Reddy P M, Sahi J, Desai G, Vidyasagar D, Rao M C
Department of Neonatology, University of Illinois at Chicago 60612-7342, USA.
Pediatr Res. 1996 Feb;39(2):287-94. doi: 10.1203/00006450-199602000-00017.
The rabbit colon was used to establish an in vitro model for examining development-related cellular changes in colonocyte function. Colonic epithelia from newborn, weanling, and adult animals were separated from the muscle and subjected to enzymatic digestion. A mixture of 0.05% Pronase, 0.015% collagenase IV, and 0.023% DTT was determined to be optimal for the isolation of newborn and weanling colonocytes. This solution yielded significantly more cells and of greater viability than a 0.1% Pronase, 0.03% collagenase IV, 0.07% DTT mixture that is optimal for adult colonocytes. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining of cytokeratins. The isolation procedure resulted in a crypt-enriched population and the cell yield/g of mucosa increased with age as did the crypt depth. Colonocyte viability of adults but not of newborns and weanlings, declined from 24 to 72 h. When grown on plastic, the newborn and weanling colonocytes show a approximately 2-fold increase in number, DNA and protein content over 48 h. In contrast, for all three parameters the adult colonocytes revealed only a approximately 10% increase. The colonocytes also showed an age-related decline in attachment to extracellular matrices. Colonocytes showed maximal attachment to Matrigel and collagen IV; newborn and weanling colonocytes show > 80% attachment, whereas adult colonocytes showed only a 45% attachment. The efficacy of attachment to Matrigel compared with that on plastic also differed with age, representing 9.3-, 5.5-, and 4.4-fold increase in adult, weanling, and newborn colonocytes, respectively. Newborn and weanling colonocytes grown on Matrigel for 48 h, showed a significant, 15% increase in cell number, DNA, and protein content compared with those grown on plastic. There was no difference in these parameters when adult colonocytes grown on Matrigel were compared with those grown on plastic. In summary, we have established an in vitro model for studying colonic epithelial cells at different stages of development.
采用兔结肠建立体外模型，以研究结肠上皮细胞功能与发育相关的细胞变化。从新生、断奶和成年动物分离结肠上皮，并去除肌肉组织，然后进行酶消化。经测定，含0.05%链霉蛋白酶、0.015%IV型胶原酶和0.023%二硫苏糖醇的混合液最适合分离新生和断奶动物的结肠上皮细胞。与最适合分离成年结肠上皮细胞的含0.1%链霉蛋白酶、0.03%IV型胶原酶、0.07%二硫苏糖醇的混合液相比，该混合液可获得更多数量且活力更强的细胞。通过细胞角蛋白免疫荧光染色证实了结肠上皮细胞的上皮来源。分离过程产生了富含隐窝的细胞群体，每克黏膜的细胞产量随年龄增长而增加，隐窝深度也随年龄增加。成年结肠上皮细胞的活力在24至72小时内下降，而新生和断奶动物的结肠上皮细胞活力则无此变化。在塑料培养皿上培养时，新生和断奶动物的结肠上皮细胞数量、DNA和蛋白质含量在48小时内增加约2倍。相比之下，成年结肠上皮细胞的这三个参数仅增加约10%。结肠上皮细胞对细胞外基质的黏附能力也呈现与年龄相关的下降。结肠上皮细胞对基质胶和IV型胶原的黏附能力最强；新生和断奶动物的结肠上皮细胞黏附率>80%，而成年结肠上皮细胞的黏附率仅为45%。与在塑料培养皿上相比，结肠上皮细胞对基质胶的黏附效率也因年龄而异，成年、断奶和新生动物的结肠上皮细胞分别增加9.3倍、5.5倍和4.4倍。在基质胶上培养48小时后，新生和断奶动物的结肠上皮细胞数量、DNA和蛋白质含量与在塑料培养皿上培养的相比显著增加15%。成年结肠上皮细胞在基质胶上培养与在塑料培养皿上培养相比，这些参数无差异。总之，我们建立了一个体外模型，用于研究不同发育阶段的结肠上皮细胞。
