体外培养新生大鼠心肌细胞的形态及其增殖能力

Morphology and proliferation of myocardiocytes cultured in vitro from neonatal rats

目的:心脏组织中心肌细胞增殖分化转变发生于出生后3～4d,有细胞周期调控机制的参与。观察新生大鼠心室肌细胞培养过程中细胞形态学和细胞周期素E表达的变化规律。方法:实验于2005-01/2006-03在新乡医学院解剖学与组织胚胎学重点学科实验室完成。①实验材料:1日龄Wistar大鼠20只,体质量7～10g。②实验方法及评估:心肌细胞分离、纯化及培养后用倒置显微镜观察其形态变化,免疫细胞化学染色及图像分析方法检测培养第1～53天新生大鼠心室肌细胞周期素E的表达。结果:纳入大鼠20只,均进入结果分析。①培养心肌细胞4h后贴壁,偶见单个细胞开始自发性搏动,部分细胞为梭形;以后细胞变为双核细胞,三角形或柱形细胞。第3天细胞聚集成片并同步搏动,第10天搏动频率最快,之后搏动频率逐渐减慢。②心肌细胞周期素E位于细胞核和核周的胞浆中。培养1～3d染色较深,以后逐渐变浅。③心肌细胞核内细胞周期素E表达量逐渐下降,第2天与第1天比较,差异无显著性(P>0.05)。从第3天开始差异有显著性(P<0.05,P<0.01)。结论:新生大鼠心肌细胞出生后早期体外培养的前3天有一定增殖能力,但以后呈下降趋势。细胞周期素E表达下调与心肌细胞增殖能力降低呈正相关。

AIM： The proliferation and differentiation of myocardiocytes in heart tissue begin since the third to fourth after born, in which the regulative mechanism of cell cycle is participated, in this study, the change rules of the morphology and cyclin E expression in cultured myocardiocytes of neonatal rats was investigated. METHODS： The experiment was carried out in the Key Laboratory of Anatomy and Histologic Embryology of Xinxiang Medical College between January 2005 and March 2006. The myocardiocytes were isolated from 20 one-day-old Wistar rats （7-10 g）, purified and cultured. The morphological changes were observed under inverted microscope, and the expression of cyclin E in cultured ventricular myocardiocytes of neonatal rats on 1^st to 53^nd days was detected by immunocytochemistry and digital image analysis technology. RESULTS： Twenty rats were involved in the result analysis. （1）The cultured myocardiocytes became adherence on hour 4, and few began to beat spontaneously; some single cells were spindle shaped. The spindle cells became gradually dicaryocytes, triangular or cylindrical cells. On the 3^nd day, they got together and beat synchronously in clusters; the fastest pulse frequency appeared on the 10^th day, and then gradually slowed down. （2）Cyclin E was located mainly at nucleus and cytoplasm round the nucleus. The staining was deeper in 1-3 days and gradually lighter then. （3）The expression amount of cyciin E was decreased significantly. There was no obvious difference between the second and first day （P 〉 0.05）, while the differences were distinct since the third day （P 〈 0.05, 0.01 ）. CONCLUSION： The early-cultured myocardiocytes of neonatal rats for 3 days display certain proliferation, but decline gradually then. It reveals that the down-regulation of cyclin E expression may be positively correlated with the decrease in myocardium proliferation ability.