、心室肌,胰酶联合EDTA充分消化心房肌细胞,利用心房肌与成纤维细胞的差速贴壁及细胞传代方法纯化心房肌细胞,免疫细胞化学染色鉴定心房肌细胞。结果:心房肌细胞培养至第3天,可见心房肌细胞覆盖率高达90%,并出现波动性,免疫细胞化学染色可见90%的心房肌细胞肌经α-肌动蛋白抗体染色阳性。结论:经酶化学消化法可成功培养出原代心房肌细胞,是一种较好的培养及鉴定乳鼠心房肌细胞的方法。     

二十二碳六烯酸对心房颤动大鼠心房肌生理改变及双孔钾通道TASK-1蛋白表达的影响

目的：探讨二十二碳六烯酸（DHA）对大鼠心房颤动（AF）模型心房肌生理特性的影响及相关机制研究。方法：80只乙酰胆碱-氯化钙混合液敏感的SD大鼠分为对照组（CTL组）、DHA处理组（DHA组）、房颤组（AF组）和房颤+DHA处理组（DHA+AF组）,观察房颤持续时间;采用全细胞膜片钳技术记录大鼠心房肌细胞动作电位时程（APD）和双孔钾通道TASK-1电流,Western blot测定大鼠心房组织TASK-1蛋白表达。结果：大鼠尾静脉注射乙酰胆碱-氯化钙混合液后,房颤持续时间随实验天数增加而逐渐延长,DHA干预缩短房颤持续时间。与CTL组相比,AF组大鼠心房肌细胞复极50%时的动作电位时程（APD₅₀）和复极90%时的动作电位时程（APD₉₀）明显缩短,心房肌细胞TASK-1电流密度升高,蛋白表达升高（P<0.05）。与AF组相比,DHA+AF组大鼠心房肌细胞APD₅₀和APD₉₀明显延长,TASK-1电流密度和蛋白表达降低（P<0.05）。结论：DHA具有延长房颤大鼠心房肌细胞APD的作用,可能与其下调心房肌TASK-1蛋白的表达从而降低心房肌细胞TASK-1电流密度有关。     

甲亢源性心房颤动与肾素-血管紧张素系统相关发病机制的研究

目的:研究高甲状腺素导致房颤与肾素-血管紧张素系统(RAS)相关的发病机制。方法:左旋甲状腺素经兔腹腔注射,制作甲亢源性房颤易患模型,同时厄贝沙坦经胃管灌胃,乳兔左心房肌细胞培养药物干预;检测房颤诱发率、左心房肌细胞凋亡情况,检测RAS相关的细胞因子,凋亡蛋白表达情况。结果:中途撤药组、厄贝沙坦组、对照组房颤诱发率低于持续给药组(P<0.05)。中途撤药组、持续给药组、厄贝沙坦组心肌细胞凋亡率、ACE m RNA相对表达量、ACE血浆浓度、表达量、AngⅡ血浆浓度、表达量均高于对照组,持续给药组高于中途撤药组、厄贝沙坦组(P<0.05)。中途撤药组、持续给药组、厄贝沙坦组PARP、caspase3相对表达量均高于对照组,持续给药组高于中途撤药组、厄贝沙坦组(P<0.05)。AngⅡ组药物干预后左心房肌细胞凋亡率高于对照组、甲状腺素组(P<0.05)。结论:高甲状腺素所致的房颤发病机制可能是间接过度激活RAS,循环、组织AngⅡ表达增高,后者诱导心房肌细胞凋亡,左心房发生电-解剖重构。     

氧化应激对原代培养乳鼠心房肌细胞中内质网应激及凋亡因子的影响

目的：研究氧化应激对原代培养乳鼠心房肌细胞凋亡、内质网应激及凋亡因子的影响。方法：实验分2组：对照组、氧化应激组。原代培养乳鼠心房肌细胞,氧化应激组在培养的原代心房肌细胞中加入终浓度为100μmol/L的H₂O₂培养2 h,检测氧化和抗氧化指标超氧化物歧化酶（SOD）活力、丙二醛（MDA）及还原型谷胱甘肽（GSH）含量;检测细胞凋亡、细胞GRP78、GRP94及chop、bax、bcl-2 mRNA表达。结果：与对照组相比较,氧化应激组心房肌细胞SOD活力和GSH含量下降、MDA含量增加（P < 0.01）,细胞凋亡增加（P < 0.01）,细胞GRP78、GRP94、chop、bax mRNA表达增加、bcl-2 mRNA表达减少（P < 0.01）。结论：氧化应激反应可能介导内质网应激反应并激活促凋亡因子表达,抑制抗凋亡因子表达,引起心房肌细胞凋亡增加。这可能与心房纤颤的发生有一定关联性。     

