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目的 通过建立分离纯化大鼠胃的主细胞培养系统 ,为进一步深入研究主细胞的生理调节和病理变化提供新的方法。方法 采用链霉蛋白酶的消化、淘析法分离纯化大鼠胃的主细胞。主细胞的纯化率在光镜下根据细胞的大小、结构和颜色计算。用血红蛋白法测定胃蛋白酶。在主细胞的短期培养系统中 ,主细胞的最佳功能状态通过比较胆囊收缩素 (CCK 8)、卡巴可林和异丙肾上腺素在不同培养时间和刺激状态下 ,主细胞分泌胃蛋白酶原的量而定。结果 在泵速为 37ml/min收集分离的主细胞化率为 83%～ 92 %。细胞的存活率 >95 %。CCK 810 -5mol/L、卡巴可林 10 -4 mol/L和异丙肾上腺素 10 -5mol/L刺激 30min后 ,主细胞分泌胃蛋白原的量在培养 2 4h明显高于培养 6、12、36h(P <0 .0 5 )。在主细胞培养 2 4h后 ,CCK 810 -5mol/L、卡巴可林 10 -4 mol/L和异丙肾上腺素 10 -5mol/L刺激主细胞 45min时胃蛋白酶原的分泌量明显高于刺激 30、6 0、90min(P <0 0 5 )。卡巴可林EC50 为 2× 10 -7mol/L ,在浓度为 10 -5mol/L时最大分泌量是对照组的 1.82倍 (P <0 .0 5 )。CCK 8的EC50 为 10 -8mol/L ,在浓度为 10 -5mol/L时最大分泌量是对照组的 1.5 2倍 (P <0 .0 5 )。结论 具有良好功能的分离纯化大鼠胃的主细胞培养系统已被建立?
OBJECTIVE: To establish a new culture system of primary cells isolated and purified from the stomach of rats to provide a new method for further study on the physiological regulation and pathological changes of the main cells. Methods The main cells of rat stomach were isolated and purified by digestion of pronase and elutriation. The purification rate of the main cells is calculated under the light microscope according to the size, structure and color of the cells. Determination of pepsin by hemoglobin method. In the short-term culture system of the main cells, the optimal functional status of the main cells is determined by comparing the activity of cholecystokinin (CCK 8), carbachol, and isoprenaline at different culture times and stimuli, and the main cells secrete pepsinogen Depending on volume. Results The main cell fractionation rate was 83% to 92% at a pump speed of 37 ml / min. Cell survival> 95%. After CCK 810 -5 mol / L, carbacholin 10 -4 mol / L and isoprenaline 10 -5 mol / L stimulated for 30 min, the amount of secreted gastrocinogen secreted by the host cells was significantly higher than that of the cultivated ones at 12 h, 36h (P <0. 05). The secretion of pepsinogen in CCK 810 -5mol / L, CaClock 10-4 mol / L and isoprenaline 10-5mol / L for 45min was significantly higher than that in stimulus 30 , 6 0,90 min (P <0 0 5). Carbocepcol EC50 was 2 × 10 -7 mol / L, and the maximum secretion rate was 1.82 times (P <0.05) at the concentration of 10 -5 mol / L. The EC50 of CCK 8 was 10 -8 mol / L, and the maximum secretion of CCK 8 was 1.5 2-fold (P <0.05) at the concentration of 10 -5 mol / L. Conclusion The main cell culture system of isolated and purified rat stomach with good function has been established.