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目的研究替莫唑胺对胶质瘤U251干细胞分化、增殖、凋亡的影响及作用机制。方法培养U251胶质瘤细胞,用磁细胞分选法分选出U251干细胞。用0、100、500μmol、1 mmol替莫唑胺分别处理对数期的U251干细胞24、48、72、96h,MTT检测各个处理对U251细胞增殖的影响;用含血清培养基诱导U251干细胞分化,分别加入0、500μmol替莫唑胺处理72 h,提取分化前及两组替莫唑胺处理后干细胞总蛋白,Western-blot检测干细胞标记物CD133及分化标记物MAP2、GFAP、MBP蛋白表达情况;分别利用0、500μmol替莫唑胺处理U251干细胞72 h,流式细胞仪检测各组细胞凋亡情况;提取0、500μmol替莫唑胺处理72 h后的U251干细胞总蛋白,Western-blot检测Notch1信号通路关键蛋白表达情况。结果替莫唑胺处理抑制U251干细胞增殖,与0μmol替莫唑胺处理相比差异显著(P<0.01),500μmol替莫唑胺处理72 h差异最显著。U251细胞分化后,CD133蛋白表达量降低,MAP2、GFAP、MBP蛋白表达量升高,与分化前相比差异显著(P<0.01),与0μmol处理相比,500μmol替莫唑胺处理后CD133、MAP2、GFAP、MBP蛋白表达差异显著(P<0.01)。500μmol替莫唑胺处理72 h后U251干细胞凋亡明显增加,与0μmol处理相比差异显著(P<0.01)。500μmol替莫唑胺处理72 h后Notch1通路关键蛋白Notch1、CBF-1、HES-1表达明显下降,与0μmol处理相比具有显著性差异(P<0.01)。结论替莫唑胺可能通过抑制Notch1信号通路抑制胶质瘤U251干细胞增殖,促进U251干细胞分化及凋亡。
Objective To study the effect of temozolomide on differentiation, proliferation and apoptosis of glioma U251 stem cells and its mechanism. Methods U251 glioma cells were cultured and the U251 stem cells were sorted by magnetic cell sorting. U251 stem cells were treated with 0, 100, 500μmol, 1mmol temozolomide for 24,48,72,96h, respectively. MTT assay was used to detect the effect of each treatment on the proliferation of U251 cells; U251 stem cells were induced to differentiate by adding 0 , And 500μmol temozolomide for 72h. The total protein of stem cells before and after temozolomide treatment was extracted, and the expression of CD133 and MAP2, GFAP and MBP proteins were detected by Western-blot. U251 stem cells were treated with 0, 500μmol temozolomide The apoptosis of U251 cells was detected by flow cytometry. The total protein of U251 stem cells treated with 0, 500 μmol of temozolomide for 72 h was detected. The expression of key proteins of Notch1 signal pathway was detected by Western-blot. Results Temozolomide treatment inhibited the proliferation of U251 stem cells, which was significantly different from that of 0μmol temozolomide treatment (P <0.01). The treatment with 500μmol temozolomide for 72h had the most significant difference. Compared with 0μmol treatment, the expression of CD133, MAP2, GFAP and MBP in U251 cells decreased and the levels of CD133, MAP2, GFAP , MBP protein expression difference was significant (P <0.01). After treated with 500μmol of temozolomide for 72h, the apoptosis of U251 stem cells was significantly increased, which was significantly different from that of 0μmol treatment (P <0.01). The expression of Notch1, CBF-1 and HES-1, the key proteins of Notch1, was significantly decreased after 500 μmol of temozolomide treatment for 72 h, which was significantly different from that of 0 μmol treatment (P <0.01). Conclusion Temozolomide can inhibit the proliferation of glioma U251 stem cells by inhibiting the Notch1 signaling pathway and promote the differentiation and apoptosis of U251 stem cells.