| Chicken cecal epithelial cells are the host cells of Eimeria tenella(E.tenella).Establishment of chicken embryo cecal epithelial cell line can provide in vitro cell model for E.tenella's mechanism of injury and anticoccidial research.The primary cecal epithelial cells of chicken embryos for the separation of digestive enzymes and subcultured digestive enzymes,and the chick embryos' age and serum type were selected.The cell viability was determined by MTT method.The purity and characteristics of cecal epithelial cells were identified by immunohistochemical staining,light microscopy,electron microscopy and karyotype analysis.The test results showed that:(1)The proliferation rate of the third generation of cecal epithelial cells of chicken embryos in the thermolysin(T group),type I collagenase(C group),and type I collagenase+hyaluronidase groups(H group)decreased sequentially;the T group cells the adherence rate was significantly higher than that of the C and H groups(P<0.01)except for the first day;the cell viability was significantly higher than the C group at the first to fifth day(P<0.01),and all the time periods were significantly higher than that of H group(P<0.01).Indicating that 50 mg/L thermolysin can be used as a digestive enzyme isolated for cecal epithelial cells..(2)The proliferation rate of chicken cecal epithelial cells from 14-day-old chick embryos(T14 group),15-day chick embryos(T15 group),16-day chick embryos(T16 group),and 17-day chick embryos(T17 group)was in turn high to low;T14 group and T15 group cell adherence rate was significantly higher than T16 group and T17 group except at the 2nd day and 8th day(P<0.05),and T15 group cell adherence rate was significantly higher(P<0.05)or significantly higher(P<0.01)T14 group from 4th day to 7th day;the cell viability of T14,T15 and T16 groups was significantly higher T17 group from 2nd day to 3rd day(P<0.01),T15 group cells vitality was significantly higher T16 and T14 groups on the 6th and 8th day(P<0.01),and there was no significant difference among the other groups at all time points(P>0.05).It indicated that the best age of cecum epithelial cells of chicken embryo was 14 to 15 days old.(3)The cecum epithelial cells of chicken embryo were not adhered to wall or only a small number of aanerent ceus were auacnea to tne ceus of me cecum epmenum using 50 g/mL tnermopnuic protease digestion,0.36%EDTA and 0.25%pancreatin digestion and 0.45 mol/m L sucrose-0.36%EDTA and 0.25%pancreatin.A large number of living cells-can adhere to the wall and proliferate-could be obtained with 0.25%pancreatin-0.02%EDTA combined digestion and 0.25%trypsin-0.02%EDTA digestion,but the adherence rate and proliferation rate of the latter were significantly lower than those of the former(P<0.01).(4)After adding 2.5%chicken serum and 2.5%bovine serum,there was no significant difference in cell proliferation rate and adherence rate between passages(P>0.05).(5)By observing the morphology of cecal epithelial cells,we found that most of the cecum epithelial cells of chicken embryo in the primary and 10 generation were polygonal or short fusiform;The cells are tightly connected to a piece of "pave stone" like,but most of the original generation are clustered in islands,and the 3?10 generation of passages of the cecum epithelial cells are distributed evenly and are not aggregated.Under electron microscope,the microvilli on the cell surface,tight junctions between cells,desmosomes and cytosolic vesicles were clear.Immunocytochemistry results showed that CK-18 protein and E-cad protein were positive.In conclusion,the cells that were passaged were cecal epithelial cells,and the purity of chick embryo cecal epithelial cells in primary and 10 generation was over 82.2%.(6)Chromosomal analysis showed that the karyotype of cecum epithelial cells of chicken embryo was 2n =78,which was transferred to 6 generation cells in vitro without transformation. |
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