^99Tc^m标记MDM2反义寡核苷酸对人前列腺癌细胞目的基因表达的影响

Effect of 99Tcm-labeled mouse double minute 2 antiseuse oligonucleotide on target gene expression of prostatic cancer cells

目的探讨99Tcm标记的小鼠双微体扩增基因2（MDM2）mRNA的ASON和错义寡核苷酸（ASONM）对前列腺癌细胞株LNCaP目的基因表达的影响，为后续的前列腺癌基因显像奠定基础。方法人工合成一段针对MDM2mRNA的ASON和ASONM，以HYNIC为螯合剂，对ASON和ASONM进行99Tcm标记，检测标记率、放化纯、稳定性及分子杂交活性。用脂质体包裹驰TcTM-HYNIC．ASON（0、100和500nmol／L）和99Tcm-HYNIC-ASONM（500nmol／L），于LNCaP细胞中培养24h，再行RT-PCR和Westernblot实验，观察探针对MDM2、p53mRNA和相应蛋白质表达的影响。各组间计量资料比较采用单因素方差分析和g检验。结果ASON的标记率为（65．15±2．05）％（n=5），ASONM的标记率为（64．93±2．18）％（n=5），两者放化纯均达90％以上。99Tcm-HYNIC-ASON稳定性好，保留了与互补链结合的能力。0、100、500nmo]／L99Tcm-HYNIC-ASON组和500nmol／LTcmHYNIC-ASONM组MDM2mRNA含量分别为0．458±0．035、0．250±0．026、0．174±0．032、0．463±0．033。除0nmol／L99Tcm．HYNIC-ASON组与500nmol／L99Tcm-HYNIC-ASONM组间外，余差异均有统计学意义（F=33．69，q=24．32-91．45，均P〈0．01）；各组MDM2蛋白的平均密度分别为90．712±3．042、71．2184-2．915、32．775±3．062、88．121±2．710，同样除0nmol／L99Tcm．HYNIC．ASON组与500nmol／L99Tcm-HYNIC-ASONM组间外，余差异均有统计学意义（F=235．93，g：6．43～19．14，均P〈0．01）。4组p53mRNA含量分别为0．185±0．046、0．203±0．040、0．213±0．027、0．163±0．049，差异无统计学意义（F=2．18，P〉0．05）；各组p53蛋白平均密度分别为33．865±2．213、70．445±2．180、99．025±3．012、38．351±3．271，除0nmol／L99Tcm-HYNIC-ASON组与500nmol／L99Tcm-HYNIC-ASONM组间外，余差异均有统计学意义（F=53．98，g=3．32-6．74，均P〈0．01）。结论MDM2反义探针能与MDM2mRNA链上的目的序列特异结合，并抑制目的基因的表达，具备活体内前列腺癌反义显像的实验条件，有望为在基因水平上诊断前列腺癌提供一种新方法。

Objective To investigate the effect of mouse double minute 2（MDM2） mRNA ASON and mismatched oligonucleotide （ASONM） radiolabeled with 99Tcm on target gene expression in LNCaP cells. Methods The ASON and ASONM targeted to MDM2 mRNA were synthesized and radiolabeled by 99Tcm with the bifunctional chelator of HYNIC. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were investigated. The different concentrations of 99Tcm-HYNIC-ASON （0, 100, 500 nmol/L） and 99Tc2-HYNIC-ASONM （500 nmol/L） coated with lipofectamin 2000 were incubated with prostate cancer cells for 24 h, then RT-PCR and Western blot were carried out to assay the MDM2, p53 mRNA and the corresponding protein level. The variables of RT-PCR and Western blot were analyzed usingone-way analysis of variance and q test. Results The labeling efficiency of ASON and ASONM were （65.15± 2.05）% （n=5） and （64.93±2.18）% （n=5）, respectively. The radioehemical purity were both more than 90%. 99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonueleotide （SON）. The contents of MDM2 mRNA in 0, 1130, 500 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC- ASONM groups were 0.458±0.035, 0.250±0.026, 0.174±0.032, 0.463±0.033, respectively, and there were significant differences between each 2 groups except between 0 nmoL/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups （F=33.69, q = 24.32-91.45, all P〈0.01 ）. The average density of MDM2 protein in the 4 groups were 90.712±3.042, 71.218-2.915, 32.775±3.062, 88.121±2.710, respectively （F= 235.93, q--6.43-19.14, all P〈0.01; except 0 nmol/L99Tem-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC- ASONM）. The contents of p53 mRNA in the 4 groups were 0.185±0.046, 0.203±0.040, 0. 213-0.027, 0. 163±0.049, respectively （ F = 2.18, P〉 0.05 ）. The average density of p53 protein was 33.865± 2.213, 70.445＋2.180, 99.025±3.012, 38.351±3.271, respectively （F=53.98, q= 3.32-6. 74, all P〈0.01; ex- cept 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmo/L 99Tcm-HYNIC-ASONM）. Conclusions The MDM2 antisense probe can accumulate in the prostate cancer cells, and specially hybridize to the MDM2 mRNA and inhibit target gene expression. This novel molecular probe has a promising potential for the diagnosis of prostate cancer at gene level.