沙利度胺联合替莫唑胺杀伤U251胶质瘤细胞机制的体外研究

Study on mechanism of thalidomide combined with temozolomide to suppress proliferation of U251 glioma cell in vitro

目的对沙利度胺联合替莫唑胺杀伤U251胶质瘤细胞的机制进行体外研究,为制订沙利度胺与替莫唑胺联合化疗方案提供理论依据。方法经体外培养的人胶质瘤细胞系U251分别接受替莫唑胺(100 μmol/L)、沙利度胺(100 μg/L)、替莫唑胺与沙利度胺联合治疗,噻唑蓝(MTT)法检测不同抗肿瘤药物处理组肿瘤细胞增殖活性;流式细胞术分析细胞增殖周期;检测经吖啶橙标记的酸性囊性细胞器数目;原位末端标记(TUNEL)法观察肿瘤细胞凋亡情况;Western blotting法检测肿瘤自噬及凋亡相关蛋白表达变化。结果与替莫唑胺和沙利度胺单药治疗相比,替莫唑胺与沙利度胺联合治疗对U251细胞生长的抑制更为明显(均P=0.000),且可诱导肿瘤细胞周期阻滞于G_0～G_1期,以及发生凋亡和自噬。两药联合治疗后,U251细胞微管相关蛋白1轻链3和Caspase-3表达水平高于替莫唑胺组和沙利度胺组(均P=0.000)。结论沙利度胺联合替莫唑胺治疗U251细胞可以上调自噬及凋亡相关基因表达水平,同时诱导凋亡及自噬性死亡,从而达到对U251胶质瘤细胞的杀伤作用。

Objective To provide a more reasonable regimen of temozolomide and thalidomide, and study the mechanism of these 2 drugs in inhibiting the proliferation and growth of U251 glioma cell in vitro. Methods Human glioma cell line U251 was cultured in vitro and divided into different treatment groups for 3 d： temozolomide group （100 μmol/L）, thalidomide group （100 μg/L）, temozolomide （100 μmol/L） with thalidomide group （100 μg/L） and vehicle control group. After different treatment for 3 d, 3-（4, 5- dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） was adopted for the determination of cell viability, and cell cycle was analysed by flow cytometry. After labeled with acridine orange （AO）, autophagy vesicles were quantitatively detected by flow cytometry. TdT-mediated dUTP-biotin nick end labeling （TUNEL） was employed in observing and detecting the apoptosis of treated cells. Western blotting was used in examining the autophagy and apoptosis-related proteins. Results Compared with the 2 drugs used alone, temozolomide with thalidomide imposed more obvious inhibition on tumor cell growth （P = 0.000, for all）. Combination of 2 drugs induced tumor cell cycle arrest in G0-Gl. Both of autophagic and apoptotic cell death could be induced by temozolomide with thalidomide in U251. In two-drug treatment group, the expression of autophagy-related microtubule-associated protein I light chain 3 （MAP1LC3） and apoptosis-related Caspase-3 was significantly higher in transcriptional level in comparing with single drug treatment group （P = 0,000, for all）. Conclusion Temozolomide combined with thalidomide may induce 2 types of programmed cell death-apoptotic and autophagic cell death by up-regulating the expression of related genes in U251 glioma cell, thereby, the combination of these 2 drugs may suppress the proliferation and growth of U251 glioma cell in vitro.