一种新型大鼠结肠纵行肌细胞分离培养方法的建立

Establishment of a new method of isolating and culturing rat colonic longitudinal muscle cells

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5.5×10-1μmol.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4.86×10-2mmol.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。

Aim To improve the method of isolating and cultu- ring 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 mus- cles. Method The colons were obtained from male Wistar rats. The outer membrane layer was scraped off. Then the longi- tudinal muscle layer was torn off and cut into small pieces. After digesting and filtering through a 100-μm mesh, the pieces re- maining on the mesh were allowed to be cultured. When the cells became confluent, they were cubcultured by trypsin diges- tion. 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 cellswith 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 acetylcho- line 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 mem- brane. 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.