一种新型大鼠结肠纵行肌细胞分离培养方法的建立

摘要

Aim To improve the method of isolating and culturing rat colonic longitudinal muscle cells, and establish a new method with easier materials-drawing and high purity-cells. To observe the effect of acetylcholine and norepinephrine on colonic longitudinal muscle cells, and provide an ideal cell model for the physiological and pathological study of colonic longitudinal muscles. Method The colons were obtained from male Wistar rats. The outer membrane layer was scraped off. Then the longitudinal muscle layer was torn off and cut into small pieces. After digesting and filtering through a 100-μm mesh, the pieces remaining on the mesh were allowed to be cultured. When the cells became confluent, they were cubcultured by trypsin digestion. The effects of acetylcholine and norepinephrine on rat colon longitudinal muscle cells were observed and analyzed with NTS-Elements BR3. 60. Result The cultured colonic longitudinal muscle cells were identified as high- purity smooth muscle cells with α-SM-actin immunohistochemistry. Acetylcholine had a contraction effect on the rat colon longitudinal muscle cells, and the effect was the strongest when the concentration of acetylcholine was 5. 5 ×10-1 μmol · L-1 . Norepinephrine had a relaxing effect on the longitudinal muscle cells, and the effect was most obvious when the concentration of norepinephrine was 4. 86 × 10-2 mmol · L-1 . Conclusion In the new method, colonic longitudinal muscularis are separated from the colon outer membrane. After digesting and filtering through a 100-μm mesh, the pieces remaining on the mesh are allowed to be cultured. This method is simple and convenient. The cells cultured in this way have high purity, and also have a good reaction to acetylcholine and norepinephrine.%目的 对大鼠结肠纵行肌细胞分离培养的方法进行改进,建立一种取材方便,纯度高的新型大鼠结肠纵行肌细胞的原代分离培养方法,并观察乙酰胆碱和去甲肾上腺素对结肠纵行肌细胞的作用,为结肠纵行肌生理、病理的研究提供细胞模型.方法 取Wistar大鼠结肠,从外侧刮去外膜层,剥离外膜下结肠纵行肌,消化后过滤所剩组织进行贴块培养.观察乙酰胆碱和去甲肾上腺素对纵行肌细胞的作用,用NTS-Elements BR3.60分析软件进行分析.结果 经α-SM-actin免疫组织化学染色确定培养的细胞为平滑肌细胞,且纯度高;乙酰胆碱对大鼠结肠纵行肌细胞具有收缩作用,当浓度为5.5×10-1 μmol·L-1时,收缩作用最强.去甲肾上腺素对纵行肌细胞具有舒张作用,当浓度为4.86×10-2 mmol·L-1时,舒张作用最强.结论 从结肠外膜分离肠纵行肌层,消化后过滤,所剩组织进行贴块培养,此方法简便易行、纯度高,且对乙酰胆碱和去甲肾上腺素反应良好.