Spraque-Dawley大鼠心室肌细胞分离和胞内钙离子浓度测定

目的 探讨Spraque-Dawley大鼠心室肌细胞分离和胞内钙离子浓度测定方法.方法 采用三步法行逆行主动脉灌流获得单个耐钙心肌细胞,然后采用胞内钙荧光测定技术测定细胞内钙离子浓度.结果 在分离过程中如整个心脏保持红润,则细胞数量在90%以上,耐钙细胞KB液中孵育后在70%左右;在分离过程中心脏局部保持红润部位的细胞数量在80%以上,耐钙细胞在60%左右,而苍白区细胞数量变异较大,但一般均在50%以下,且耐钙细胞较少;在分离过程中如整个心脏始终苍白,则细胞数量不仅低于30%,且几乎没有耐钙细胞.采用荧光探针Fura-2/AM,可准确测定细胞内钙离子浓度.结论 通过本研究采用的方法,不仅可获得大量耐钙心肌细胞,而且可准确测定细胞内钙离子浓度.     

膜片钳实验中心肌细胞快速分离方法分析

目的介绍一种膜片钳实验中简便、快速分离大鼠和豚鼠心肌细胞的方法,探讨影响细胞分离产量和质量的原因。方法迅速取出动物心脏,挂在Langendorff装置上,分别用无钙台式液和酶液进行灌流一段时间,然后剪取心室部分,置于KB液中将其剪碎吹打分散,筛网过滤将滤液放置在2℃～6℃冰箱中冷藏4h后可进行膜片钳实验。结果用此方法分离出的心肌细胞存活率高,结构完整,具有良好的耐钙性和较长的存活时间。结论控制好分离心肌细胞分离中的各种实验条件和熟练的实验操作,是分离出适合于膜片钳实验的心肌细胞的重要因素。     

大鼠心肌细胞急性分离方法及影响因素探讨

目的探讨酶解法急性分离大鼠心肌细胞的相关影响因素。方法采用改良Langendorff主动脉逆行灌流法对大鼠全心工作细胞进行酶解分离,分析分离过程中的诸多条件,从中优化最佳的分离方案,为膜片钳实验提供合格细胞。结果分离所用灌流液pH值为7.35~7.40,分离细胞的存活率最高。不同复钙方法对后续细胞存活有明显影响。三步梯度复钙后的细胞存活率明显高于一步、二步复钙法。细胞分离时,灌流速度对细胞存活率有较大影响,3mL/min流速时细胞存活率最高,不同灌流流程对分离细胞的质和量较大影响。采用钙-无钙台氏液先后灌流后再行酶解分离方式可获得长杆状、棱角分明、立体感强的心肌细胞,该细胞复钙后存活率为(56.8±2.6)%;直接使用无钙台氏液灌流后再行酶解分离方法获得(57.2±3.3)%的心肌细胞,其复钙后存活率为(36.7±3.5)%,前述方法明显优于后者。结论灌流液的pH值、灌流速度、细胞分离后的复钙方法及不同的灌流流程等因素可对大鼠心肌细胞的急性分离结果产生显著影响。     

成年大鼠心室肌细胞的急性分离方法

目的介绍一种新型的成年大鼠心肌细胞的急性分离方法。方法麻醉大鼠后快速取心脏,为心脏先循环灌流无钙台氏液,后循环灌流酶液,酶解完成后取左心室,迅速置于含0.5 mg/ml BSA的KB液(恒温37℃)中拉碎,吹打数次后离心,去上清,温育在含0.5 mg/ml BSA的KB溶液中35 min,梯度复钙。结果首次分离细胞存活率在80%~90%,复钙完成仍有40%~50%的细胞维持杆状,横纹清晰且90%以上细胞处于静止状态。结论通过该方法可获得稳定、高比例的存活心肌细胞,能够为成年大鼠心肌细胞原代培养及电生理学研究提供高品质细胞。     

