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目的证实豚鼠胆囊Cajal间质细胞(ICC)是胆囊平滑肌动力的起搏细胞。方法采用在体记录和组织钳细胞内记录的方式,研究胆囊自发电活动。亚甲蓝+光照的方法破坏胆囊ICC。胶原酶分离方法分离胆囊平滑肌细胞,并对分离的单个细胞进行形态学分析,联合激光共聚焦显微镜检测c-kit蛋白表达的方法鉴定ICC。膜片钳用于证明胆囊ICC能产生具有起搏活性的自发去极化电流。结果豚鼠胆囊在体记录的慢波每分钟(58±3.2)次,细胞内记录时可记录到慢波和动作电位。采用亚甲蓝+光照的方法后,慢波和动作电位明显减弱或消失。豚鼠胆囊新鲜分离的细胞经过贴壁之后,可见少量具有典型ICC光镜特征的细胞,占细胞总数的(5±1.2)%(n=12),(21±4)%细胞的c-kit蛋白表达阳性。全细胞膜片钳记录胆囊ICC的自发去极化电流,发现豚鼠胆囊ICC自发去极化电流频率每分钟(58.4±3.5)次(n=21),幅度约-(72±3.5)pA。结论豚鼠胆囊平滑肌中存在ICC起搏细胞,参与起搏胆囊自发节律性电活动和自动节律性收缩活动。
Objective To confirm that the guinea pig gallbladder Cajal stromal cells (ICC) are gallbladder smooth muscle motility pacemaker cells. Methods In vivo recording and tissue-clamp recordings were used to study the spontaneous activity of the gallbladder. Methylene blue + light method to destroy the gallbladder ICC. Collagenase separation was used to separate the gallbladder smooth muscle cells. Morphological analysis was performed on the isolated single cells. ICC was identified by laser confocal microscopy in detecting the expression of c-kit protein. Patch-clamp was used to demonstrate that gallbladder ICCs produce a spontaneous depolarization current with pacing activity. Results Guinea pig gallbladder recorded in vivo slow wave per minute (58 ± 3.2) times, intracellular recording can be recorded when the slow wave and action potential. Using methylene blue + light method, the slow wave and action potential significantly weakened or disappeared. After attachment of freshly isolated cells from guinea pig gallbladder, a small number of cells with typical characteristics of ICC light microscopy were observed, which accounted for (5 ± 1.2)% (n = 12) and (21 ± 4)% of cells Positive expression. Whole-cell patch-clamp recordings of spontaneous depolarization currents in gallbladder ICCs revealed a spontaneous depolarization current frequency of 58.4 ± 3.5 times per minute (n = 21) in guinea pig gallbladder ICCs with an amplitude of approximately ± (72 ± 3.5) pA. Conclusion There are ICC pacemaker cells in the gallbladder smooth muscle of guinea pigs, which participate in spontaneous rhythmic electrical activity and rhythmic contractile activity of the gallbladder.