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目的 利用流式细胞仪 (flowcytometry ,FCM)探讨反义IGF IR基因片段诱导胶质瘤细胞凋亡的作用。方法 根据IGF IRcDNA序列设计其正义、反义基因片段 ,并对部分碱基进行硫代磷酸修饰。实验分为空白对照组、正义核酸组和反义核酸组 ,应用流式细胞技术检测DNA含量 ,根据DNA直方图分析凋亡细胞情况。应用透射电镜观察了凋亡细胞的形态学变化。结果 经反义寡核苷酸 5、10、15、2 0μmol·L-1处理的胶质瘤细胞其凋亡率分别为 16 0 6 %±3 86 %、2 4 5 2 %± 4 84%、36 6 9%± 5 5 3%和 43 85 %±5 72 % ,而用正义寡核苷酸 5、10、15、2 0 μmol·L-1处理的胶质瘤细胞其凋亡率分别为 3 76 %± 1 2 2 %、3 14%±1 15 %、2 5 1%± 0 93%和 3 2 6 %± 1 15 % ,空白对照组(野生型 )自然凋亡率为 1 0 1%± 0 89%。反义核酸组与对照组相比有显著性差异 (P <0 0 5 ) ,而正义核酸组与空白对照组无明显差异 (P >0 0 5 )。凋亡细胞形态上表现为细胞体积缩小 ,染色质凝聚 ,并聚集于核膜下成新月状或块状 ,胞膜内馅形成膜包裹的核碎片或细胞器 ,最后出泡形成凋亡小体。结论 反义IGF IR基因片段诱导胶质瘤细胞凋亡是其抑制细胞增殖机制之一。通过FCM分析可较准确地判定肿瘤细胞凋亡情况
Objective To investigate the effect of antisense IGF IR gene fragment on glioma cell apoptosis by flow cytometry (FCM). Methods According to the sequence of IGF IR cDNA, the sense and antisense gene fragments were designed and phosphorothioate was modified to some bases. The experiment was divided into the blank control group, the sense nucleic acid group and the antisense nucleic acid group. The DNA content was detected by flow cytometry, and the apoptotic cells were analyzed by DNA histogram. The morphological changes of apoptotic cells were observed by transmission electron microscopy. Results The apoptotic rate of glioma cells treated with antisense oligodeoxynucleotides 5,10,15,2Oμmol·L-1 were 16 0% ± 3 86%, 2 452% ± 4 84% , 36 6 9% ± 5 5 3% and 43 85% ± 5 72%, while the apoptotic rates of glioma cells treated with 5,10,15,2 0 μmol·L-1 of sense oligonucleotide The percentages of apoptosis in the blank control group (WT) were 10 76% ± 122%, 3 14% ± 1 15%, 25 1% ± 0 93% and 32 6% ± 1 15% 1% ± 0 89%. There was a significant difference between the antisense nucleic acid group and the control group (P <0.05), but there was no significant difference between the positive nucleic acid group and the blank control group (P> 0.05). Apoptotic cells morphologically characterized by reduced cell size, chromatin condensation, and gathered in the nuclear membrane into a crescent or lumps, membrane formation of membrane-filled nuclear debris or organelles, the final bubble formation of apoptotic bodies . Conclusion Antisense IGF-IR gene fragment induces glioma cell apoptosis is one of the mechanisms of its inhibition of cell proliferation. FCM analysis can be more accurately determine the apoptosis of tumor cells