Establishment Of Goat Intestinal Epithelial Cell Line And Nitrogen Metabolism Of IEC

Culture of intestinal epithelial cells is one of major means to research on intestine function, pathology and cell differentiation,nutrient absorption and regulatory mechanism, it also can reduce the loss of feeding animals. On the other side, many kinds of interactional intestinal cells make the experiment much more difficult.As a result, Culture of intestinal epithelial cells is a simple, fast and exact tool to research on digestion, absorption, immunization barrier and stress reaction, and also provide an ideal model of intestinal nutriment effect on intestinal epithelial cells.Enzymatic digestion were used in this study to separate epithelial cells from neonatal goat intestine.The result showed that the viable epithelial cells which had high capacity of proliferation could be observed by means of 200U/mL collagenaseâ… and 100U/mL hyaluronidase digestion technique,and the second digestion could get intact units (villi and crypt units). The epithelium in these organoid units remained as polarized intact layers and proliferated well. Light microscope confirmed the cultures had the same cellular morphology as IEC and grew as monolayers of closely opposed polygonal cells. 80% IECs could be got through scraping and twice digestion and adherence.The collagenaseâ…ª/dispaseâ… and thermolysin digestion methods could generated viable epithelium,which were hard to attach and proliferate after passage.The cells obtained by collagenaseâ… digestion were mostly fibroblast.Infinite dilution was used to clone single cell which came from 3 generation. Large coalescing colonies of epithelium in 96-well plate made the cell lines successfully, which could passage up to 10 generation steadily.To detect if the cell line was IEC,we mensurated immunocytochemical characterization, cell growing kinetics, different effect of three stock methods.The result were as follows: the attached cells were positive for cytokeratin; the best effect for cells frozen storage and resuscitation was obtained by a combination of the frozen solutionâ… and frozen approachâ…¢,and the cell survival rate reached to 85%;After cell resuscitation,cell morphous and multiplication were the same as they were before frozen storage .MTT colorimetric assay was developed to allow a reproducible and quantitative measurement to determine the effects of glutamine (Gln),arginine (Arg) and combine of both on the growth of IECs.The results indicated that both Gln and Arg could accelerate IECs'growth , Gln boosted cell differentiation while Arg restrained cell differentiation.Compare to the single Gln factor, nourishment to IEC of combined one decresed distinctly.To study the metabolism of anima acid in goat intestinal epithelial cells in vitro,the changes of animo acid compositions in cultured medium were observed respectively.The result showed that concentration of arginine,histidine, threomine and leucine decreased significantly,concentration of glutamic acid,alanine and praline increased.The other animo acid remained consuming state slightly.