犬心房肌细胞分离的方法学探讨

目的建立稳定的适于膜片钳实验研究的犬心房肌细胞分离方法。方法取健康成年犬心脏12个,采用Langendorff灌流,行回旋支插管,正常台式液灌流10 s,使心脏自主收缩排除残留余血。持续高钾液灌流,同时结扎其他血管及分支,剪去其余心脏组织,待左房及左心耳充盈,无钙台氏液灌流5 min,125 U/ml胶原酶Ⅱ200ml反复灌流消化约30~45 min,后用无钙台氏液冲洗心脏5 min,剪下心房肌组织,KB液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,逐步复钙法复钙后,室温静置1 h,应用于膜片钳记录。结果分离的活性心肌细胞比率约90%,形态呈杆状、横纹清晰、膜周边光滑完整。结论采用本方法可以获得高产量与高质量的用于膜片钳离子流检测的心房肌细胞。     

成年小鼠心肌细胞的分离及电生理特性

目的 :摸索成年小鼠心肌细胞分离方法并观察其电生理特性。方法 :采用酶消化法分离单个心肌细胞 ,应用膜片钳全细胞记录技术记录钙电流和钾电流。结果 :本法分离所得心肌细胞横纹清晰 ,一次性复钙 ,可获得 5 0 %～6 0 %的耐钙细胞 ;并具有正常电生理活性 ,易于形成高阻封接 ,可用于钙电流和钾电流记录。结论 :本实验所采用的分离方法经济、简便、成功率高 ,所获细胞具有正常的电生理活性     

胚胎期及出生后不同年龄段小鼠心肌细胞体外分离方法及优化

目的:基于目前已有报道的心肌细胞体外消化及分离方法,对胚胎期及出生后不同年龄段小鼠心肌细胞体外分离的方法进行系统探索和优化。方法:取胚胎期及出生后不同年龄段小鼠心脏,运用不同方法分离心肌细胞。利用Count Star仪器检测心肌细胞大小及活性。取细胞悬液涂片,针对心肌细胞标志物α辅肌动蛋白(α-actinin)表达进行免疫荧光染色,用共聚焦显微镜观察心肌细胞形态。结果:用胰酶消化法分离胚胎期小鼠心肌细胞,获得存活率大于94%的心肌细胞。用心脏解离试剂盒法(Gentle MACS法)体外分离新生小鼠(出生后1～3 d)心肌细胞,获得存活率约为97%的心肌细胞。分别用Gentle MACS法、非Langendorff灌流法分离出生后4 d和7 d的小鼠心肌细胞,前者仅获得存活率为58%的心肌细胞,而后者获得的心肌细胞存活率为70%～80%。对于成年小鼠,非Langendorff灌流法和Langendorff灌流法均可获得杆状率、存活率都在70%左右的心肌细胞。结论:结合文献以及本实验结果发现,分离胚胎期小鼠心肌细胞可用胰酶消化法;分离出生后1～3 d小鼠心肌细胞可用Gentle MACS法。对于出生后4 d和7 d的小鼠,非Langendorff灌流法可获得存活率及杆状率均较高的心肌细胞,分离成年小鼠心肌细胞则适合用非Langendorff灌流法和Langendorff灌流法。     

Wistar大鼠乳鼠与成年鼠心室肌细胞的分离与性质鉴定

目的 探索和优化稳定的适于电生理实验研究的乳鼠及成年大鼠心室肌细胞分离方法。方法 切碎乳鼠心室肌,胰蛋白酶消化,差速贴壁2 h纯化心室肌细胞,台盼蓝染色判定心肌细胞活力,体外培养48 h后分别行倒置显微镜观察细胞形态,免疫组化鉴定,微电极阵列记录细胞搏动频率和场电位。采用Langendorff灌流成年大鼠心脏,主动脉逆行插管,胶原酶域反复灌流消化约30 min,无钙台氏液冲洗心脏5 min,剪下心室肌组织,台氏液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,将细胞悬液用逐步复钙法复钙后,室温静置1 h,用于膜片钳记录。结果 经4 -6次消化后,乳鼠心室组织消化完全,细胞存活率大于80%。倒置显微镜下观察,细胞呈梭形、多角形。 12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30 - 80次/分。 琢鄄辅肌动蛋白（琢鄄actin）经免疫组化检测,纯度达96%。 Langendorff灌流酶解法可获得形态呈杆状、横纹清晰、膜周边光滑完整、立体感强的单个成年鼠心肌细胞,存活率85%,复钙后存活率50%,可用于膜片钳记录。结论 采用本方法可以获得高产量与高质量的用于电生理检测的心室肌细胞。     

