ASPP1基因mRNA在肿瘤细胞株中的表达初探

A preliminary study on ASPP1 mRNA expression level in tumor cell line

目的建立p53凋亡刺激蛋白基因(ASPP1)mRNA表达水平的RT-PCR测定方法;并应用该方法测定ASPP1 mRNA在正常人单核细胞与白血病细胞株Jurkat、HL-60、K562中的表达水平。方法应用单核细胞分离液分离正常人血液单核细胞,用RPMI 1640培养液培养细胞株Jurkat、HL-60、K562,所得细胞用Tripure分离试剂提取总RNA,用RT-PCR扩增。取ASPP1基因和β-actin基因的扩增产物各10μl,行溴钇锭染色,在琼脂糖凝胶上进行电泳,检测ASPP1 mRNA的表达水平。对ASPP1基因表达量与β-actin表达量的比值进行比较,从而计算ASPP1在各细胞株的相对表达量。结果在正常人单核细胞和各白血病细胞株中均有ASPP1基因的mRNA表达,ASPP1与β-actin的比值在正常人单核细胞中为0.545±0.019,而在白血病细胞株Jurkat、HL-60、K-562中分别为0.155±0.081、0.199±0.016、0.191±0.015,可见ASPP1 mRNA在正常细胞中的表达水平比在白血病细胞株Jurkat、HL-60、K-562中的表达水平高(P<0.01)。结论在肿瘤的发生、发展中,ASPP1与p53共同作用,形成ASPP-p53复合物作用于原凋亡基因启动子,能特异性增强p53结合DNA的能力,促进p53诱导细胞凋亡的作用。ASPP1的这种功能在抑制正常细胞恶性转化的过程中有重要作用。

Objective To establish a reverse transcription PCR （RT-PCR） assay for detecting ASPP1 mRNA expression levels in normal blood monocyte and leukemia jurkat, HL-60 and K-562 cell lines. And to analyze the differences in relative expression levels of ASPP1 in respective cell line by calculating the value of ASPP1/β-actin for evaluating the effects of ASPP1 on inhibiting the malignant trans- formation of normal cells. Methods The monocyte isolation reagent was used to isolate normal blood monocyte, and the RPMI 1640 medi um was used to culture jurkat, HL-60 and K562 cell lines. The cell RNA was extracted with Tripure^TM reagent and amplified by RT PCR. The amplified products were E.B. stained and 10μl of the products were used for agarose gel eleetrophoresis. The expression levels of ASPP1 mRNA and β-actin in respective cell line were measured and then compared. Result The ASPP1 mRNA was expressed both in normal blood monocyte and all the leukemic cells. The value of ASPP1/β-actin was 0. 545±0. 019 in normal blood monocyte, and it was 0. 155±0. 081, 0. 199±0. 016 and 0. 191±0. 015, respectively, in the leukemic cells jurkat, HL-60, and K562. The expression level of ASPP1 mRNA was higher in normal cells than that in the three leukemic cell lines. Conclusion On the process of tumor development, an ASPP1 p53 compound is formed by collaborated effects of ASPP1 and p53, which may act on the areh-apoptosis gene promoter, specifically en hance the ability of p53 binding DNA, and reinforce the function of p53 in enhancing apoptosis. The declined expression and/or deprived function of ASPP1 might lead to tumorigenesis, implying that the function of ASPP1 in enhancing p53 apoptosis may play an important role on inhibiting the malignant transformation of normal cells.