一种离体心房肌细胞压应力超负荷模型的建立方法

A method for establishing compressive stress overload model of in vitro atrial myocytes

目的心房颤动(简称房颤)常见而高危,其发病机制未完全阐明。心房力学环境改变常见于易发房颤的多种基础疾病,其中压应力是心房应力的重要构成。建立心房肌细胞压应力超负荷模型,为研究心房机械-电反馈过程和房颤力学机制奠定方法学基础。方法选择出生0~3 d的SPF级SD大鼠乳鼠30只,体质量5~10 g,雌雄不限。设计压应力刺激装置;提取、消化、分离、纯化SD大鼠乳鼠心房肌细胞并体外培养;在培养环境中通过375 r/min(低压应力组,n=4)和735 r/min(高压应力组,n=4)的恒速离心对心房肌细胞施加不同强度的持续压应力刺激48 h,对照组细胞不施加压应力刺激(n=4);通过细胞骨架染色计算细胞面积,3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲酯基)-2-(4-磺苯基)-2H-四唑(金翁)(内盐)(MTS)法检测细胞活性,分析由压应力超负荷导致的细胞形态和活性变化。结果成功分离、培养SD大鼠乳鼠心房肌细胞并通过鉴定;接种48 h后细胞贴壁良好,密度70%~80%,呈长梭形、三角形或不规则多边形,可见细胞搏动,心肌细胞纯度达90%~95%。设计的压应力装置在确保湿度、氧和二氧化碳浓度的培养箱环境中对细胞施加压应力刺激。MTS法检测到低压应力组细胞活性高于对照组和高压应力组(OD值0.57±0.03 vs 0.50±0.02、0.50±0.01。P<0.05)。高压应力组细胞活性与对照组差异无统计学意义(OD值0.50±0.01 vs 0.50±0.02。P=0.69)。细胞骨架染色以对照组细胞平均面积为基准,对照组、低压应力组、高压应力组细胞的相对面积依次增大(1.00±0.23 vs 1.20±0.16 vs 1.59±0.20。P<0.05)。离心仓与培养箱的温度差随离心过程逐渐升高,并于6 h后趋于恒定,与培养箱最高温差≤2℃,通过实时调控培养箱温度,可稳定维持离心仓温度于37.0℃~37.5℃。结论实验构建的离体心房肌细胞压应力超负荷模型具有良好的安全性、实验效率和可重复性,通过离心向培养环境中的离体心房肌细胞施加定量、定时压应力刺激后,心房肌细胞可存活,细胞形态和活性随压应力强度变化。该模型可为进一步探索压应力相关的心房机械-电反馈机制提供方法学基础。

Objective To establish a pressure stress overload model of atrial myocytes,and lay methodological foundation for mechanical mechanism study of atrial electro-anatomical remodeling,based on the consideration that atrial fibrillation(AF)is a common and high-risk condition without complete clarified pathogenesis,changes in atrial mechanical environment are com-mon in variety of basic diseases that cause AF,and compressive stress is an important component of atrial stress.Methods Thirty male or female SPF neonatal SD rats aged 0-3 days with body mass of 5-10 g were sacrificed.The compressive stress stimula-tion device was designed to extract,digest,separate and purify neonatal SD rat atrial myocytes and culture them in vitro.Constant speed centrifugation of 375 r/min(low-pressure stress group,n=4)and 735 r/min(high-pressure stress group,n=4)were used to apply continuous compressive stress stimulation with different intensities to atrial myocytes for 48-hour,control group with out compressive stress stimulation(n=4).The cytoskeleton staining was used to measure cell area,cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(inner salt)(MTS)method,and changes in cell morphology and viability caused by compressive stress overload were analyzed.Results Atrial myocytes of neonatal SD rats were successfully isolated,cultured and identified;48-hour after inoculation,the cells adhered with density of 70%-80%,and in shape of long spindle,triangle or irregular polygon.Cell pulsation was observed and purity was 90%-95%.The cells were stimulated by compressive stress device in incubator environment that ensured humidity,oxygen and carbon dioxide concentrations.The MTS test results showed that cell viability of low-pressure stress group was higher than that of control group and high-pressure stress group(OD value 0.57±0.03 vs 0.50±0.02,0.50±0.01.P<0.05).There was no significant difference in cell viability between high-pressure stress group and control group(OD value 0.50±0.01 vs 0.50±0.02.P=0.69).The cytoskeleton staining was based on mean cell area of control group,and relative areas of control group,low-pressure stress group and high-pressure stress group increased sequentially(1.00±0.23 vs 1.20±0.16 vs 1.59±0.20.P<0.05).The temperature difference between centrifuge chamber and incubator gradually increased with centrifugation process,and was constant after 6-hour.The maximum temperature difference was≤2℃,and the centrifuge chamber temperature stably maintained at 37.0℃-37.5℃by real-time regulation of incubator temperature.Conclusion It is demonstrated that the in vitro compressive stress overload model constructed shows satisfactory safety,experimental efficiency and repeatability.After quantitative and timed compressive stress stimulation to isolated atrial myocytes in culture environment by centrifugation,atrial myocytes of neonatal SD rats can survive,the cell morphology and activity changes with compressive stress intensity,which provides novel method for further research on mechano-electrical feedback mechanism of cardiomyocytes induced by compressive stress.