腺病毒过表达TNNI3K基因对成年大鼠心肌细胞肌丝钙敏感度的影响

目的检测TNNI3K基因过表达对成年大鼠心肌细胞肌丝钙敏感度变化的影响,为进一步研究TNNI3K基因在心肌收缩功能、心肌肥厚、心衰及其它心脏疾病中的作用提供了可靠有效的研究模型。方法恒压Langedoff灌流装置逆向灌流法分离成年大鼠心肌细胞,之后对该心肌细胞进行逐级复钙。然后,将心肌细胞接种于改良的M199培养基中。分别感染Ad.GFP病毒和Ad.TNNI3K病毒,24小时后,以IonOptix单细胞可视化动缘系统检测心肌细胞肌丝钙敏感度。结果成功分离培养了成年大鼠心肌细胞,并在该细胞中成功过表达了TNNI3K基因。钙敏感度测定结果表明,TNNI3K基因能够增强心肌细胞肌丝钙敏感度。结论在本实验成功分离培养的成年大鼠心肌细胞中以腺病毒载体过表达TNNI3K基因,可增强心肌细胞肌丝的钙敏感度,该模型为进一步研究TNNI3K基因在心肌收缩功能中的作用及机制奠定了基础。     

溶血磷脂酸对幼年大鼠心肌细胞收缩和钙瞬变的影响

目的我们前期的研究发现LPA受体在幼年大鼠心脏的表达显著高于成年的表达,提示LPA信号在心脏收缩功能尚未成熟时可能具有更为重要的调节作用。本研究通过观察LPA对此未成熟阶段心肌细胞钙瞬变和收缩力的作用,探讨LPA信号对幼年心肌兴奋-收缩耦联的影响。方法 Langendorff装置逆向灌流胶原酶分离获得不同发育阶段大鼠心肌细胞;采用IonOptix细胞收缩和钙离子浓度同步测定系统进行心肌细胞收缩力和钙瞬变的测定;Western blot检测不同发育阶段心肌细胞LPA受体的表达。结果 LPA对出生后14天和21天大鼠心肌细胞的收缩和钙瞬变均没有显著影响,表明LPA信号并不参与出生后14-21天大鼠心肌细胞的兴奋-收缩耦联过程和心肌细胞收缩。在大鼠出生后发育过程中,LPA受体在心肌细胞的表达在出生后14天已显著下调,不同于在整体心脏表达下调的时间点(P21d),这可能是LPA对此发育阶段心肌细胞的收缩和钙瞬变均不产生影响的原因,提示在出生后14天LPA信号对心肌细胞的发育调节功能可能即已减弱或消失。结论 LPA信号对出生后14天及之后心肌收缩和钙瞬变无显著影响,但尚不能排除LPA对出生后更早期的未成熟心肌细胞收缩和兴奋-收缩耦联的作用。     

Effect of calcium on the dissociation of the mature rat heart into individual and paired myocytes: electrical properties of cell pairs

The dissociation of adult rat heart into individual, functionally intact, calcium-tolerant myocytes requires precise manipulation of extracellular calcium levels. Dissociation of intercellular connections is achieved by lowering extracellular calcium to micromolar levels for a short period. By imposing a very small increment in free calcium activity (from 14 to 17 microM) during this period, we achieve a significant yield of functionally intact pairs of myocytes still joined at the intercalated disc. We obtain fewer intact cells, but many of these are paired end to end. These findings permit us to describe some structural characteristics of intercellular connections between cardiac cells and to report unambiguous measurements of electrotonic coupling and dye transfer between rat cardiac cell pairs. We find that the strength of electrical coupling between cells isolated as pairs with intact junctional contacts is much greater than that measured between cell pairs that have formed new junctional contacts.     

