| Experiment I:Culture and Identification of Small Intestinal Epithelial Cells in Chicken EmbryosObjective:Cultured the small intestinal epithelium cells(IECs) of chicken embryos.Methods:Small intestinal crypt was isolated from the tissue mass which was collected from the duodenum in chicken embryos 18 days old and digested with 50μg·ml-1 thermolysin and 0.2 mg·ml-1 collagenaseⅠ. The IECs was cultured from small intenstinal crypt with complete medium containing 5% fetal bovine serum at 37.5℃in a cell incubator with 5% CO2 and was purified by the methods of differential digestion, differential adhesion and mechanical curettage. IECs was identified by immunocyto-chemistry assay for keratin. Results:The tissue mass culture and the combined enzyme digestion were both fit for the culture of IECs. These cultured cells had ideal purity after purified. Conclusion:The results confirmed that IECs culture was successful. This method was simple, efficient and well reproducible. These cultured cells had ideal purity and stable nature. This provided experimental material for further research.ExperimentⅡ:Effect of Ipriflavone on the Proliferation and Differentiation of Chicken Embryonic Intestinal Epithelial Cells In VitroObjective:Based upon the culture method of intestinal epithelial cells established from chicken embryos in vitro, we studied the proliferation, differentiation and metabolic function effect of ipriflavone on chicken embryonic intestinal epithelial cells. Methods: The good second generation of the intestinal epithelial cells were inoculated in 96-well plates. After adding the different concentrations of ipriflavone to the plates, and adding DMEM with 1% FBS instate of ipriflavone in control group. The MTT and the PNPP methods were used for the determination of the proliferation rate and alkaline phosphatase (ALP) activity of the intestinal epithelial cells. Results:(1) Compared with the control group,10-6 mol·L-1,10-7 mol·L-1 and 10-8mol·L-1 of ipriflavone significantly promoted the proliferation of intestinal epithelial cells (P<0.01).10-5 mol·L-1 of ipriflavone significantly inhibited the proliferation of intestinal epithelial cells (P<0.01). (2) Compared with the control group,10-9 mol·L-1 of ipriflavone significantly promoted the ALP activity of the intestinal epithelial cells (P<0.05),10-6 mol·L-1,10-7 mol·L-1 and 10-8 mol·L-1 of were more significantly (P<0.01).10-5 mol·L-1 of ipriflavone were significantly inhibited ALP activity of the intestinal epithelial cells (P<0.01). Conclusion: Ipriflavone can significantly promote the proliferation of the intestinal epithelial cells; but at the high concentration, it shows inhibition.ExperimentⅢ:Influence of Ipriflavone on the Calcium Channel Gene Expression of Chicken Embryonic Small Intestinal Epithelial CellsObjective:Based upon the culture method of intestinal epithelial cells established from chicken embryos in vitro, we studied the location of TRPV6 and influence of Ipriflavone on TRPV6 mRNA, CaBP-D28k mRNA and PMCAlb mRNA expression. Methods:The location of TRPV6 was detected based on immunohistochemical method; The expression of TRPV6 mRNA, CaBP-D28k mRNA and PMCAlb mRNA were detected using RT-PCR on the second generation of the intestinal epithelial cells which were treated in different concentrations of ipriflavone(10-6 mol·L-1,10-7mol·L-1,10-8 mol·L-1,10-9 mol·L-1 and 10-10 mol·L-1) and DMEM with 1% FBS in control group for 72 h. Results:(1) TRPV6 was located in the membrane of small intestinal epithelial cells; (2) Compared with control group,10-6 mol·L-1,10-7 mol·L-1 and 10-8 mol·L-1 Ipriflavone significantly higher CaBP-D28k mRNA and PMCAlb mRNA expression(P<0.01),10-9 mol·L-1 higher TRPV6 mRNA expression(P<0.05),10-6 mol·L-1,10-7 mol·L-1 and 10-8 mol·L-1 significantly higher TRPV6 mRNA expression(P<0.01). Conclusion:TRPV6 was expressed in the membrane of small intestinal epithelial cells; Ipriflavone could significantly higher TRPV6 mRNA, CaBP-D28k mRNA and PMCAlb mRNA expression. |
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