Benya R V, Schmidt L N, Sahi J, Layden T J, Rao M C
Department of Medicine, University of Illinois, Chicago.
Gastroenterology. 1991 Sep;101(3):692-702. doi: 10.1016/0016-5085(91)90527-r.
The authors investigated various enzymatic digestion procedures for isolating epithelial cells from the distal colon of New Zealand White male rabbits. Rabbit mucosa was washed, diced, and digested for 90 minutes in one of five different solutions, including a new combination consisting of 0.03% collagenase IV and 0.1% pronase (solution V). Solution I (0.3% dispase) yielded 14.2 +/- 8.2 x 10(6) colonocytes/g mucosa, solution II (0.15% dispase and 0.03% collagenase) yielded 7.7 +/- 2.8 x 10(6) colonocytes/g mucosa, and solution III (0.03% collagenase IV) yielded 15.4 +/- 10(6) cells/g mucosa. Solutions I-III have previously been described for the isolation of colonocytes. Solution IV (0.1% pronase and 325 U/mL DNAase) was originally described for the isolation of nasal epithelial cells but yielded only 2.5 +/- 1.2 x 10(6) cells/g mucosa when applied to the isolation of colonocytes. The new combination of pronase and collagenase, solution V, yielded significantly more colonocytes, 34.5 +/- 3.0 x 10(6) cells/g mucosa, than previously described methods (P less than 0.01). Inclusion of 5 mmol/L ethylenediaminetetraacetic acid in any of the solutions enhanced neither viability nor yield. The digestion product of solution V could be enriched for crypts by serial low-speed centrifugations. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining for cytokeratins. Functional viability was tested by determining the presence of a Na+/H+ exchanger, using the pH fluorescent dye bis(carboxymethyl)-5(6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH. The authors document that sodium-dependent restoration of intracellular pH in colonocytes acid-loaded to a pH of 6.30 occurred at a rate of 0.19 +/- 0.02 pH U/min. Amiloride at concentrations of 1 mmol/L completely inhibited operation of the exchanger, as did sodium substitution with choline or tetramethylammonium. Lineweaver-Burke analysis at this intracellular pH showed a Michaelis constant of 10.71 mmol/L Na+ and a maximum velocity of 0.12 pH U/min. Exposing the colonocytes to 100 nmol/L phorbol 12,13-dibutyrate increased antiporter activity by 62.0%. Finally, the authors describe the synthesis of a new biomatrix composed of the basement membrane of 3T3 NIH fibroblasts that permits significantly improved colonocyte attachment than to glass, plastic, collagen types I or IV, or matrigel.
作者研究了从新西兰白兔雄性远端结肠分离上皮细胞的各种酶消化程序。兔黏膜经冲洗、切块后，在五种不同溶液之一中消化90分钟，其中一种新组合为0.03%的IV型胶原酶和0.1%的链霉蛋白酶（溶液V）。溶液I（0.3%的分散酶）每克黏膜产生(14.2±8.2)×10⁶个结肠细胞，溶液II（0.15%的分散酶和0.03%的胶原酶）每克黏膜产生(7.7±2.8)×10⁶个结肠细胞，溶液III（0.03%的IV型胶原酶）每克黏膜产生(15.4±10)×10⁶个细胞。溶液I - III先前已被描述用于分离结肠细胞。溶液IV（0.1%的链霉蛋白酶和325 U/mL的脱氧核糖核酸酶）最初被描述用于分离鼻上皮细胞，但应用于结肠细胞分离时每克黏膜仅产生(2.5±1.2)×10⁶个细胞。链霉蛋白酶和胶原酶的新组合，即溶液V，产生的结肠细胞显著多于先前描述的方法，每克黏膜有(34.5±3.0)×10⁶个细胞（P＜0.01）。在任何一种溶液中加入5 mmol/L的乙二胺四乙酸既未提高细胞活力也未增加产量。溶液V的消化产物可通过连续低速离心富集隐窝。通过细胞角蛋白免疫荧光染色证实了结肠细胞的上皮来源。通过使用pH荧光染料双（羧甲基）-5（6）-羧基荧光素乙酰氧基甲酯测量细胞内pH来确定Na⁺/H⁺交换体的存在，以此测试功能活力。作者记录到，酸加载至pH 6.30的结肠细胞中，细胞内pH的钠依赖性恢复速率为0.19±0.02 pH单位/分钟。1 mmol/L浓度的氨氯地平完全抑制了该交换体的运作，用胆碱或四甲基铵替代钠时也是如此。在此细胞内pH下的Lineweaver - Burke分析显示米氏常数为10.71 mmol/L的Na⁺，最大速度为0.12 pH单位/分钟。将结肠细胞暴露于100 nmol/L的佛波醇12,13 - 二丁酸酯可使反向转运体活性增加62.0%。最后，作者描述了一种由3T3 NIH成纤维细胞基底膜组成的新型生物基质的合成，与玻璃、塑料、I型或IV型胶原或基质胶相比，该生物基质能显著改善结肠细胞的附着。
