沼泽型水牛乳腺上皮细胞体外培养体系建立及永生化研究-动物遗传育种与繁殖专业论文.docx

NCBI上发表的奶牛雌激素受体Q基因和催乳素受体基因cDNA序列，设计两 对特异性引物，用RT-PCR从永生化水牛乳腺上皮细胞系扩增水牛雌激素受 体Q基因和催乳素受体基因部分序列。结果扩增出雌激素受体Q基因序列 为171bp，催乳素受体基因序列为253bp，与奶牛的同源性分别为100％和 99．2％。证实了永生化水牛乳腺上皮细胞与原代细胞一样，仍能表达雌激素 受体和催乳素受体。采用MTT法检测不同浓度雌二醇对水牛乳腺上皮细胞增 殖的影响，结果得到浓度为5mg／L处理组的增殖效果最佳，差异显著(P0．05)。以上结果表明：(1)用组织块接种法比酶消化法更易获得原代的水牛乳 腺上皮细胞；(2)经过荧光免疫细胞染色、Q一酪蛋白PCR鉴定和染色体核型 分析证实所获得的细胞为水牛乳腺上皮细胞，且具有正常分泌酪蛋白的功 能；(3)通过DNA-磷酸钙沉淀法转染使SV40在水牛乳腺上皮细胞中得到表 达，细胞的端粒酶活性高于未转染前，成功建立了永生化沼泽型水牛乳腺 上皮细胞体外培养体系；(4)经过SV40转染后水牛乳腺上皮细胞仍表达雌激 素受体Q和催乳素受体；(5)适当浓度的雌二醇可以促进水牛乳腺上皮细胞 的增殖，而高浓度的雌二醇则会抑制水牛乳腺上皮细胞增殖，以浓度为 5mg／L时细胞的增殖效果最佳。关键词：水牛乳腺上皮细胞SV40端粒酶活性检测MTT雌激素受体 催乳素受体STUDIES ON THE CU删RE SYSTEM AND IMMORTALIZATIONOF MAMMARY EPITHELL气L CELLS IN SWAMP BUFFALOESABSTRACTThe purpose of the study was to establish an in vitro culture system of immortalization mammary epithelial cells in swamp buffaloes，SO as to provide more evidences for further studies on development of mammary gland and itsmilking mechanism．The mid—laction mammary gland tissue in swamp buffaloeswas used as the research material in the study as follows：The first part of the experiment was to isolate，purify，characterize and culture the swamp buffalo mamma巧epithelial cells．Primary buffalo mammary epithelial cells were cultivated from the mid·laction mammary gland tissue by either gland explant or separation by trypsin-collagenase．The method of thedifferent adhesive and the sensitivity of trypsin-collagenase can be used to isolate and purify swamp buffalo mammary epithelial cells．After purification， Morphological observation indicates that the major morphology of epithelial cells are cobblestone，and the cell growth curve assumes j‘S”shape．Analysis showed that the normal chromosome number(2n=48)and the type also possed inthe cultured cells；The results for immunochemical identification of cytokeratin8 and amplification of a-casein gene in mammary epithelial cells indicated that the cells were swamp buffalo mammary epithelial cells．The method of swamp buffalo mammary epithelial cells immortalizatio