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本文旨在应用膜片钳技术结合单细胞逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)技术研究小鼠胚胎心肌细胞的不同电生理学特点的分子生物学基础。应用胶原酶B消化分离得到小鼠胚胎早期(E10.5)单个心肌细胞,先用膜片钳技术记录单个胚胎心肌细胞的动作电位,随即应用玻璃微电极吸取该细胞内容物,进一步应用RT-PCR技术检测该单个心肌细胞离子通道基因Kir2.1、SCN5a和SCN1b的表达,以GAPDH为内参基因。全细胞电流钳技术记录到心室肌样细胞、心房肌样细胞和起搏样细胞的动作电位。心室肌样细胞和心房肌样细胞动作电位的最大舒张期电位(maximum diastolic potential,MDP)较起搏样细胞更负,0期最大除极速度(maximum velocity of depolarization,Vmax)更快;心室肌样细胞动作电位的MDP相对心房肌样细胞更负。相对应的,心室肌样细胞的Kir2.1、SCN5a、SCN1b三种基因表达均较强;心房肌样细胞的SCN5a表达较强,而SCN1b和Kir2.1相对心室肌样细胞表达稍弱;而起搏样细胞的上述三种基因表达均很弱。不同类型心肌细胞动作电位的基本特点与其基因表达密切相关,应用膜片钳技术结合单细胞RT-PCR技术可以敏锐检测到特定电生理特点的分子生物学基础。
The purpose of this study was to investigate the molecular biology of different electrophysiological characteristics of mouse embryonic cardiomyocytes using patch-clamp technique and single-cell reverse transcriptase polymerase chain reaction (RT-PCR). Single cardiomyocytes were harvested by early digestion of collagenase B (E10.5). Patch-clamp technique was used to record the action potentials of single embryonic cardiomyocytes. Then the cell contents were extracted by glass microelectrodes and further analyzed by RT- PCR technique to detect the single cardiomyocyte ion channel genes Kir2.1, SCN5a and SCN1b expression, GAPDH as an internal control gene. Whole-cell current-clamp technique recorded the action potentials of ventricular myocytes, atrial myofibroblasts and pacemaker-like cells. The maximum diastolic potential (MDP) of ventricular myocytes and atrial myocyte-like cells was more negative than that of paced cells, and the maximum velocity of depolarization (Vmax) was faster in ventricular myocytes MDP - like cell action potential is more negative than atrial myocyte - like cells. Correspondingly, the expressions of Kir2.1, SCN5a and SCN1b genes in cardiac ventricular myocytes were stronger than those in normal control group. The expression of SCN5a was stronger in atrial myofibroblasts, but weaker in SCN1b and Kir2.1 than in ventricular myocytes. Pacemaker-like cells of these three genes were weakened. The basic characteristics of action potentials of different types of cardiomyocytes are closely related to their gene expression. The molecular biology basis of specific electrophysiological characteristics can be detected acutely using patch clamp technique combined with single cell RT-PCR.