【作者】 王磊；

【导师】 侯加法；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2009， 硕士

【摘要】 试验I、鸡胚小肠上皮细胞培养与鉴定目的：培养鸡胚小肠上皮细胞。方法：从18日龄鸡胚中取一段十二指肠段,用组织块培养法和以50μg·mL-1嗜热菌蛋白酶与0.2μg·mL-1胶原酶I型联合消化获取的肠隐窝,在37.5℃、5% CO2的条件下,用含5%胎牛血清的完全培养基培养小肠上皮细胞。用差速消化法和差速贴壁法,进一步纯化、传代。采用形态学观察、Giemsa染色和免疫化学染色法进行小肠上皮细胞鉴定。结果：组织块培养法和酶联合消化培养法都可以得到鸡胚小肠上皮细胞,纯化后纯度理想。结论：鸡胚小肠上皮细胞培养成功,培养的细胞纯度理想,为下一步研究提供了实验材料。试验Ⅱ、依普拉芬对鸡胚小肠上皮细胞生长的影响目的：在建立鸡胚小肠上皮细胞体外培养方法的基础上,研究依普拉芬对鸡胚小肠上皮细胞增殖、分化和代谢功能的影响。方法：取生长状态良好的第二代小肠上皮细胞接种于96孔板,添加含1%FBS的DMEM对照组和含不同浓度的依普拉芬实验组,72 h后分别用MTT法、PNPP法测定小肠上皮细胞增殖率和小肠上皮细胞内碱性磷酸酶(ALP)活性。结果：(1)与对照组相比,10-6 mol·L-1,10-7 mol·L-1和10-8 mol·L-的依普拉芬均显著促进小肠上皮细胞增殖(P<0.01); 10-5mol·L-1依普拉芬显著抑制小肠上皮细胞的增殖(P<0.01)；(2)与对照组相比,10-6 mol·L-1、10-7 mol·L-1和10-8 mol·L-1依普拉芬极显著促进小肠上皮细胞ALP活性(P<0.01),10-9mol·L-1显著促进小肠上皮细胞ALP活性(P<0.05); 10-5mol·L-1(?)勺依普拉芬显著抑制小肠上皮细胞内ALP活性(P<0.01)。结论：依普拉芬能显著促进小肠上皮细胞的增殖；使小肠上皮细胞内的ALP显著增加；但依普拉芬浓度越大,其上皮细胞则呈现明显的抑制作用。试验Ⅲ、依普拉芬对鸡胚小肠上皮细胞钙离子通道基因表达的影响目的：在建立鸡胚小肠上皮细胞体外培养方法的基础上,研究TRPV6在小肠上皮细胞上表达的定位以及依普拉芬对TRPV6、CaBP-D28k和PMCA1b mRNA表达的影响。方法：免疫细胞化学方法研究TRPV6表达定位；用含10-6mol·L-1、10-7mol·L-1、10-8mol·L-1、10-9mol·L-1和10-10mol·L-1不同浓度的依普拉芬以及含1% FBS的DMEM对照组处理第二代鸡胚小肠上皮细胞72 h后,用RT-PCR方法检测其TRPV6、CaBP-D28k和PMCA1b mRNA表达量变化。结果：(1) TRPV6表达定位于鸡胚小肠上皮细胞胞膜上；(2)与对照组相比,10-6 mol·L-1,10-7 mo·L-1、10-8 mol·L-1依普拉芬显著上调PMCA1b和CaBP-D28k mRNA的表达(P<0.01),10-9 mol·L-1依普拉芬作用下TRPV6的表达出现明显上升(P<0.05),10-6 mol·L-1、10-7mol·L-1、10-8 mol·L-1依普拉芬能够显著上调TRPV6 mRNA表达(P<0.01)。结论：鸡胚小肠上皮细胞胞膜上存在TRPV6表达；依普拉芬能显著上调小肠上皮细胞TRPV6、CaBP-D28k和PMCA1b mRNA表达量。更多还原

【Abstract】 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.更多还原