中华核医学与分子影像杂志
中華覈醫學與分子影像雜誌
중화핵의학여분자영상잡지
Chinese Journal of Nuclear Medicine and Molecular Imaging
2014年
2期
125-129
,共5页
吴琼%张月红%付鹏%田国梅%赵长久
吳瓊%張月紅%付鵬%田國梅%趙長久
오경%장월홍%부붕%전국매%조장구
前列腺肿瘤%基因表达%寡核苷酸类,反义%锝%小鼠双微体扩增基因2
前列腺腫瘤%基因錶達%寡覈苷痠類,反義%锝%小鼠雙微體擴增基因2
전렬선종류%기인표체%과핵감산류,반의%득%소서쌍미체확증기인2
Prostatic neoplasms%Gene expression%Oligonucleotides,antisense%Technetium%Mouse double minute 2
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MDM2 mRNA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99Tcm-HYNIC-ASON(0、100和500 nmol/L)和99Tcm-HYNIC-ASONM(500 nmol/L),于LNCaP细胞中培养24h,再行RT-PCR和Westem blot实验,观察探针对MDM2、p53 mRNA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q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、500 nmol/L 99Tcm-HYNIC-ASON组和500 nmol/L99Tcm-HYNIC-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0 nmol/L 99Tcm-HYNIC-ASON组与500 nmol/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、71.218±2.915、32.775±3.062、88.121±2.710,同样除0 nmol/L 99Tcm-HYNIC-ASON组与500 nmol/L99 Tcm-HYNIC-ASONM组间外,余差异均有统计学意义(F=235.93,q=6.43～ 19.14,均P＜0.01).4组p53 mRNA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0 nmol/L 99Tcm-HYNIC-ASON组与500 nmol/L99Tcm-HYNIC-ASONM组间外,余差异均有统计学意义(F=53.98,q=3.32～6.74,均P＜0.01).结论 MDM2反义探针能与MDM2 mRNA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.
目的 探討99Tcm標記的小鼠雙微體擴增基因2(MDM2) mRNA的ASON和錯義寡覈苷痠(ASONM)對前列腺癌細胞株LNCaP目的基因錶達的影響,為後續的前列腺癌基因顯像奠定基礎.方法 人工閤成一段針對MDM2 mRNA的ASON和ASONM,以HYNIC為螯閤劑,對ASON和ASONM進行99Tcm標記,檢測標記率、放化純、穩定性及分子雜交活性.用脂質體包裹99Tcm-HYNIC-ASON(0、100和500 nmol/L)和99Tcm-HYNIC-ASONM(500 nmol/L),于LNCaP細胞中培養24h,再行RT-PCR和Westem blot實驗,觀察探針對MDM2、p53 mRNA和相應蛋白質錶達的影響.各組間計量資料比較採用單因素方差分析和q檢驗.結果 ASON的標記率為(65.15±2.05)％(n=5),ASONM的標記率為(64.93±2.18)％(n=5),兩者放化純均達90％以上.99Tcm-HYNIC-ASON穩定性好,保留瞭與互補鏈結閤的能力.0、100、500 nmol/L 99Tcm-HYNIC-ASON組和500 nmol/L99Tcm-HYNIC-ASONM組MDM2mRNA含量分彆為0.458±0.035、0.250±0.026、0.174±0.032、0.463±0.033,除0 nmol/L 99Tcm-HYNIC-ASON組與500 nmol/L99Tcm-HYNIC-ASONM組間外,餘差異均有統計學意義(F=33.69,q=24.32～ 91.45,均P＜0.01);各組MDM2蛋白的平均密度分彆為90.712±3.042、71.218±2.915、32.775±3.062、88.121±2.710,同樣除0 nmol/L 99Tcm-HYNIC-ASON組與500 nmol/L99 Tcm-HYNIC-ASONM組間外,餘差異均有統計學意義(F=235.93,q=6.43～ 19.14,均P＜0.01).4組p53 mRNA含量分彆為0.185±0.046、0.203±0.040、0.213±0.027、0.163±0.049,差異無統計學意義(F=2.18,P＞0.05);各組p53蛋白平均密度分彆為33.865±2.213、70.445±2.180、99.025±3.012、38.351±3.271,除0 nmol/L 99Tcm-HYNIC-ASON組與500 nmol/L99Tcm-HYNIC-ASONM組間外,餘差異均有統計學意義(F=53.98,q=3.32～6.74,均P＜0.01).結論 MDM2反義探針能與MDM2 mRNA鏈上的目的序列特異結閤,併抑製目的基因的錶達,具備活體內前列腺癌反義顯像的實驗條件,有望為在基因水平上診斷前列腺癌提供一種新方法.
목적 탐토99Tcm표기적소서쌍미체확증기인2(MDM2) mRNA적ASON화착의과핵감산(ASONM)대전렬선암세포주LNCaP목적기인표체적영향,위후속적전렬선암기인현상전정기출.방법 인공합성일단침대MDM2 mRNA적ASON화ASONM,이HYNIC위오합제,대ASON화ASONM진행99Tcm표기,검측표기솔、방화순、은정성급분자잡교활성.용지질체포과99Tcm-HYNIC-ASON(0、100화500 nmol/L)화99Tcm-HYNIC-ASONM(500 nmol/L),우LNCaP세포중배양24h,재행RT-PCR화Westem blot실험,관찰탐침대MDM2、p53 mRNA화상응단백질표체적영향.각조간계량자료비교채용단인소방차분석화q검험.결과 ASON적표기솔위(65.15±2.05)％(n=5),ASONM적표기솔위(64.93±2.18)％(n=5),량자방화순균체90％이상.99Tcm-HYNIC-ASON은정성호,보류료여호보련결합적능력.0、100、500 nmol/L 99Tcm-HYNIC-ASON조화500 nmol/L99Tcm-HYNIC-ASONM조MDM2mRNA함량분별위0.458±0.035、0.250±0.026、0.174±0.032、0.463±0.033,제0 nmol/L 99Tcm-HYNIC-ASON조여500 nmol/L99Tcm-HYNIC-ASONM조간외,여차이균유통계학의의(F=33.69,q=24.32～ 91.45,균P＜0.01);각조MDM2단백적평균밀도분별위90.712±3.042、71.218±2.915、32.775±3.062、88.121±2.710,동양제0 nmol/L 99Tcm-HYNIC-ASON조여500 nmol/L99 Tcm-HYNIC-ASONM조간외,여차이균유통계학의의(F=235.93,q=6.43～ 19.14,균P＜0.01).4조p53 mRNA함량분별위0.185±0.046、0.203±0.040、0.213±0.027、0.163±0.049,차이무통계학의의(F=2.18,P＞0.05);각조p53단백평균밀도분별위33.865±2.213、70.445±2.180、99.025±3.012、38.351±3.271,제0 nmol/L 99Tcm-HYNIC-ASON조여500 nmol/L99Tcm-HYNIC-ASONM조간외,여차이균유통계학의의(F=53.98,q=3.32～6.74,균P＜0.01).결론 MDM2반의탐침능여MDM2 mRNA련상적목적서렬특이결합,병억제목적기인적표체,구비활체내전렬선암반의현상적실험조건,유망위재기인수평상진단전렬선암제공일충신방법.
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 99Tcm-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 using one-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 radiochemical purity were both more than 90％.99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonucleotide (SON).The contents of MDM2 mRNA in 0,100,500 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/L99Tcm-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 ; except 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/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.
