小鼠乳腺上皮细胞培养体系的建立及Heregulin-α的调控作用  

Establishment of Mouse Mammary Epithelial Cell Culture System and Study on the Modulation of Heregulin-α Factor

作　　者：耿丽晶[1,2] 张以涛[2] 林叶[2] 李庆章[2] 

机构地区：[1]辽宁医学院食品科学与工程学院,锦州121001 [2]东北农业大学,乳品科学教育部重点实验室,哈尔滨150030

出　　处：《中国科学：生命科学》2011年第4期296-303,共8页Scientia Sinica(Vitae)

基　　金：国家自然科学基金(批准号:30671538)资助项目

摘　　要：旨在建立完善的小鼠乳腺上皮细胞体外培养体系,探讨Heregulin-α(HRG-α)对该体系乳腺上皮细胞增殖及代谢的影响,为进一步深入研究乳腺上皮细胞的形态、结构和功能,揭示HRG-α在小鼠乳腺发育中的作用及其规律提供理论依据.通过比较不同pH和不同血清浓度配比的生长培养液对小鼠乳腺细胞生长的影响,得出pH7.4,添加10%血清的生长培养液为乳腺上皮细胞生长的最佳条件.通过运用相差显微镜技术、MTT等研究方法进行不同取材时期乳腺上皮细胞的形态、接种存活率、倍增时间、生长曲线等生物学特性比较,获得妊娠15天为较好的乳腺细胞取材时期.采用免疫组化和RT-PCR方法对细胞重要的标志蛋白角蛋白-18进行检测,鉴定所获得的细胞为乳腺上皮细胞,成功建立小鼠乳腺上皮细胞体外培养体系.利用所建立的小鼠乳腺上皮细胞体外培养体系,采用液相色谱技术及MTT方法,初步探讨了HRG-α对小鼠乳腺上皮细胞的影响.结果表明,适宜量HRG-α对小鼠乳腺上皮细胞生长和分泌总蛋白、乳糖均有显著促进作用,0.1～20ng/mL的HRG-α可促进小鼠乳腺上皮细胞的增殖和总蛋白、乳糖含量的增加,20ng/mL时达到最大值(P<0.01),50和100ng/mLHRG-α抑制小鼠乳腺上皮细胞的增殖,降低小鼠乳腺上皮细胞的总蛋白、乳糖含量(P<0.05).This study amis to establish and perfect the mouse mammary epithelial cell culture system,and to discuss the effect of Heregulin-α （HRG-α） on the mammary epithelial cell proliferation and metabolism within this cell system. It provides important theoretical basis for further research on mammary epithelial cell morphology,structure and function,and reveals the effect and laws of HRG-α in mouse mammary gland development. By comparing the mouse mammary cell growth in different culture media of different pH,serum concentration ratio,we have found that the culture medium of pH 7.4,supplemented with 10% serum was the best conditions for the growth of mammary epithelial cells. Through the use of phase contrast microscopy technology,MTT assay,and other techniques and research methods,this study compared mammary epithelial cell morphology,inoculation survival rate,doubling time,growth curves of biological characteristics at different periods derived. We have found that the good breast cells from the period was the 15 d pregnant mouse mammary. On this basis,important cell marker protein keratin-18 was detected by immunohistochemistry and RT-PCR,to further identify the cells obtained by mammary epithelial cells. Mouse mammary epithelial cells （MMECs） were successfully established in vitro culture system. By liquid chromatography and MTT,the study discusses the effects of HRG-α on MMECs in the established cell culture system in vitro. The result showes that MMECs’ cell growth and total secretion proteins and lactose were significantly promoted by the appropriate amount of HRG-α. 0.1～20 ng/mL HRG-α could promote MMECs to proliferate and total proteins and lactose content to increase. The maximum value was HRG-α at the concentration of 20 ng/mL. 50 ng/mL,100 ng/mL HRG-α inhibited the proliferation of MMECs,and decreased the total proteins and lactose content of MMECs.

关 键 词：小鼠 乳腺上皮细胞 细胞培养体系 Heregulin-α 生物学特性 

分 类 号：Q813[生物学—生物工程]