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梭囊的存在限制了人们对肌梭功能及其机制的深入研究,本研究旨在建立将梭内肌纤维从梭囊中分离出来的方法。应用混合酶液消化的方法,分离大鼠比目鱼肌的梭内肌纤维,使用不同的培养基溶液进行培养,用台盼蓝染色法检测细胞活性,用膜片钳技术检测静息膜电位。结果显示,氨基酸-生理盐溶液中梭内肌纤维几乎全部坏死,DMEM培养基虽能较好地维持细胞状态,但是对CO2含量要求较高,而Leiboviz’s 15(L-15)培养基能够维持梭内肌纤维的正常形态和功能达1～2h;和常规处理过的盖玻片相比,用明胶-多聚赖氨酸-血清处理过的盖玻片上梭内肌纤维更易贴壁;分离出的梭内肌纤维静息膜电位为(45.3±5.1)mV,显示纤维的功能状态良好,能够满足电生理实验的要求。本研究为进一步研究梭内肌纤维的功能及其机制奠定了良好的方法学基础。
The existence of the shuttle limited the further study on the function and mechanism of the muscle spindle. The purpose of this study was to establish a method to separate the intrafusal myofibers from the shuttle. The mixed muscle enzyme digestion method was used to separate the intrasternal muscle fibers of the soleus muscle of rats. The fibroblasts were cultured in different media. The cell viability was measured by trypan blue staining. The resting membrane potential was measured by patch clamp technique. The results showed that almost all of the intrafusal myofibers were necrotic in the amino acid-physiological saline solution. Although DMEM medium could maintain the cell state better, its requirement for CO2 content was higher. However, Leiboviz’s 15 (L-15) The normal morphology and function of muscle fibers reached 1 ~ 2 h. Compared with the conventional treated coverslips, the intracelal muscle fibers on the coverslips treated with gelatin-polylysine-serum were more adherent; The resting membrane potential of muscle fibers was (45.3 ± 5.1) mV, indicating that the functional status of the fibers was good and could meet the requirements of electrophysiological experiments. This study laid a good methodological basis for further study on the function and mechanism of intrascleal muscle fibers.