犬三层心室肌细胞的分离和鉴别

目的探讨有效分离和鉴别犬三层心室肌细胞的方法。方法用带有左室前降支的犬心肌组织块灌流分离心室肌细胞,得到的心肌细胞先根据解剖部位大致分成三层,再采用膜片钳技术,在电流钳模式下,随机选择各层15个细胞记录动作电位(AP),根据AP的形态、时程、频率依赖性进一步判断是否为相应层的心室肌细胞。结果经左室前降支插管灌流心肌组织块,可以得到数量多、状态好的心肌细胞。在15个心室肌细胞中,能准确判断其层次的有:外层7个、中层5个、内层8个。结论经冠状动脉插管灌流分离犬心室肌细胞是可行的,结合解剖部位和动作电位特点,能有效鉴别不同层的心室肌细胞。     

A method for detecting chaos in canine myocardial microcirculatory red cell flux

OBJECTIVE: To determine whether red cell movement, as measured by laser Doppler velocimetry, in the capillary net of the beating heart is chaotic. METHODS: Using two dog hearts, in situ red blood cell flux was measured at many sites. Simultaneously, epicardial arterial flow and left ventricular pressure were recorded via transit-time flowmeter and catheter manometer, respectively. The presence or absence of chaos was tested by two methods: Lyapunov exponents and correlation dimension. RESULTS: For capillary red cell flux, the Lyapunov was strongly positive at most sites. It was less so for coronary arterial flow and least for left ventricular pressure. Correlation dimension calculation was less able to distinguish the presence or absence of chaos in capillary red cell tissue flux, coronary arterial flow, and left ventricular pressure. CONCLUSIONS: Capillary red cell flux (movement of red cells in capillaries) is nonlinear, (i.e., chaotic). This complexity suggests that the primary control for oxygen delivery to cardiac myocytes by red blood cells resides in the microcirculation. Also, capillary red cell flux is bifractal, suggesting an ordering of control.     

Identification and properties of ATP-sensitive potassium channels in myocytes from rabbit Purkinje fibres

OBJECTIVE: Our goal was to identify the ATP-sensitive potassium (KATP) channels in cardiac Purkinje cells and to document the functional properties that might distinguish them from KATP channels in other parts of the heart. METHODS: Single Purkinje cells and ventricular myocytes were isolated from rabbit heart. Standard patch-clamp techniques were used to record action potential waveforms. and whole-cell and single-channel currents. RESULTS: The KATP channel opener levcromakalim (10 microM) caused marked shortening of the Purkinje cell action potential. Under whole-cell voltage-clamp, levcromakalim induced an outward current, which was blocked by glibenclamide (5 microM), in both Purkinje cells and ventricular myocytes. Metabolic poisoning of Purkinje cells with NaCN and 2-deoxyglucose caused a significant shortening of the action potential (control 376 +/- 51 ms; 6 min NaCN/2-deoxyglucose 153 +/- 21 ms). This effect was reversed with the application of glibenclamide. Inside-out membrane patches from Purkinje cells showed unitary current fluctuations which were inhibited by cytoplasmic ATP with an IC50 of 119 microM and a Hill coefficient of 2.1. This reflects approximately five-fold lower sensitivity to ATP inhibition than for KATP channels from ventricular myocytes under the same conditions. The slope conductance of Purkinje cell KATP channels, with symmetric, 140 mM K+, was 60.1 +/- 2.0 pS (mean +/- SEM). Single-channel fluctuations showed mean open and closed times of 3.6 +/- 1.5 ms and 0.41 +/- 0.2 ms, respectively, at -60 mV and approximately 21 degrees C. At positive potentials. KATP channels exhibited weak inward rectification that was dependent on the concentration of internal Mg2+. Computer simulations, based on the above results, predict significant shortening of the Purkinje cell action potential via activation of KATP channels in the range 1-5 mM cytoplasmic ATP. CONCLUSIONS: Purkinje cell KATP channels may represent a molecular isoform distinct from that present in ventricular myocytes. The presence of KATP channels in the Purkinje network suggests that they may have an important influence on cardiac rhythm and conduction during periods of ischemia.     

