GM-CSF在替莫唑胺抗高级别胶质瘤中的作用

GM-CSF Enhances Effect of TMZ on High-grade Glioma Cells

目的探讨粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GMCSF)在替莫唑胺(temozolomide,TMZ)抗高级别胶质瘤中的作用及机制。方法选取6例高级别胶质瘤患者来源的肿瘤组织培养肿瘤细胞,待细胞状态稳定后采用MTT法进行细胞增殖毒性实验,流式细胞仪检测细胞周期变化和细胞凋亡情况,甲基化特异性PCR和免疫组织化学染色法分别检测六氧甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine-DNA methyltransferase,MGMT)基因启动子甲基化状态和MGMT蛋白表达水平。结果 MTT实验显示,GM-CSF处理组的细胞存活率与对照组相比均有不同程度的增加。MGMT基因启动子甲基化的3例细胞,GM-CSF+TMZ组的细胞存活率比TMZ组显著降低(P<0.05),另3例MGMT启动子非甲基化细胞,GM-CSF+TMZ组与TMZ组相比,其存活率差异均无统计学意义(P>0.05)。6例细胞GM-CSF组的G1期细胞比例均比对照组降低,而S期细胞比例GMCSF处理组较对照组显著增加(P<0.05)。流式细胞仪凋亡检测显示,MGMT启动子甲基化的3例细胞,GM-CSF+TMZ组凋亡率与单药TMZ组凋亡率相比均显著增加(P<0.05),而MGMT非甲基化细胞GM-CSF+TMZ组与单药TMZ组凋亡率相比无统计学差异。结论 GM-CSF可通过诱导高级别胶质瘤细胞快速进入细胞周期,显著提高TMZ对MGMT基因启动子甲基化的高级别胶质瘤细胞的杀伤作用,而对MGMT基因启动子非甲基化高级别胶质瘤细胞的作用不明显。

Objective To investigate the effect of granulocyte-macrophage colony-stimulating factor （GM- CSF） combined with temozolomide （TMZ） on high-grade glioma cells and related mechanism. Methods We cultured six cases of high-grade glioma cells from patient＇s tumor tissues. MTT assay was used to detect cell proliferation and toxicity. Flow cytometry was used to detect cell cycle and apoptosis rate. Specific PCR and immunofluorescence were used to detect the expression of O6-methylguanine-DNA methyltransferase （MGMT） methylation status and MGMT protein respectively. Results MTT assay suggested that compared with the control group, GM-CSF group had increased cell viability in varying degrees. In three cases of cells （MGMT gene methylation）, the cell viability of the combination group [（67.67±1.16）, （68.13±1.06）, （68.42± 1.73）]was significantly lower than that of the corresponding TMZ group [（90.00±1.73）, （82.33±1.53）, （82.67 ±2.11）] （P〈0.05）. However, there was no significant difference between the two groups in another three cases （MGMT gene unmethylated） （P〉0.05）. For the MGMT methylation cells, the apoptosis rate in combination group was higher than that in the corresponding TMZ group（P〈0.05）, which coincided with MTT assay results. In all 6 cases of primary glioma cells, GM-CSF treated group showed significant reduction in the fraction of cells in G1 phase with concomitant increase in S phase（P〈0.05）. Conclusion GM-CSF could induce high-grade glioma cells entering the cell cycle rapidly, which could enhance the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation.However this effect is not ideal on glioma cells with MGMT unmethylation.