体外诱导牛骨髓间充质干细胞向乳腺样上皮细胞分化的初步研究  

Preliminary Study on Differentiation of Bovine BMSC into Mammary Gland-like Epithelial Cells in vitro

作　　者：朱孟芝[1,2] 董雅娟[1,2,3] 龚宜超[1,2] 王文明[1,2] 刘玮[1,2] 柏学进[1,2,3] 

机构地区：[1]青岛农业大学动物胚胎工程中心,山东青岛266109 [2]山东省黑牛繁育工程技术研究中心,山东青岛266109 [3]山东布莱凯特黑牛科技股份有限公司,山东淄博256306

出　　处：《中国畜牧兽医》2012年第1期92-97,共6页China Animal Husbandry & Veterinary Medicine

基　　金：山东省自主创新成果转化重大专项资助(2007ZHZX11101);国家农业科技成果转化资金资助(2010GB2C600245)

摘　　要：为培育转基因肉牛提供种子细胞及进一步丰富牛骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)的多向分化潜能,本试验利用细胞免疫荧光染色方法和分子生物学方法初步探讨在表皮生长因子、胰岛素、催产素和孕酮等条件下,体外诱导牛BMSC向乳腺样上皮细胞分化的可能性。利用不同浓度的诱导液对纯化稳定的P4、P8和P12代牛BM-SC进行体外诱导,并对诱导后的细胞进行细胞免疫荧光观察和RT-PCR鉴定。结果显示,诱导后部分BMSC细胞呈多角形,而不再呈明显的梭形和纺锤形,经细胞角蛋白18免疫荧光染色后出现明显的荧光。P4代牛BMSC在诱导液Ⅱ的诱导作用下分化效果显著。RT-PCR结果显示,诱导分化后细胞角蛋白19基因、β-酪蛋白基因及αs1-酪蛋白基因在细胞中表达。因此,在体外,多因子联合诱导可使牛BMSC初步分化为乳腺样上皮细胞并且在诱导液Ⅱ的联合诱导下分化作用最明显。To provide seed cells for breeding genetically modified beef cattle and to enrich multi-differentiation potential of the bovine BMSC,immunofluorescence staining and molecular biology technology were used to explore the possibility of BMSC differentiating into mammary gland-like epithelial cells in vitro in the presence of the epidermal growth factor（EGF）,insulin,prolactin,progesterone and so on.Purified and stable P4,P8 and P12 generation bovine BMSC were utilized to induce in vitro in medium with different cytokines and different concentrations.We used immunofluorescence and RT-PCR to observate and identify the cells after inducing.The results suggested that some BMSC changed from Spindle-like cells into polygon-like cells after induction;meanwhile they were clearly showed fluorescence by cytokeratin 18 staining.The bovine P4 generation BMSC remarkably differentiated under the action of induction medium Ⅱ.RT-PCR results demonstrated that cells after induction clearly expressed cytokeratin 19 gene,β-casein gene and αs1-casein gene.Consequently,BMSC can differentiate into mammary gland-like epithelial cells by using multi-factor to induce in vitro and the induction medium Ⅱshow good effects.

关 键 词：布莱凯特黑牛 骨髓间充质干细胞 表皮生长因子 胰岛素 催乳素 孕酮 乳腺样上皮细胞 

分 类 号：Q813[生物学—生物工程]