Energy dependent insulin binding, internalization and degradation in isolated cardiac myocytes from normal and diabetic rats

Insulin binding to isolated cardiac myocytes from normal and streptozotocin-induced diabetic rats was investigated. We found that at high affinity sites, the maximum numbers of insulin binding sites per cell are 33 000 and 22 000 for normal and diabetic myocytes, respectively with no discernible difference in receptor affinity. However, since the yield of myocytes from the diabetic heart was only 1/3 of the normal heart, it is suspected that the insulin function in the diabetic heart may be significantly lower than that in the normal heart. Chloroquine was found to markedly decrease insulin degradation with concomitant increase in net insulin uptake by isolated myocytes. This suggests that insulin degradation may take place within lysosomes after insulin is internalized. To determine whether internalization of insulin in myocytes is an energy dependent process, insulin binding and subsequent degradation were assessed in cells depleted of ATP by treatment with various metabolic inhibitors (2,4-dinitrophenol, NaF and iodoacetic acid). Depletion of the cellular ATP level resulted in a decrease in both insulin uptake and degradation. In diabetic myocytes, the general relationship between cellular ATP level and insulin uptake and degradation was similar to that found in normal myocytes. However, in diabetic myocytes, the cellular ATP level and insulin uptake were lower, but insulin degradation was greater than in normal myocytes. Insulin uptake by normal and ATP depleted cells at 4 degrees C (16 h) was lower than at 37 degrees C (1 h), while the ATP level was almost the same at both temperatures. This suggests that the internalization of insulin is a temperature as well as an ATP dependent process.     

The cytoplasmic free energy of ATP hydrolysis in isolated rod-shaped rat ventricular myocytes

The relationship between the percentage of rod-shaped rat heart myocytes and ATP, creatine phosphate, creatine and inorganic phosphate content was determined. With these values the free energy of ATP hydrolysis was calculated and found to be 59.2 kJ/mol, a much higher value than found for the perfused rat heart. When, during the isolation procedure, creatine was present in the perfusion medium during the low-calcium period, the total creatine content of the myocytes after isolation was comparable to that found in the perfused rat heart. However, when creatine was absent during this low-calcium perfusion period, total creatine content of the myocytes was significantly lower. This difference is caused by leakage of creatine from healthy cells. The free energy of ATP hydrolysis was not affected by the absence of creatine during the low-calcium perfusion period.     

The aging of the heart: weight and structural changes in the left ventricle with age]

Structural alterations of the cardiovascular system with aging are difficult to differentiate from superimposed pathologic processes. To determine whether aging "per se" affects the dimension of the heart, the weight of the heart, and the left ventricle and their rations to body weight, left ventricle wall thickness, the number of myocyte nuclei in the ventricle and the myocyte cell volume per nucleus were measured in 67 autopsies of subjects, 45 males and 22 females, who died from causes independent of cardiovascular diseases, from 17 to 90 years old. With aging, total heart weight increased slightly, while left ventricular and interventricular septum weights after dissection of the subepicardial fat decreased significantly (r = 0.44; p less than 0.001). Although left ventricular wall thickness remained constant with time, left ventricular weight to body weight ratio decreased progressively. At the structural level the number of myocyte nuclei within the left ventricle decreased (r = 0.45; p less than 0.001), whereas myocyte cell volume per nucleus increased (r = 0.30; p greater than 0.05) with age. Thus, the aging process of the heart is associated with a reduction in volume of the myocardial mass resulting from myocyte cell loss and reactive hypertrophy of the spared myocytes.     

Aging aggravates heterogeneities in cell-size and stress-intolerance of cardiac ventricular myocytes

RATIONALE: Heterogeneity of ventricular myocytes from old murine hearts was tested in regard to cell volume and tolerance to stress induced by ROS generated by photoactivation of tetramethylrhodamine methylester. RESULTS: During repetitive linescans, ROS generated 'scars' of depolarized mitochondria. All young and 60% of old myocytes had type1 scars that were small and remained stable with time. Forty percent cells from old hearts responded with type2 scars that were wide and expanded with time. Depolarization of individual mitochondria was characterized by its half decay time. HDT was 6.9s in young mice (type1 mitochondria). HDT was 5.6s in old mice due to a 30% population of mitochondria with reduced ROS-tolerance (type2, HDT=3.0 s). Type1 and type2 mitochondria did not coexist in the same cell, however, in the same old heart (different myocytes). In a different set of myocytes the volumes were measured to be larger (83+/-29 pl) and more heterogeneous than in young myocytes (49+/-13 pl). CONCLUSION: Hearts of old mice contain a subpopulation of myocytes with reduced mitochondrial stress-tolerance that is attributed to an age-dependent reduction of cellular ROS defence capacity. We discuss how heterogeneities in stress-tolerance and cell volume could be causally linked.