摘要
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 This study was undertaken to establish n ew isolated and purified rat chief cell preparation. Methods Pure isola ted chief cell preparation was developed by pronase digestion, elutriation. The c ell purity was determined by 1∶1 mixture of 0.1% Toluidine Blue in 0.1 mol/L Na _2HPO _4 and 0.15% Basic Fuchsin in 5% Ethenol according to size, structure and color under light microscope. In the culture system best working condition was determ ined by secretory quantity of pepsinogen from isolated rat chief cells stimulate d by CCK-8, carbachol and isoproterenol in different culture and stimulated tim e. Results Chief cells was collected by harvested at pump speed of 37 ml/min which were in 83%-92%. The secretory quantity of pepsinogen stimulated by CCK-8, carbachol and isoproterenol in the group cultured with 24 hours is much higher than in other groups ( P<0.05) . In the group cultured with 24 hours t he highest secretory quantity of pepsinogen was obtained when chief cells were s timulated by CCK-8 or carbachol or isoproterenol for 45 minutes. A dose respons e to carbachol was evident (EC_\{50\} 2×10\+\{-7\} mol/L; P<0 05 ). Maximu n stim ulation (1.82-fold) was noted at 10\+\{-5\} mol/L. CCK-8 stimulation resulted in a significant increase in pepsinogen secretion (EC_\{50\} 10\+\{-8\} mol/L; P<0 .05 ). Maximun response was 1.5-fold at 10\+\{-5\}M. Conclusion Isolate d pure rat gastric chief cell preparation was successfully established. Chief ce lls have a good function in short culture.\;
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
2000年第5期339-341,共3页
Journal of Nanjing Medical University(Natural Sciences)
关键词
主细胞
胃蛋白酶原
分离
培养
功能测定
chief cell
pepsinogen
isolation
cultured
animals, lab oratory
