CAJ | 학술논문

目的初步探讨14-3-3β基因对胶质瘤细胞生物学行为的影响及其作用机制. 方法收集常规培养的SVGp12及U251、U87、SHG-44以及用14-3-3β特异性siRNA(14-3-3β-siRNA)沉默的U251细胞等各胶质瘤细胞株,应用实时定量PCR (RT-PCR)和Westernblotting检测各胶质瘤细胞中14-3-3β基因及蛋白表达情况.将常规培养生长至对数期的U251细胞分为实验组(14-3-3β-siRNA转染)、阴性对照组(对照siRNA转染)和空白对照组(未经任何转染),MTT法测定各组细胞增殖率,流式细胞术检测细胞凋亡情况,Transwell小室法检测细胞侵袭能力,RT-PCR检测细胞p53mRNA水平. 结果与正常胶质细胞SVGp12相比,14-3-3β基因及蛋白在胶质瘤细胞中均呈现高表达,差异均有统计学意义(P＜0.05).与阴性对照组及空白对照组相比,实验组U251细胞14-3-3β基因及蛋白表达量明显降低,细胞增殖率明显下降,凋亡率显著增加,侵袭能力明显降低,p53mRNA水平显著升高,差异均有统计学意义(P＜0.05). 结论选择性沉默14-3-3β基因可有效抑制U251细胞的增殖及侵袭能力、促进其凋亡,这一过程主要是通过14-3-3β基因调控p53基因及其下游调控因子来实现的,表明14-3-3β基因可作为神经胶质瘤治疗药物研发的潜在靶点.
목적 초보탐토14-3-3β기인대효질류세포생물학행위적영향급기작용궤제. 방법 수집상규배양적SVGp12급U251、U87、SHG-44이급용14-3-3β특이성siRNA(14-3-3β-siRNA)침묵적U251세포등각효질류세포주,응용실시정량PCR (RT-PCR)화Westernblotting검측각효질류세포중14-3-3β기인급단백표체정황.장상규배양생장지대수기적U251세포분위실험조(14-3-3β-siRNA전염)、음성대조조(대조siRNA전염)화공백대조조(미경임하전염),MTT법측정각조세포증식솔,류식세포술검측세포조망정황,Transwell소실법검측세포침습능력,RT-PCR검측세포p53mRNA수평. 결과 여정상효질세포SVGp12상비,14-3-3β기인급단백재효질류세포중균정현고표체,차이균유통계학의의(P＜0.05).여음성대조조급공백대조조상비,실험조U251세포14-3-3β기인급단백표체량명현강저,세포증식솔명현하강,조망솔현저증가,침습능력명현강저,p53mRNA수평현저승고,차이균유통계학의의(P＜0.05). 결론 선택성침묵14-3-3β기인가유효억제U251세포적증식급침습능력、촉진기조망,저일과정주요시통과14-3-3β기인조공p53기인급기하유조공인자래실현적,표명14-3-3β기인가작위신경효질류치료약물연발적잠재파점.
Objective To explore the effect of 14-3-3 β gene on biological behavior ofglioma cell line and its mechanism.Methods Conventional cultured SVGp12,U251,U87 and SHG-44 cell lines and U251 cells silenced by 14-3-3[β-small interfering RNA (siRNA) were collected; real time-PCR and Western blotting were used to detect the 14-3-3β gene and protein expressions in these cells.Conventional cultured U251 cells at logarithmic phase were divided into three groups:experimental group (14-3-3β-siRNA transfection),negative control group (siRNA transfection) and blank control group; 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to assess the proliferation of U251 cells,flow cytometry was used to test the cell apoptosis,and cell migration was analyzed by Transwell chamber assay.Results As compared with those in the normal glial cells,14-3-3β gene and protein expression levels in the glioma cells were significantly higher (P＜0.05); as compared with negative control and blank control groups,U251 cells in the experimental group had significantly decreased gene and protein expressions of 14-3-3β,decreased proliferation and migration abilities,significantly increased apoptosis rate and p53 mRNA level (P＜0.05).Conclusion Silence of 14-3-3 β gene decreases U251 cells proliferation and migration through p53 mediated pathway; consequently,a new explanation about how 14-3-3 β regulates glioma cells proliferation and migration can be clarified,and a potential target for glioma treatment can be provided.