CAJ | 학술논문

目的观察胶质瘤细胞中异柠檬酸脱氢酶1(IDH1)基因突变对肿瘤细胞的生长抑制作用并探讨其机制.方法构建表达IDH1 R132H突变体的慢病毒载体,转染U87胶质瘤细胞,通过免疫荧光染色观察细胞增殖,原位缺口末端标记法(TUNEL)法检测细胞凋亡,水溶性四唑盐(WST)法绘制细胞生长曲线,比色法检测细胞内还原性烟酰胺腺嘌呤二核苷酸磷酸(NADPH)、还原性谷胱甘肽(GSH)含量,荧光探针检测细胞内活性氧(ROS)含量;构建裸鼠动物模型,绘制肿瘤生长体积曲线与重量柱形图.结果表达IDH1 R132H突变体的胶质瘤细胞生长明显抑制,免疫荧光染色以及TUNEL法证实细胞增殖减少、凋亡增多;细胞内NADPH水平降低伴随GSH含量降低和ROS蓄积,H2O2(1 mmol/L)可明显增加胶质瘤细胞的生长抑制作用,GSH(1 mmol/L)则可减弱抑制作用.结论IDH1基因R132H突变可显著抑制胶质瘤细胞生长,细胞内GSH含量降低和ROS蓄积可能是其重要机制.
목적 관찰효질류세포중이저몽산탈경매1(IDH1)기인돌변대종류세포적생장억제작용병탐토기궤제.방법 구건표체IDH1 R132H돌변체적만병독재체,전염U87효질류세포,통과면역형광염색관찰세포증식,원위결구말단표기법(TUNEL)법검측세포조망,수용성사서염(WST)법회제세포생장곡선,비색법검측세포내환원성연선알선표령이핵감산린산(NADPH)、환원성곡광감태(GSH)함량,형광탐침검측세포내활성양(ROS)함량;구건라서동물모형,회제종류생장체적곡선여중량주형도.결과 표체IDH1 R132H돌변체적효질류세포생장명현억제,면역형광염색이급TUNEL법증실세포증식감소、조망증다;세포내NADPH수평강저반수GSH함량강저화ROS축적,H2O2(1 mmol/L)가명현증가효질류세포적생장억제작용,GSH(1 mmol/L)칙가감약억제작용.결론IDH1기인R132H돌변가현저억제효질류세포생장,세포내GSH함량강저화ROS축적가능시기중요궤제.
Objective To explore the growth influence of isocitrate dehydrogenase 1 (IDH1) mutations on glioma cells and the mechanism.Methods The lentivirus vector expressing IDH1 R132H mutant gene,and transfected into U87 cells.The proliferation of U87 cells was measured with proliferating cell nuclear antigen (PCNA) immunofluorescent staining,and TdT-mediated dUTP nick end labeling (TUNEL) method was used to evaluate apoptosis.The growth curve of U87 cells was drawn by using WST assay kit.Cellular nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) levels,and intracellular reactive oxygen species (ROS) were measured by fluorescence microplate.A animal model of nude mice was established,and the tumor volume curves and the bar chart of the weight were drawn.Results The growth was significantly inhibited in glioma cells overexpressing IDH1R132H.Moreover,in the glioma cells overexpressing IDH1 R132H,proliferation was reduced,and apoptosis was increased.These cells were characterized by decreased intracellular NADPH levels accompanied by GSH depletion and ROS generation.Accordingly,H2O2(1 mmol/L) could obviously increase the inhibition of mutant IDH1 glioma cells growth,nevertheless GSH (1 mmol/L) had the opposite result.Conclusion Our study provides direct evidence that R132H mutation of IDH1 profoundly inhibits the growth of glioma cells,and depletion of GSH and generation of ROS are the primary cellular events associated with this mutation.