MGMT-04多肽在人脑胶质瘤原代细胞中模拟抗TMZ耐药作用的实验研究

Experimental study of MGMT-04 polypeptide mimicking anti-TMZ drug resistance in primary human glioma cells

目的研究多肽MGMT-04模拟O 6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)在人脑胶质瘤原代细胞系SHG-140中抗替莫唑胺(TMZ)耐药的实验。方法首先设计穿膜肽MGMT-04,并加以FITC示踪,再通过免疫荧光定位MGMT-04多肽可以到达细胞核发挥作用。通过流式细胞术检测各处理组SHG-140细胞凋亡的比例,包括正常对照组,MGMT-04多肽组,TMZ组,TMZ+MGMT-04多肽组。通过免疫荧光技术检测上述四组SHG-140细胞BCL2及C-PARP的表达。使用Western Blot法检测上述四组SHG-140细胞中凋亡相关蛋白PARP、Cleaved-PARP、BCL2、Caspase-3、Cleaved-Caspase-3、BAX、BCL2的表达。结果流式细胞术检测结果示正常对照组与MGMT-04多肽组的凋亡无明显差异,TMZ组凋亡细胞比例明显增加,而TMZ+MGMT-04多肽组较TMZ组的凋亡细胞比例下降。免疫荧光染色结果示TMZ+MGMT-04多肽组较TMZ单组BCL2表达高、Cleaved-PARP表达量降低。Western Blot结果进一步验证与正常对照组相比,经MGMT-04多肽单独处理的相关凋亡蛋白的表达无明显差异,而TMZ组较正常对照组的Cleaved-PARP、Cleaved-Caspase-3、BAX表达量增加,BCL2的表达量下降,两组之间的差异有统计学意义(P<0.05),而TMZ+MGMT-04多肽组较TMZ组Cleaved-PARP、Cleaved-Caspase-3、BAX表达量降低,BCL2表达量升高,两组之间的差异有统计学意义(P<0.05)。结论多肽MGMT-04在人脑胶质瘤原代细胞中具有抗TMZ而增强耐药作用。

Objective To study the resistance of the peptide MGMT-04 mimicking O 6-methylguanine-DNA methltransferase(MGMT)to temozolomide(TMZ)in human glioma primary cell line SHG-140.Methods Firstly,the transmembrane peptide MGMT-04 was designed and traced by FITC,and then the MGMT-04 peptide could reach the nucleus by immunofluorescence.Flow cytometry was used to detect the early apoptosis rate of SHG-140 cells in each treatment group,including normal control group,MGMT-04 polypeptide group,TMZ group,and TMZ+MGMT-04 polypeptide group.The expression of BCL2 and Cleaved-PARP in the above four groups of SHG-140 cells was detected by immunofluorescence technology.Western blot was used to detect the expression of the aforementioned four groups of SHG-140 apoptosis-related proteins PARP,Cleaved-PARP,BCL2,Caspase-3,Cleaved-Caspase-3,BAX,and BCL2.Results The results of flow cytometry showed that there was no significant difference in apoptosis between the normal control group and the MGMT-04 group.The proportion of premature withering cells in the TMZ group was significantly increased,while the proportion of premature withering cells in the TMZ+MGMT-04 group was lower than that in the TMZ group.The results of immunofluorescence staining showed that the expression of BCL2 was higher in the TMZ+MGMT-04 group and the expression of Cleaved-PARP was lower than that in the TMZ single group.The Western Blot results further verified that compared with the normal control group,there was no significant difference in the expression of related apoptosis proteins treated with MGMT-04 polypeptide alone,while the TMZ group increased the expression of Cleaved-PARP,Cleaved-Caspase-3,and BAX compared with the normal control group.The expression of protein BCL2 decreased,and the difference between the two groups was statistically significant(P<0.05).Compared with the TMZ group,the expression of Cleaved-PARP,Cleaved-Caspase-3,and BAX decreased in the TMZ+MGMT-04 group,and the expression of BCL2 increased.The difference between the two groups was statistically significant(P<0.05).Conclusion The polypeptide MGMT-04 has anti-TMZ drug resistance in primary human glioma cells.