BrdU标记小鼠视网膜祖细胞的研究  

Investigation of 5-Bromo-2′-deoxyuridine Labelling Mice Retinal Progenitor Cells

作　　者：孙雪荣[1,2] 董志章[2] 邓飞[2] 胡惠玲[2] 葛坚[2] 

机构地区：[1]广东医学院广东省医学分子诊断重点实验室衰老研究所,东莞523000 [2]中山大学中山眼科中心眼科学国家重点实验室,广州510000

出　　处：《生物医学工程学杂志》2013年第1期125-130,共6页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(309766;81170846);眼科学国家重点实验室专项经费资助项目(QN-01);广东医学院科技创新基金资助项目(STIF201102);广东省医学科研基金资助项目(A2012420)

摘　　要：为了探讨BrdU(5-Bromo-2′-deoxyuridine)体外标记视网膜祖细胞(RPCs)的适宜浓度,将不同浓度BrdU(0.2、1、5和10μmol/L)与小鼠孕龄(E)17.5dRPCs共培养48h后,进行增殖或分化培养。用免疫荧光检测BrdU标记百分率及细胞分化潜能,用细胞计数法检测其增殖能力,用乳酸脱氢酶(LDH)法检测其细胞毒性。结果发现,0.2μmol/L BrdU不能明显标记RPCs,而1、5和10μmol/L 3种浓度BrdU均可有效标记RPCs,且三者的标记率相近(P>0.05),1μmol/L BrdU无明显细胞毒性,对RPCs增殖、分化亦无明显影响,而5μmol/L和10μmol/L Br-dU均能抑制细胞增殖,并引起LDH释放增加,说明有明显的细胞毒性,10μmol/L BrdU还会阻碍RPCs向MAP2+神经元方向分化,但对其向GFAP+或glutamine synthetase+神经胶质细胞分化无明显影响。本研究表明,1μmol/L BrdU既能有效标记E17.5RPCs,又无显著毒副作用,是较适宜的浓度。BrdU （5-Bromo-2＇-deoxyuridine） is usually used to label the mitotic cells as well as to trace reagent in cell transplation. However, BrdU could also exert some side effect on cellular biological characteristics upon inappropri- ate use. To explore the appropriate concentration of BrdU for labelling retinal progenitor cells （RPCs）, we co-cul- tured Embryonic day （E） 17.5 RPCs with different concentrations of BrdU, which were 0.2, 1, 5 and 10μmol/L, respectively. After 48 hours, the RPCs were proliferation- or differentiation-cultured. Immunofluorescence was used to detect the BrdU-positive ratio and differentiation potential. Cell count was used to evaluate proliferation ability, and lactate dehydrogenase （LDH） release assay was used to monitor eytotoxicity. The results showed that 0.2 μmol/ L BrdU could not label RPCs clearly, while BrdU of 1, 5 or 10 μmol/L could label the RPCs with similar ratios. 1 μmol/L BrdU displayed no obvious cytotoxicity and showed no obvious effect on the proliferation and differentiation ability. However, 5 μmol/L or 10μmol/L BrdU could evidently inhibit RPCs proliferation, partly due to the eyto- toxicity effect. Furthermore, 10 μmol/L BrdU could inhibit the differentiation of RPCs towards MAP2-positive nerve cells, but showed no influence on the differentiation of RPCs towards GFAP- and glutamine synthetase- posi- tive glial cells. This study suggested that 1 μmol/L BrdU could be an appropriate concentration for RPCs labelling and could efficiently label RPCs without obvious side effect.

关 键 词：视网膜祖细胞 BRDU 细胞毒性 分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]