牵张应变对小肠Cajal间质细胞起搏电流的作用

Effect of membrane stretch on the pacemaker current in cultured interstitial cells of Cajal from murine small intestine

目的研究牵张应变刺激对体外培养的小肠Cajal间质细胞(ICC)起搏电流的影响。方法利用II型胶原酶消化并在含有干细胞因子的SmGM培养基中培养ICC,72h后采用免疫荧光细胞化学方法鉴定培养的ICC。利用传统全细胞记录模式膜片钳技术记录正常小肠ICC起搏电流,然后采用对细胞直接施加正压和灌流低渗溶液的两种方法给细胞膜施加张应变刺激,以观察牵张应变对小肠ICC起搏电流的影响。结果培养72h后的ICC在光镜下,胞体呈三角形或梭形,且自胞体发出2￣4条长突起,并与邻近ICC突起相互连接成网络状;荧光显微镜下观察ICC胞体和突起酪氨酸激酶受体c-kit表达呈阳性;在膜电位钳制在-60mV的条件下,可以记录到自发而节律性内向电流,即起搏电流;在传统全细胞记录模式下,两种张应变刺激均可以激活一种内向电流同时明显增加起搏电流的振幅及频率。结论牵张应变对胃肠壁的刺激可能作用于胃肠平滑肌节律性运动的起搏细胞ICC并改变其电生理特性,从而调节胃肠平滑肌运动的基础张力和频率。

Objective To investigate the role of membrane stretch on the pacemaker current in cultured interstitial cells of Cajal. Methods ICC was isolated from murine small intestine and then cultured in the medium with stem cell factors. After 72 hours, ICC was identified by immunohistochemical technique with monoclonal antibody for labeling c-kit protein and the pacemaker current in intestinal ICC were recoded under the conventional whole-cell patch clamp configuration. The membrane stretch was achieved by the application of direct pressure through the patch pipette and perfusion of hypotonic solution. Results At the 72nd 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. The spontaneous and rhythmic inward currents （pacemaker currents） were elicited under cells were voltage clamped in the whole-cell patch-clamp configuration and held at -60 mV. The sustained inward current was activated and the amplitudes and frequencies of pacemaker currents increased by the application of membrane stretch. Conclusion The mechanical stretch can regulate basic tone and frequency of gastrointestinal smooth muscle motility via activating or potentiating ICC pacemaker currents.