鸡源CD40L基因克隆、蛋白表达及生物活性检测

为获得鸡源CD40L (chCD40L) 蛋白,以鸡脾细胞制备cDNA并以之为模板扩增chCD40L基因,构建pFastBac-chCD40L供体重组质粒,转化感受态细胞DH10Bac,通过筛选及鉴定获得Bacmid-chCD40L重组质粒,转入真核表达系统sf9昆虫细胞进行蛋白表达与纯化,获得His-chCD40L蛋白。此外,构建pQM01-chCD40L质粒,转染HEK 293T细胞进行蛋白表达与纯化,获得Strep-chCD40L蛋白。亲和层析纯化的chCD40L蛋白浓度为0.01 mg/mL。为检测chCD40L蛋白的生物活性,分离和培养3周龄SPF雏鸡的法氏囊组织原代细胞,将chCD40L加入细胞培养液刺激细胞增殖,通过Western blotting试验、间接免疫荧光试验、流式细胞术检测,发现该蛋白能够与法氏囊B淋巴细胞表面的CD40结合,说明chCD40L具有生物活性。成功获得chCD40L蛋白,为原代B淋巴细胞体外培养及IBDV野毒分离与诊断奠定了基础。     

埃博拉病毒(EBOV)蛋白的最新研究进展

埃博拉病毒(EBOV)是一种高致死性的病毒,在其RNA编码的7种蛋白中,糖蛋白GP、基质蛋白VP40以及膜蛋白VP24在病毒粒子的装配、出芽以及致病过程中起着关键的作用。详细介绍了这三种蛋白的结构、功能以及作用机制的最新研究进展,并对EBOV蛋白的研究前景作出了展望。     

荧光定量PCR观察鸭胚肝细胞培养上清DHBV载量的动态变化

通过实时荧光定量PCR(FQ-PCR)观察后天感染和先天感染鸭乙型肝炎病毒(DHBV)的鸭胚肝细胞培养上清中DHBV载量的动态变化,探讨适宜于抗HBV药物的体外实验、HBV生物学特性及乙型肝炎发病机制研究的鸭胚肝细胞模型.筛选先天感染DHBV的阳性鸭胚并进行肝细胞原代培养,对DHBV阴性鸭胚肝细胞原代培养,并以不同浓度的DHBV强阳性血清感染细胞.于细胞接种后不同时间点收集培养上清,FQ-PCR检测分析培养上清DHBV DNA的含量.结果显示:先天感染的鸭胚肝细胞接种第2 d上清中DHBV载量最高,第3 d大幅下降,然后缓慢上升,第8 d达到高峰,至第15 d时仍然与高峰时接近,DHBV载量的数量级都在10<'8>copies/mL没有变化;后天感染不同浓度的DHBV阳性血清的鸭胚肝细胞培养上清中的DHBV载量动态变化趋势基本一致,DHBV感染后第3 d大幅下降,以后逐渐上升,至第11 d达高峰,第15 d又略有下降.结论:先天感染DHBV的鸭胚肝细胞的培养上清病毒载量上升至峰值时间短,病毒载量稳定,更适合于抗HBV药物的体外实验研究.     

糖尿病大鼠心房肌细胞心房特殊颗粒和心房肽免疫反应强度的观察

本研究运用透射电镜及立体计量学方法结合免疫组化技术对糖尿病大鼠右心耳肌细胞心房特殊颗粒(ASG)和心房肽(ANP)免疫反应强度进行了观察和定量研究.实验动物为体重150～250克之间的SD大鼠,糖尿病的诱导采用四氧嘧啶(150mg/kg)右腹股沟区皮下注射.电镜超微结构发现糖尿病未治疗组均出现线粒体肿胀、肌原纤维缩短等病理性改变.ASG膜溶解、排空等现象多见,ASG体密度明显减少.免疫组化分析表明糖尿病未治疗组心肌细胞ANP免疫反应强度减弱.胰岛素治疗组各指标与对照组相比均无显著差别.     

高甘油三酯血症动物模型研究进展

高甘油三酯血症(HTG)与肥胖、代谢综合征和糖尿病密切相关,同时也是诱发急性胰腺炎(AP)和增加动脉粥样硬化性心血管疾病(ASCVD)发病风险的重要影响因素之一,是当前的研究热点.成功建立符合要求的动物模型是开展相关实验研究的关键,本文对HTG动物模型的研究现状进行了系统的总结,以期为深入研究HTG的发病机制和降脂药物...     

