培养的小鼠小肠和胃Cajal间质细胞起搏电流的特性

Characteristics of the pacemaker current in cultured interstitial cells of Cajal from murine stomach and small intestine

目的:探讨培养的小鼠小肠和胃Cajal间质细胞(ICC)起搏电流的特性.方法:用二型胶原酶分离ICC并在含有干细胞因子的培养液中培养,72h后通过免疫组织化学方法进行鉴定.利用传统全细胞模式膜片钳技术记录胃窦和小肠ICC的起搏电流.把电极内液的钙螯合剂EGTA浓度由0.1mmol/L增加到10mmol/L,使游离钙浓度降低,观察细胞内低钙对起搏电流的影响.结果:培养72h后的ICC在光镜下表现出自胞体发出2-4条长突起,并与邻近ICC突起相互连接成网络状;激光共聚焦显微镜下观察到ICC表面酪氨酸激酶受体c-kit表达呈阳性;膜电位钳制在-60mV的条件下,ICC上可以记录到自发而节律性内向电流,即起搏电流.在胃和小肠ICC之间以及单个ICC和ICC网络之间,电流幅度、频率有差异.在传统的全细胞膜片钳模式下,通过增加电极内液中EGTA浓度使细胞内游离钙降低时,可以激活ICC的内向电流;在细胞外给予钙调蛋白抑制剂W-7时,可以激活ICC内向电流的同时明显增加起搏电流的幅度.结论:小鼠胃和小肠ICC产生的起搏电流频率与小鼠胃和小肠自律运动的频率非常接近;网络ICC产生的起搏电流比单个ICC稳定、幅度高、频率快;ICC内游离钙浓度的降低是产生起搏电流的重要因素;钙调蛋白在ICC内参与起搏电流的抑制性调节.

AIM： To investigate the characteristics of the pacemaker current in cultured interstitial cells of Cajal （ICC） from murine stomach and small intestine. METHODS： ICC was isolated from murine stomach and small intestine and then cultured in the medium with stern cell factors. After 72 hours, ICC was identified by immunohistochemical technique with monoclonal antibody for c-kit protein labelling and the pacemaker currents in gastric and intestinal ICC were recorded under the conventional whole-cell patchclamp configuration. When the concentration of calcium-chelating agent EGTA was increased from 0.1 to 10 mmol/L, the effects of low intracellular calcium on the pacemaker currents were observed. RESULTS： At the 72na hour, 2 to 4 projections were stacked out from the body of ICC, which interconnected with neighboring cells and formed cell networks under light microscope. Immunohistochemistry showed that c-kit protein was positively expressed on the surface of ICC. Under the conventional whole-cell configuration, the spontaneous and rhythmic inward currents （pacemaker currents） were elicited in cultured gastric and small intestinal ICC, respectively, as the membrane potential was held at -60 mV. There were differences in the amplitudes and frequencies between single ICC and ICC networks or between gastric ICC and small intestinal ICC. Under the conventional whole-cell configuration, an inward current was activated in ICC by increasing the concentration of EGTA in pipette solution W-7, a calmodulin-inhibitor, which activated the inward current and increased the amplitude of pacemaker currents in ICC. CONCLUSION： The frequencies of the pacemaker currents from gastric and small intestinal ICC are very similar to the frequencies of rhythmic contraction in murine gastric and small intestinal smooth muscles; ICC network generates higheramplitude, stabler and more rhythmic pacemaker currents than single ICC does; low concentration of intracellular free calcium is an important factor for generating pacemaker current; calmodulin is involved in the inhibitory regulation of the pacemaker current.