CAJ | 학술논문

目的探讨NPM1基因突变对THP-1细胞体外增殖和侵袭的影响及其机制.方法将THP-1细胞分为THP-1-mA组、空载体转染组和未处理组,THP-1-mA组使用携带人NPM1-mA的重组质粒pEGFPC1-NPM 1-mA转染THP-1细胞,建立稳定表达NPM1-mA的白血病细胞系(THP-1-mA)；空载体转染组使用空载体质粒pEGFPC1转染THP-1细胞；未处理组不进行质粒转染.采用反转录PCR、免疫细胞化学检测3组细胞NPM1-mA基因和蛋白的表达；使用细胞生长曲线观察细胞体外增殖能力；流式细胞术检测细胞周期分布；细胞体外侵袭实验观察细胞体外侵袭能力；实时荧光定量PCR检测血管生成素1 (Ang-1)、Ang-2 mRNA的表达.结果成功构建了稳定表达NPM1-mA的白血病细胞株.与空载体转染组和未处理组比较,稳定表达NPM1-mA蛋白的THP-1-mA细胞体外增殖能力明显增强,G1期细胞比例减少,S期细胞比例增加(均P＜0.01).与空载体转染组和未处理组比较,细胞体外侵袭实验显示THP-1-mA组细胞体外侵袭能力增强,实时荧光定量PCR显示THP-1-mA组细胞Ang-1 mRNA表达增高(均P＜0.01).结论 NPM1突变基因的表达能够促进THP-1细胞体外增殖和侵袭,而Ang-1可能在其中发挥重要作用.
목적 탐토NPM1기인돌변대THP-1세포체외증식화침습적영향급기궤제.방법 장THP-1세포분위THP-1-mA조、공재체전염조화미처리조,THP-1-mA조사용휴대인NPM1-mA적중조질립pEGFPC1-NPM 1-mA전염THP-1세포,건립은정표체NPM1-mA적백혈병세포계(THP-1-mA)；공재체전염조사용공재체질립pEGFPC1전염THP-1세포；미처리조불진행질립전염.채용반전록PCR、면역세포화학검측3조세포NPM1-mA기인화단백적표체；사용세포생장곡선관찰세포체외증식능력；류식세포술검측세포주기분포；세포체외침습실험관찰세포체외침습능력；실시형광정량PCR검측혈관생성소1 (Ang-1)、Ang-2 mRNA적표체.결과 성공구건료은정표체NPM1-mA적백혈병세포주.여공재체전염조화미처리조비교,은정표체NPM1-mA단백적THP-1-mA세포체외증식능력명현증강,G1기세포비례감소,S기세포비례증가(균P＜0.01).여공재체전염조화미처리조비교,세포체외침습실험현시THP-1-mA조세포체외침습능력증강,실시형광정량PCR현시THP-1-mA조세포Ang-1 mRNA표체증고(균P＜0.01).결론 NPM1돌변기인적표체능구촉진THP-1세포체외증식화침습,이Ang-1가능재기중발휘중요작용.
Objective To explore the effect of NPM 1 mutations on proliferation and invasion of THP-1 cells in vitro and its associated mechanism.Methods The THP-1 cells were allocated to THP-1-mA,nil vector transfection and nil treatment groups,respectively.In group THP-1-mA,the pEGFPC1-NPM1-mA plasmid vector with NPM 1 mutation A (NPM 1-mA) was transfected into THP-1 cells for constructing the THP-1-mA cells,the leukemia cell line,with stable expression of NPM1-mA protein.In nil vector transfection group,the pseudo vector pEGFPC1 was employed to transfect the THP-1 cells.Transfection was not processed in nil treatment group.Reverse transcriptase polymerase chain reaction and immunohistochemistry assay were employed to assay the expression of NPM-mA gene and protein,and the cell growth curve was used to determine the proliferation potential in vitro.Flow cytometry was applied to detect the distribution of cell cycles.The invasiveness of cells in vitro was also assayed.The expression of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) mRNA were assayed by quantitative real-time polymerase chain reaction.Results The THP-l-mA cell strain stably expressing NPM 1-mA protein was established successfully.Compared with nil vector transfection and nil treatment groups,the THP-1-mA cells stably expressing NPM-mA protein were characterized by significantly augmented capacity of proliferation in vitro,reduced percentage of cells in phase G1 yet increased proportion in phase S (both P＜0.01).These cells demonstrated,as evidenced by invasion assay,markedly augmented ability of invasion in vitro and,as suggested by fluorescent real-time polymerase chain reaction substantially increased expression of Ang-1 mRNA (both P＜0.01).Conclusion NPM 1 mutations may promote THP-1 cell proliferation and invasion in vitro,in which Ang-1 may play a crucial role.