BrdU作为脂肪干细胞标记示踪法的可行性  

Bromodeoxyuridine labeling of swine adipose-derived stem cells in vitro

作　　者：顾广祥[1] 施晓雷[2] 杭化莲[2] 丁义涛[1] 

机构地区：[1]南京医科大学鼓楼临床医学院肝胆外科,江苏省南京市210008 [2]南京大学附属鼓楼医院肝胆外科,江苏省南京市210008

出　　处：《世界华人消化杂志》2010年第1期14-19,共6页World Chinese Journal of Digestology

摘　　要：目的:研究BrdU标记猪脂肪干细胞(adipose-derived stem cells,ADSCs)的最佳剂量及时间,探讨其作为干细胞标记示踪方法的可行性.方法:自中华实验猪背部提取脂肪组织,采用贴壁法分离培养ADSCs,取第3代细胞以终浓度分别为10、15、20、25及30μmol/LBrdU进行标记;另一孔不含BrdU作为对照组,分别培养12、24、48、72及96h.免疫荧光法检测各组细胞BrdU标记率,找出BrdU的最佳标记方法,通过台盼蓝排斥试验、MTT及细胞凋亡检测,观察BrdU对ADSCs生长情况的影响.对第3代的ADSCs采用最佳标记方法后更换普通培养基继续培养,适时传代,连续检测第4、5、6、7、8代ADSCs的BrdU标记率.结果:原代培养的ADSCs的形态主要为长梭形,第3代ADSCs经BrdU标记后胞核呈红色荧光,随浓度的升高及时间的延长,BrdU阳性标记率逐渐升高,以终浓度20μmol/LBrdU标记48h后阳性率达90%以上,且连续传5代标记率仍达40%.MTT、台盼蓝排斥试验及细胞凋亡检测发现BrdU对ADSCs生长增殖基本无影响.结论:BrdU标记ADSCs的最佳剂量和时间为20μmol/L和48h,该方法标记率高,对细胞影响小,可用于动态研究ADSCs在移植体内生长、分化.AIM： To investigate the optimal dosage and treatment duration of bromodeoxyuridine （BrdU） for labeling of porcine adipose-derived stem cells （ADSCs） in vitro and explore the feasibility of using BrdU as a tracer to label stem cells. METHODS： ADSCs were isolated from swine fat tissue by type I collagenase digestion and purified by differential adhesion. ADSCs at passage 3 were labeled with BrdU at different concentrations （10, 15, 20, 25 and 30 μmol/L）. The labeling rates were determined by immunofluorescence at 12, 24, 48, 72 and 96 h after incubation with BrdU to find the optimal labeling parameters.The impact of BrdU on the growth of ADSCs was examined by trypan blue exclusion, methyl thiazoly tetrazolium （MTT） assay and cell apoptosis assay. ADSCs at passage 3 were labeled with BrdU using the optimal labeling parameters and then cultured and passaged in essential medium without BrdU. The labeling rates in ADSCs between 4-8 passages were determined to observe the attenuation of BrdU incorporation. RESULTS： The majority of primary ADSCs were fusiform in shape. After BrdU labeling, the nuclei of ADSCs showed red fluorescence. With the increase in labeling concentration and duration, the BrdU labeling rate gradually increased. After labeling with 20 μmol/L BrdU for 48 hours, the labeling rate exceeded 90%. The labeling rate decreased with increased passages, declining to 40% after 5 passages. MTT assay, trypan blue exclusion and cell apoptosis assay showed that BrdU labeling had no significant impact on the growth of ADSCs. CONCLUSION： The optimal labeling dosage and duration of BrdU are 20 μmol/L and 48 hours, respectively. The BrdU-labeling method achieves a high labeling rate, has little impact on the growth of ADSCs and can therefore be used for dynamic observation of the survival, growth and differentiation of implanted ADSCs.

关 键 词：脂肪干细胞 5-溴脱氧尿嘧啶核苷 标记 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]