Horster M, Fabritius J, Büttner M, Maul R, Weckwerth P
Physiologisches Institut, Universitt München, Germany.
Pflugers Arch. 1994 Jan;426(1-2):110-20. doi: 10.1007/BF00374678.
The processes of transport differentiation from stem cell to the terminally differentiated cell in intact colonic crypts are difficult to study because access to the lumen is limited. Colonocytes were isolated from the lower two-thirds of rat distal colon crypts and grown to confluence on reconstituted basement membranes and permeable support in primary culture. Crypt and surface cells were distinguished by the uptake of [3H]thymidine and [3H]leucine and by brushborder fluorescence binding. Ion concentrations in apical and basolateral compartments of filter monolayer cultures after 48 h of incubation on days 16-18 were (in mM): apical, Na+ 116 +/- 4 (n = 48) and K+ 6 +/- 1 (n = 48); basolateral, Na+ 151 +/- 3 and K+ 3.7 +/- 0.5, respectively (mean +/- SE). Aldosterone (10(-8) M), added to the basolateral compartment from days 10-18, changed apical Na+ to 72 +/- 6 mM and apical K+ to 13 +/- 4 mM (n = 23). Dexamethasone (10(-8) M) changed apical Na+ to 84 +/- 7 mM but did not influence apical K+ (n = 22). Transmonolayer electrical potential difference (VtM; control medium; days 8-10) was 5 +/- 1 mV (n = 16; apical compartment negative); electrical resistance (RtM) was 217 +/- 21 omega.cm2 and short circuit current (ISC) was 21 +/- 5 microA.cm-2. Amiloride (0.1 mM; n = 12) in the apical medium decreased VtM to 2 +/- 1 mV and ISC to 11 +/- 4 microA.cm-2. Aldosterone (10(-8) M) after 1 week in the basolateral compartment (n = 21) changed VtM to 12.3 +/- 3 mV, RtM to 92 +/- 9 omega.cm2, and ISC to 138 +/- 23 microA.cm-2. Apical amiloride (0.1 mM; n = 9) decreased the induced VtM to -3 +/- 1 mV and ISC to -13 +/- 7 microA.cm-2. Colonic-crypt-derived epithelial cells proliferate and differentiate in primary culture, when grown on reconstituted basement membrane substratum and in supplemented medium, to form monolayers that express net Na+ absorption and net K+ secretion after 1 week. Na+ and K+ vectorial transport differentiation is primarily regulated by aldosterone, which specifically induces apical conductive Na+ transfer. Mineralocorticoid and glucocorticoid hormones appear to have differing actions on ion transport in functionally surface-type colonocytes derived in culture from isolated crypt-type cells.
由于进入肠腔的途径有限，完整结肠隐窝中从干细胞到终末分化细胞的转运分化过程很难研究。从大鼠远端结肠隐窝的下三分之二分离结肠上皮细胞，并在重组基底膜和可渗透支持物上进行原代培养，使其生长至汇合。通过[3H]胸腺嘧啶核苷和[3H]亮氨酸的摄取以及刷状缘荧光结合来区分隐窝细胞和表面细胞。在第16 - 18天孵育48小时后，滤膜单层培养物顶端和基底外侧隔室中的离子浓度（以mM计）为：顶端，Na + 116±4（n = 48），K + 6±1（n = 48）；基底外侧，Na + 151±3，K + 3.7±0.5。从第10 - 18天向基底外侧隔室添加醛固酮（10(-8) M），使顶端Na +变为72±6 mM，顶端K +变为13±4 mM（n = 23）。地塞米松（10(-8) M）使顶端Na +变为84±7 mM，但不影响顶端K +（n = 22）。跨单层电位差（VtM；对照培养基；第8 - 10天）为5±1 mV（n = 16；顶端隔室为负）；电阻（RtM）为217±21Ω·cm2，短路电流（ISC）为21±5μA·cm-2。顶端培养基中加入氨氯吡脒（0.1 mM；n = 12）使VtM降至2±1 mV，ISC降至11±4μA·cm-2。基底外侧隔室中加入醛固酮（10(-8) M）1周后（n = 21），VtM变为12.3±3 mV，RtM变为92±9Ω·cm2，ISC变为138±23μA·cm-2。顶端加入氨氯吡脒（0.1 mM；n = 9）使诱导的VtM降至 - 3±1 mV，ISC降至 - 13±7μA·cm-2。当在重组基底膜基质上并在补充培养基中生长时，结肠隐窝来源的上皮细胞在原代培养中增殖并分化，形成在1周后表达净Na +吸收和净K +分泌的单层。Na +和K +的向量转运分化主要受醛固酮调节，醛固酮特异性诱导顶端传导性Na +转运。盐皮质激素和糖皮质激素似乎对从分离的隐窝型细胞培养而来的功能性表面型结肠上皮细胞中的离子转运有不同作用。
