摘要
目的研究针对增殖细胞核抗原(PCNA)mRNA的反义寡核苷酸的标记方法,标记后探针在卵巢癌细胞株HO8910中的摄取情况及其对细胞增殖和目的基因表达的影响。方法人工合成一段针对PCNAmRNA的反义寡核苷酸(ASON),以放射性核素99mTc进行标记,研究标记物的标记率,放射化学纯度,与互补正义链寡核苷酸(SON)的结合能力,不同温度条件下在卵巢癌细胞中的摄取情况,以及反义探针对细胞增殖和细胞PCNAmRNA表达的影响。结果在双功能螯和剂NHSMAG3的作用下,ASON的标记率为(60.12±3.01)%(n=5),经纯化后放射化学纯度可达95%以上;标记物在体外能稳定存在,且仍保持与互补链的结合能力;37℃和22℃条件下,细胞对ASON的摄取率高于对SON,对ASON的清除速度缓于对SON,而37℃时,细胞对ASON的摄取明显高于22℃时,清除速度则明显快于22℃。标记的ASON与细胞孵育48h后,HO8910细胞中PCNAmRNA的表达显著减少。结论在双功能螯和剂的作用下,寡核苷酸能成功地得到标记,与SON相比,反义探针能被代谢旺盛的HO8910细胞特异性摄取并抑制目的基因的表达。
Objective To investigate the radiolabeling method of the oligonucleotide targeted to proliferating cell nucleus antigen (PCNA), and the uptake kinetics of the radiolabeled probe in human ovary carcinoma cell strain HO8910, and its effect on cell proliferation and gene expression.Methods An antisense oligonucleotide (ASON) targeted to PCNA mRNA was synthesized and radiolabeled with ^(99m)Tc. The labeling efficiency, radiochemical purity, in vitro stability, the ability of the labeled ASON to hybridize to the sense oligonucleotide (SON), the uptake kinetics of the oligonucleotides in HO8910 at different temperature, and the influence of the antisense probe on cell proliferation and PCNA mRNA expression were analyzed.Results The oligonucleotides was radiolabeled with the bifunctional chelator at the labeling efficiency of (60.12±3.01) % (n=5). The radiochemical purity was over 95 % after purification. ASON showed significantly higher accumulation and effluxed much slowly than SON both at 37 ℃ and at 22 ℃. At 37 ℃, the accumulation of ASON was higher than that at 22 ℃, but the efflux was more rapid than that at 22 ℃. After incubation with HO8910 cells for 48 h, the radiolabeled ASON could decrease PCNA mRNA level of the proliferating HO8910 cells significantly.Conclusion With the effect of bifunctional chelator, ASON can be successfully radiolabeled, and the antisense probe can accumulate in the proliferating HO8910 cells more specifically and inhibit the gene expression intensively compared with SON.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2005年第3期348-352,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30070310)
