调控抗凋亡基因survivin表达的因素研究

Factors Regulating Expression of Antiapoptosis Gene Survivin

为了探讨调控存活蛋白(survivin)基因表达的影响因素,以NB4和HL60细胞株作为实验对象,对细胞进行体外细胞培养、形态学观察,并用半定量RTPCR检测survivinmRNA的表达。结果显示:NB4细胞株细胞survivin基因表达阳性,经1μmol/LATRA处理后,随着作用时间延长,其细胞分化抗原CD11b表达量逐渐增高(χ=47.002,P=0.000),而CD33表达量逐渐减低(χ=1.614,P=0.806),并见survivin基因表达下调,细胞周期阻滞于G0G1期(χ=58.566,P=0.000);ATRA对HL60细胞株细胞survivinmRNA表达有下调作用;GCSF、GMCSF和植物血凝素(PHA)均能使NB4和HL60细胞株细胞survivinmRNA表达上调,并呈时效关系;100-1000nmol/Lsurvivin反义寡聚核苷酸(ASODN)抑制NB4细胞株细胞survivinmRNA表达,随着浓度增加其抑制率增加,600nmol/L时其抑制率最低为38%,增加浓度未见抑制率继续下降,而对照组、脂质体组和正义链组中NB4细胞株细胞survivinmRNA表达几乎未受到抑制;在细胞形态学上,NB4细胞株细胞经survivinASODN处理后出现典型的凋亡形态学变化。结论:PHA、GMCSF和GCSF对HL60和NB4细胞株细胞survivin基因表达起正向调节作用,而survivinASODN和ATRA起负向调节作用;ATRA下调survivin基因表达,且细胞周期阻滞于G0G1期,这说明survivin基因表达与细胞周期之间关系密切,抑制NB4细胞株细胞survivin基因的表达可促使细胞发生凋亡。

To explore the regulation mechanism of survivin gene, the NB4 and HL-60 cells were used in experiments, the cell culture in vitro and cell morphological observection were performed and survivin mRNA expression was detected by semi-quantitative RT-PCR. The results showed that the survivin expression in NB4 cell was positive. By treatment of 1 μmol/L ATRA, cell differentiation antigen CD11b was gradually increased （-↑x = 47. 002, P = 0. 000 ） and CD33 was gradually decreased （-↑x = 1. 614, P = 0. 806 ） with time. Simultaneously, survivin mRNA expression was down-regulated and the cell cycle was arrested at G0-G1 phase （-↑x = 58. 566 ,P = 0. 000 ）. ATRA could down-regulate the survivin mRNA expression of HL-60 cell, but G-CSF, GM-CSF and PHA could up-regulate the survivin expression of HL-60 cell. The cytokine could regulate survivin expression in gene transcription level. The up-regulation of survivin expression was observed while HL-60 cell was stimulated by PHA. The survivin gene expression could be blocked by the survivin antisense oligonucleotide. The survivin mRNA expression of NB4 cell was inhibited by 100 nmol/L - 1000 nmol/L survivin antisense oligonucleotide in a dose-dependent manner. The survivin mRNA expression in the NB4 cell was obviously inhibited in 600 nmol/L survivin AS-ODN groups （38%） while the AS-ODN dose increases, the inhibition rate does not descend, but was not inhibited in the control groups, liposomes groups and ODN groups. After NB4 cell was treated by survivin AS-ODN, the typical morphological changes for the apoptosis emerged in NB4 cell. These changes were not found in control groups. It is concluded that PHA, GM-CSF and G-CSF can up-regulate the survivin gene expression, but survivin AS-ODN and ATRA can down-regulate survivin gene expression. The cell cycle arrest at G0-G1 phase while the survivin gene expression was down-regulated by ATRA. It suggested that the survivin gene expression is very related to cell cycle. The morphological changes of cell apoptosis can be observed when the survivin gene expression of NB4 cell was surpressed.