摘要
背景:到目前为止已建立了数百个小鼠胚胎干细胞系,一般情况下实验室培养的胚胎干细胞有3个细胞来源,即胚泡内的内细胞群、胚胎生殖嵴的原始生殖细胞和应用克隆技术制造人体胚胎并提取干细胞。目的:尝试用自制的小鼠胚胎成纤维细胞饲养层分离培养小鼠胚胎干细胞。设计、时间及地点:细胞学体外观察,于2005-12/2007-09在辽宁医学院完成。材料:妊娠14.5d的孕鼠40只,由辽宁医学院实验动物中心提供。方法:无菌条件下剪开孕鼠子宫壁,取出胚胎,去除头、尾、内脏和四肢后采用胰蛋白酶消化法分离培养小鼠胚胎成纤维细胞,经丝裂霉素C处理3.5h后接种于6孔板中,细胞贴壁后即为成纤维细胞饲养层。收集3.5d的小鼠囊胚,在显微镜下将囊胚置于上述6孔板的中央,五六天后取隆起生长的内细胞团块,分离后再培养。主要观察指标:成纤维细胞饲养层的制备情况,胚胎干细胞的生长状态、碱性磷酸酶染色结果及分化趋势。结果:光镜下小鼠胚胎成纤维细胞呈不规则形,生长较快,传代比例为1∶4,经丝裂霉素C处理后细胞贴壁较慢,失去分裂增殖能力。小鼠囊胚孵出透明带的时间为24～72h,从胞膜中孵出约为1d,3～5d后可形成胚胎干细胞集落,7d后集落呈"鸟巢"状。组织化学染色后可见细胞内有大量的蓝紫色颗粒沉积,连续培养20d,期间不换培养液,可见胚胎干细胞团分化为亮度较小、界限清楚的单核细胞。结论:小鼠囊胚在胚胎成纤维细胞饲养层上可发育成胚胎干细胞,并能进行传代。
BACKGROUND： Hundreds of mouse embryonic stem cell line have been created in recent years. Generally, there are 3 cell sources： inner cell mass in blastocyst, primordial germ cells in embryo genital ridge and human embryo made by cloning technique. OBJECTIVE： To isolate and culture mouse embryonic stem cell from embryos of mouse using self-made mouse embryonic fibroblast feeder layer. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Liaoning Medical University from December 2005 to September 2007. MATERIALS： A total of 40 rats at gestational day 14.5 were provided by Animal Experimental Center, Liaoning Medical University. METHODS： Uterine wall of pregnant rats were sterilely incised to obtain embryo. After removal of head, tail, internal organs and limbs, mouse embryonic fibroblasts were isolated and cultured by the trypsin digestion, treated with mitomycin C for 3.5 hours, incubated in a 6-well plate. After cell adherence, fibroblast feeder layer was created. The blastocytes of 3.5 days from mouse were cultured in the middle of the mouse fibroblast feeder layers for 5-6 days, and the cells from inner cell mass were isolated and subsequently cultured in vitro. MAIN OUTCOME MEASURES： Preparation of fibroblast feeder layer; growth of embryonic stem cells; results of alkaline phosphatase staining; cell differentiation tendency. RESULTS： Under the optical microscope, mouse embryonic fibroblasts were irregular, rapidly grew, passaged at 1：4. Following mitomycin C treatment, cells slowly adhered and could not proliferate. Mouse blastula showed clear band in 24-72 hours, came out from cell membrane in 1 day. After 3-5 days, it become embryonic stem cell mass. At 7 days, the cell mass showed ＂bird nest＂ shape. Following histochemical staining, lots of amethyst particle deposit in cells. Continual to culture the embryonic stem cells for 20 days without changing the medium, the embryonic stem cell changed into single nucleus cells with dark lightness and clear boundary. CONCL
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第14期2779-2782,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
