【作者】 匡伟；

【导师】 赵国琦；

【作者基本信息】 扬州大学 ， 动物营养与饲料科学， 2007， 硕士

【摘要】 动物细胞和组织培养已经被广泛的运用于细胞和分子生物学领域,其中更多的应用于细胞分化机制的研究。小肠是食物进行消化吸收的主要场所,而小肠黏膜上皮细胞(Intestinal epithelial cells,IEC)是肠道主要的功能细胞,参与肠道食糜的消化、吸收、免疫屏障和应激反应等,并与肠道的内、外分泌功能有密切关系。因此,进行小肠上皮细胞的分离培养是研究小肠功能、病理、分化,营养物质吸收机制及其调控的主要手段之一。本试验使用了组织块和酶消化等多种方法分离新生山羊小肠上皮细胞,结果发现组织块培养可以分离纯化出增殖能力较强的小肠上皮细胞,通过刮除等方法纯化获得纯的上皮细胞系,并稳定传代至12代。300U/ml胶原酶Ⅺ和0.1mg/ml中性蛋白酶Ⅰ联合使用可获得完整的绒毛隐窝单位,其中的上皮细胞结构完整,24h内贴壁,增殖能力较强,48h内形成明显的集落。但传代后细胞难贴壁,增殖能力明显下降。0.25%胰蛋白酶和胶原酶Ⅰ消化获得的细胞大多为成纤维细胞,大部分细胞无法贴壁。50μg/ml嗜热菌蛋白酶消化所得的消化液二次贴壁后,可获得增殖能力较强的上皮细胞,但是传代后细胞难以贴壁,增殖能力明显减弱。本试验采用MTT显色法定量研究了不同浓度的FBS(0%~10%)、Gln(0~8 mmol/mL)、EGF(0~20 ng/mL)和胰岛素(0~20μg/mL)对新生山羊IEC生长的影响,研究发现其他营养条件一致,FBS为10%,Gln浓度为2 mmol/mL,EGF浓度为10 ng/mL,胰岛素浓度为5μg/mL分别培养时,IEC的生长最为迅速。试验中采用了两种冻存液对获得的山羊IEC进行冷冻保存,发现冻存液中添加葡聚糖对冻存的山羊IEC具有保护作用,能显著提高冻存细胞的存活率。更多还原

【Abstract】 Animal cell and tissue culture has been widely used in cell biology and molecular biology, especially on the study of mechanism of cell differentiation. Small intestine is the main place for digestion and absorption of nutrient. Intestinal epithelial cells (IEC) are the primary functional cells in intestinal tract. Besides participating in digestion, absorption, immunization barrier and stress reaction, IECs are closely related to secretory function internal and external intestine. As a result, isolation and culture of intestinal epithelial cells is one of major means to study on intestine function, pathology and cell differentiation,nutrient absorption and regulatory mechanism.Both methods of explant culture and enzymatic digestion were used in this study to separate epithelial cells from intestine of neonatal goat. The result showed that the viable epithelial cells which have high capacity of proliferation can be observed by means of explant culture method. And the epithelial cell line can be obtained through scraping, which could passage up to 12 generation steadily. Isolation of epithelia and preservation of its integrity was well achieved by using collagenase (300U/ml)/dispase (0.1mg/ml) digestion technique, the epithelial cell attached as intact units (villi and crypt units). The epithelium in these organoid units remained as polarized intact layers, and attached readily to collagen coated dishes within 24h, forming large coalescing colonies of epithelium within 48h. But, it was difficult for the epithelial cells to attach and the multiplication capacity decreased after passage. The cells obtained by 0.25% trypsin and collagenase digestion were mostly fibroblasts which were difficult to attach. The thermolysin digestion method could generated viable epithelium, but it was hard to attach and proliferate after passage.MTT colorimetric assay was developed to allow a reproducible and quantitative measurement to determine the effects of fetal bovine serum (FBS), glutamine (Gln), epidermal growth factor (EGF) and insulin on the growth of IEC from neonate goat The results indicated that the optimum condition for IEC culture were: 10% FBS,2 mmol/ml Gln,10 ng/ml EGF and 5μg/ml insulin. In this study, two kinds of frozen stock solution were used to stock IEC, result showed that supplement of dextran could protect IEC during the process of stock, and increase the livability of stock cells.更多还原