Hitchin B W, Dobson P R, Brown B L, Hardcastle J, Hardcastle P T, Taylor C J
Department of Human Metabolism and Clinical Biochemistry, University Medical School, Sheffield.
Gut. 1991 Aug;32(8):893-9. doi: 10.1136/gut.32.8.893.
A method that maximises the yield of viable enterocytes has been developed for the isolation of enterocytes from human jejunal biopsy specimens. These enterocytes have been used to study the values of intracellular free calcium and the rises in adenosine 3'5'-cyclic monophosphate (cAMP) induced by secretagogues in normal and cystic fibrosis cells. Basal intracellular free calcium of cystic fibrosis enterocytes, measured fluorimetrically with fura-2, was within the range of the basal intracellular free calcium of non-cystic fibrosis enterocytes (cystic fibrosis 263 nmol/l; non-cystic fibrosis 287 nmol/l). Changes in intracellular free calcium were observed after exposure to ionomycin: a 100 nmol/l solution induced a 2.5 fold increase in intracellular free calcium in the cystic fibrosis enterocytes and a 2.2 fold increase in the intracellular free calcium concentration of the non-cystic fibrosis enterocytes. Basal cAMP values were not significantly different between cystic fibrosis and non-cystic fibrosis enterocytes (cystic fibrosis 575 fmol/100,000 cells; non-cystic fibrosis 716 fmol/100,000 cells, p greater than 0.05) and the enterocyte cAMP value increased in response to stimulation with prostaglandin E2 (7 mumol/l) (cystic fibrosis 2.2 fold increase over basal, p less than 0.05; non-cystic fibrosis 1.9 fold stimulation over basal, p less than 0.05) and vasoactive intestinal polypeptide (100 nmol/l) (cystic fibrosis 7.1 fold increase over basal, p less than 0.05; non-cystic fibrosis 5.8 fold increase over basal, p less than 0.05). There was no significant difference in the magnitude of the response between cystic fibrosis and non-cystic fibrosis enterocytes (p greater than 0.05). These results indicate that the cystic fibrosis defect in the small intestine, as in other affected epithelia, seems to be distal to the production of second messengers. The small intestine is therefore an appropriate model in which to study the biochemical defect in cystic fibrosis.
已开发出一种可使活肠上皮细胞产量最大化的方法，用于从人空肠活检标本中分离肠上皮细胞。这些肠上皮细胞已被用于研究正常细胞和囊性纤维化细胞中细胞内游离钙的水平，以及促分泌剂诱导的3',5'-环磷酸腺苷（cAMP）升高情况。用fura-2荧光法测定，囊性纤维化肠上皮细胞的基础细胞内游离钙在非囊性纤维化肠上皮细胞基础细胞内游离钙范围内（囊性纤维化263 nmol/L；非囊性纤维化287 nmol/L）。暴露于离子霉素后观察到细胞内游离钙的变化：100 nmol/L溶液使囊性纤维化肠上皮细胞内游离钙增加2.5倍，使非囊性纤维化肠上皮细胞内游离钙浓度增加2.2倍。囊性纤维化和非囊性纤维化肠上皮细胞的基础cAMP值无显著差异（囊性纤维化575 fmol/100,000细胞；非囊性纤维化716 fmol/100,000细胞，p>0.05），肠上皮细胞cAMP值在前列腺素E2（7 μmol/L）刺激下升高（囊性纤维化比基础值增加2.2倍，p<0.05；非囊性纤维化比基础值增加1.9倍，p<0.05）以及在血管活性肠肽（100 nmol/L）刺激下升高（囊性纤维化比基础值增加7.1倍，p<0.05；非囊性纤维化比基础值增加5.8倍，p<0.05）。囊性纤维化和非囊性纤维化肠上皮细胞之间反应幅度无显著差异（p>0.05）。这些结果表明，与其他受影响的上皮一样，小肠中的囊性纤维化缺陷似乎位于第二信使产生的下游。因此，小肠是研究囊性纤维化生化缺陷的合适模型。
