摘要
目的观察增殖细胞核抗原(PCNA)基因反义脱氧寡核苷酸对人多形性胶质母细胞瘤细胞增殖的影响.方法采用人工合成的PCNA反义和正义脱氧寡核苷酸经阳离子脂质体包裹后转染人多形性胶质母细胞瘤细胞BY325.用MTT比色法检测转染后瘤细胞的生长抑制率;3H-TdR法检测细胞DNA合成速率;流式细胞仪检测细胞周期分布;RT-PCR检测PCNA mRNA表达;免疫组化方法检测PCNA蛋白表达水平.结果PCNA反义寡核苷酸可明显抑制BT325细胞的生长;明显减慢其DNA合成速率,阻滞细胞由G0/Gl期→S期,PCNA mRNA及其蛋白质表达也同时受到抑制.抑制效应在转染后12 h即出现,24 h达到高峰,48 h后逐渐减弱.抑制作用随反义寡核苷酸浓度的升高而增强,0.8μM PCNA反义寡核苷酸的细胞生长抑制率达84.6%.同样浓度的正义寡核苷酸和脂质体则无明显的细胞生长抑制作用.结论PCNA反义寡核苷酸在体外能明显抑制人脑多形性胶质母细胞瘤细胞BT325生长、DNA合成、mRNA和PCNA蛋白表达.PCNA基因可望成为胶质瘤基因治疗的新靶点.
Objective To observe the effect of proliferating cells nuclear antigen (PCNA) antisense oligodeoxynucleotides on the proliferation of human multiform glioblastoma cells. Methods Antisense and sense PCNA phosphorothioate oligodeoxynucleotides were synthesized and transfected to BT325 cells mediated by lipofection. The effect on cells growth rate, mRNA, protein expression and DNA contents in cell cycle phase were examined respectively by MTT assay, RT-PCR, immunohistochemical technique, 3H thymidine incorporation and flow cytometric analysis. Results PCNA antisense oligodeoxynucleotides could significantly inhibit the growth of BT325 cells line. The inhibition started at 12 hours, peaked at 24 hours and decreased at 48 hours after the transfection .The inhibition effect was dose dependent. The cell growth inhibition rate was 84.6% at 0.8 pmol/L. Antisense PCNA oligodeoxynucleotide also inhibited BT325 cell DNA, mRNA syntheses and PCNA protein expression. However, the same concentration of sense oligodeoxynucleotide and lipofection showed insignificant inhibition of effect on BT325 cells growth. Conclusions Antisense PCNA oligodeoxynucleotide can inhibit the cell DNA, mRNA syntheses and expression of PCNA protein and markly inhibit the cell growth of human multiform glioblastoma BT325 cells line. PCNA gene can be used as the target of gene therapy for malignant gliomas.
出处
《中国临床神经外科杂志》
2002年第2期103-106,共4页
Chinese Journal of Clinical Neurosurgery
关键词
增殖细胞核抗原
神经胶质瘤
反义寡核苷酸
细胞增殖
基因治疗
Proliferating cell nuclear antigen
Glioraa
Atltisense oligodeoxynucleotides
Cell proliferation
Gene therapy
