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目的应用小干扰RNA(siRNA)质粒载体抑制人脑胶质瘤T98细胞系中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基本的表达,了解其对胶质瘤细胞化疗敏感性的影响。方法通过LipofectimineTM2000将针对MG-MT的siRNA质粒载体转入T98细胞中。采用实时定量PCR法测定MGMT mRNA的表达,Western blot测定MGMT蛋白的表达。MTT法测定转染前后T98细胞系对卡氮芥(BCNU)的敏感性变化。结果成功构建针对MGMT基因的siRNA质粒载体;质粒载体转染T98细胞后,对其MGMT mRNA的抑制率达87%,MGMT蛋白表达量明显减少,转染后的T98细胞对BCNU的敏感性增加,提高了约6倍。结论针对MGMT基因的siRNA质粒载体可以靶向抑制MGMT基因在T98细胞系中的表达,增加T98细胞对BCNU的敏感性。
OBJECTIVE: To study the basic expression of O6-methylguanine-DNA methyltransferase (MGMT) in human glioma T98 cell line by using small interfering RNA (siRNA) plasmid vector and to investigate its chemosensitivity to glioma cells influences. Methods siRNA plasmid vector targeting MG-MT was transfected into T98 cells by LipofectimineTM2000. The expression of MGMT mRNA was detected by real-time PCR and MGMT protein was detected by Western blot. The sensitivity of BCNU to T98 cell line before and after transfection was measured by MTT assay. Results The siRNA plasmid vector targeting MGMT gene was successfully constructed. After transfected into T98 cells, the inhibition rate of MGMT mRNA was 87% and the expression of MGMT protein was significantly decreased. The sensitivity of T98 cells after transfection to BCNU increased, Increased by about 6 times. Conclusion siRNA plasmid vector targeting MGMT gene can inhibit the expression of MGMT gene in T98 cell line and increase the sensitivity of T98 cells to BCNU.