夏 飞 刘胜武 魏雅稚 肖 凌
【摘要】 目的 比较不同DNA提取方法对凋亡细胞内断裂DNA片段的提取效果及其影响因素。方法 采用试剂盒法、磷酸盐/柠檬酸缓冲液抽提法提取地塞米松诱导5h胸腺细胞内的DNA片段，后者分别经真空离心浓缩法和异丙醇及70％乙醇沉淀漂洗法提取DNA片段，1％琼脂糖凝胶电泳，紫外灯下观察。结果 磷酸盐/柠檬酸缓冲液抽提法对低浓度细胞内的小DNA片段有显著的抽提效果，紫外灯下可见明显的小DNA片段条带，改用异丙醇和70%乙醇抽提法以代替真空离心浓缩后仍然可见代表小DNA片段的条带；试剂盒提取法可提取凋亡细胞内的各种DNA片段，紫外灯下可见明显的“梯状条带”。结论 以上二种方法实验所需时间较少，且都避免了酚等有毒试剂的使用。试剂盒提取法对凋亡细胞DNA片段的提取效果较好，但费用较高；磷酸盐/柠檬酸缓冲液抽提法可高效地提取凋亡细胞中的小片段DNA，且异丙醇和70％乙醇沉淀抽提法可替代真空离心浓缩，更易于此种方法地推广。
【关键词】 细胞凋亡；磷酸盐/柠檬酸缓冲液法；DNA片段化
The Improvement of DNA Extract Method for Apoptosis Detection XIA Fei, LIU Sheng-wu, WEI Ya-zhi,
XIAO Ling. Department of Immunology, Medical School of Wuhan University, Wuhan 430071
【Abstract】 Objective To study the application of two different DNA extraction methods for the detection of apoptosis. Methods The DNA fragment in thymocytes cultured with dexamethasone were extracted respectively by kit and phosphate-citric acid buffer, and subsequently fragment in the buffer were concentrated by vacuum pump or precipitated by isopropyl alcohol, and washed by 70% alcohol solution. Results The phosphate-citric acid buffer could apparently extract the small DNA fragment. DNA ladder could be observed by 1% agarose gel electrophoresis after precipitation by isopropyl alcohol and 70% alcohol solution in stead of vacuum pump. The kit could obviously acquire various DNA fragment. Conclusions The kit could extract various DNA fragment at the advantage of avoiding toxic reagents, but the average cost is expensive. Phosphate-citric acid buffer could efficiently extract small DNA fragment, even though isopropyl alcohol and 70% alcohol substitute for the vacuum concentrate, which is more practical to be spread.
【Key words】 Apoptosis; Phosphate-citric acid buffer method; DNA fragmentation
凋亡是一种不同于坏死的细胞死亡方式，是细胞受基因调控的一种自然死亡过程。细胞凋亡在形态学、生化等方面有其显著的特征[1]。其检测方法也分为形态学观察、DNA片段化的凝胶电泳分析、凋亡相关酶学及流式细胞术等。但就确定凋亡发生而言，通过凝胶电泳获得DNA片段化的“梯状条带” 是其重要生物化学依据。因此，提取凋亡DNA片段成为凝胶电泳成败的关键。本文分别参照DNA提取试剂盒的方法与磷酸盐/柠檬酸缓冲液法提取同一批样本中的DNA片段，并对磷酸盐/柠檬酸缓冲液法加以改进优化，使之能更广泛地得到运用。
