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目的:研究促红细胞生成素(erythropoietin,EPO)对胶质母细胞瘤体外增殖的影响,并探讨其机制。方法:体外培养胶质母细胞瘤U87细胞株,实验分为对照组、EPO处理组及EPO+细胞周期素D1(Cyclin D1)拮抗剂组。对照组:用等体积PBS替代EPO;EPO处理组:在U87细胞的培养基中加入EPO(0.1 U/ml)处理7d;EPO+cyclin D1拮抗剂组:U87细胞的培养基中同时加入EPO(0.1 U/ml)及Cyclin D1拮抗剂(5μmol/L)共处理7 d。用Cell Counting Kit-8(CCK-8)法测细胞增殖情况,细胞倍增时间检测各组细胞增殖速度,RT-PCR、Western blot和细胞免疫荧光法(immunofluorescence,IF)测定细胞周期关键蛋白Cyclin D1的m RNA和蛋白表达变化,流式细胞仪检测各组细胞周期。结果:与对照组相比,EPO处理组中U87细胞生长速度明显增快(CCK8:第3天P=0.014,第5天P=0.029,倍增时间:P=0.012),细胞中Cyclin D1的m RNA及蛋白表达水平也明显升高,差异有统计学意义(m RNA:P=0.001,蛋白:P=0.002,荧光定量:P=0.005),流式细胞仪检测发现U87细胞的增殖指数也明显升高(P=0.000);与EPO处理组相比,EPO+cyclin D1拮抗剂组细胞Cyclin D1 m RNA及蛋白的表达水平明显降低(m RNA:P=0.009,蛋白:P=0.000,荧光定量:P=0.013),流式细胞仪检测细胞增殖指数则有显著下降(P=0.000),同时细胞增殖能力和速度较EPO处理组明显降低(CCK8:第3天P=0.000,第5天P=0.000,倍增时间:P=0.001)。结论:本研究发现EPO可显著促进胶质母细胞瘤的体外增殖,上调细胞周期关键蛋白Cyclin D1的表达,加快细胞增殖周期可能是其重要的分子机制。
Objective: To study the effect of erythropoietin (EPO) on the proliferation of glioblastoma in vitro and its mechanism. Methods: The glioblastoma U87 cell line was cultured in vitro. The experiment was divided into control group, EPO-treated group and EPO + Cyclin D1 antagonist group. In the control group, EPO was replaced by an equal volume of PBS. EPO group: EPO (0.1 U / ml) was added to U87 cells for 7 days; EPO + cyclin D1 antagonist group: U87 cells were treated with EPO 0.1 U / ml) and Cyclin D1 antagonist (5μmol / L) for 7 days. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Cell proliferation rate was detected by cell doubling time. The expression of Cyclin D1, the key protein of cell cycle was detected by RT-PCR, Western blot and immunofluorescence (IF) The changes of m RNA and protein expression were detected by flow cytometry. Results: Compared with the control group, the growth rate of U87 cells was significantly increased in EPO-treated group (CCK8: P = 0.014 on the 3rd day, P = 0.029 on the 5th day, doubling time: P = 0.012) RNA and protein expression levels were significantly increased, the difference was statistically significant (m RNA: P = 0.001, protein: P = 0.002, fluorescence quantitative: P = 0.005), flow cytometry showed that the proliferation index of U87 cells was also significantly (P = 0.000). Compared with EPO group, the expression of Cyclin D1 m RNA and protein in EPO + cyclin D1 antagonist group was significantly decreased (m RNA: P = 0.009, P = 0.000, : P = 0.013). The cell proliferation index was significantly decreased by flow cytometry (P = 0.000), while the cell proliferation rate and speed were significantly lower than those of EPO treatment group (P = 0.000 on the third day, P = 0.000, doubling time: P = 0.001). Conclusion: This study found that EPO can significantly promote glioblastoma cell proliferation in vitro, upregulate the expression of Cyclin D1, a key protein in the cell cycle, and accelerate the cell cycle may be an important molecular mechanism.