Synaptobrevin-2促进心房肌SK2通道转运的机制研究

小电导钙激活钾通道亚型2(SK2通道)主要在心房表达,与心房颤动有关.高频刺激模拟心房快速起搏可以增加SK2电流,但是其中的机制并不完全清楚.本研究旨在研究转运调节蛋白Synaptobrevin-2(VAMP2)在调节心房肌细胞膜SK2通道转运过程中的作用.以培养的新生大鼠(Rattus norvegicus)心房肌细胞(NRAMs)和表达有SK2通道的HEK293细胞为研究对象,采用蛋白质印迹、膜片钳、共聚焦显微成像、流式细胞术和全内荧光反射等技术研究VAMP2对SK2通道转运的影响.结果发现,快速起搏可上调VAMP2蛋白和增加SK2通道蛋白在细胞膜上的表达,而敲低VAMP2可降低NRAMs细胞膜上SK2通道蛋白的表达.在HEK293细胞上共表达VAMP2和SK2,增加了SK2的表达,加速了SK2向细胞膜的转运,并通过抑制SK2内吞来稳定SK2通道蛋白在细胞膜上表达.由以上结果可知,VAMP2可能通过促进SK2通道的表达,转运和稳定细胞膜上的表达,参与快速起搏心房肌细胞SK2功能的增强.     

The WATCHMAN Left Atrial Appendage Closure Device for Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States ¹. Since AF affects primarily elderly people, its prevalence increases parallel with age. As such, it is expected that 15.9 million Americans will be affected by the year 2050 ². Ischemic stroke occurs in 5% of non-anticoagulated AF patients each year. Current treatments for AF include rate control, rhythm control and prevention of stroke ³.The American College of Cardiology, American Heart Association, and European Society of Cardiology currently recommended rate control as the first course of therapy for AF ³. Rate control is achieved by administration of pharmacological agents, such as β-blockers, that lower the heart rate until it reaches a less symptomatic state ³. Rhythm control aims to return the heart to its normal sinus rhythm and is typically achieved through administration of antiarrhythmic drugs such as amiodarone, electrical cardioversion or ablation therapy. Rhythm control methods, however, have not been demonstrated to be superior to rate-control methods ^4-6. In fact, certain antiarrhythmic drugs have been shown to be associated with higher hospitalization rates, serious adverse effects ³, or even increases in mortality in patients with structural heart defects ⁷. Thus, treatment with antiarrhythmics is more often used when rate-control drugs are ineffective or contraindicated. Rate-control and antiarrhythmic agents relieve the symptoms of AF, including palpitations, shortness of breath, and fatigue ⁸, but don''t reliably prevent thromboembolic events ⁶.Treatment with the anticoagulant drug warfarin significantly reduces the rate of stroke or embolism ^9,10. However, because of problems associated with its use, fewer than 50% of patients are treated with it. The therapeutic dose is affected by drug, dietary, and metabolic interactions, and thus requires detailed monitoring. In addition, warfarin has the potential to cause severe, sometimes lethal, bleeding ². As an alternative, aspirin is commonly prescribed. While aspirin is typically well tolerated, it is far less effective at preventing stroke ¹⁰. Other alternatives to warfarin, such as dabigatran ¹¹ or rivaroxaban ¹² demonstrate non-inferiority to warfarin with respect to thromboembolic events (in fact, dabigatran given as a high dose of 150 mg twice a day has shown superiority). While these drugs have the advantage of eliminating dietary concerns and eliminating the need for regular blood monitoring, major bleeding and associated complications, while somewhat less so than with warfarin, remain an issue ^13-15.Since 90% of AF-associated strokes result from emboli that arise from the left atrial appendage (LAA) ², one alternative approach to warfarin therapy has been to exclude the LAA using an implanted device to trap blood clots before they exit. Here, we demonstrate a procedure for implanting the WATCHMAN Left Atrial Appendage Closure Device. A transseptal cannula is inserted through the femoral vein, and under fluoroscopic guidance, inter-atrial septum is crossed. Once access to the left atrium has been achieved, a guidewire is placed in the upper pulmonary vein and the WATCHMAN Access Sheath and dilator are advanced over the wire into the left atrium. The guidewire is removed, and the access sheath is carefully advanced into the distal portion of the LAA over a pigtail catheter. The WATCHMAN Delivery System is prepped, inserted into the access sheath, and slowly advanced. The WATCHMAN device is then deployed into the LAA. The device release criteria are confirmed via fluoroscopy and transesophageal echocardiography (TEE) and the device is released.     

Atrial natriuretic factor

Mammalian atria contain different peptides with potent diuretic, natriuretic, smooth muscle relaxing and blood pressure lowering properties. A preprohormone of these peptides is synthetized and stored in specific granules in atrial myocytes. Different peptides have been isolated, analyzed and in vitro synthetized. Their biological activity indicates a potential role in the regulation of volume and sodium homeostasis as well as in blood pressure regulation.