中文摘要：

目的 采用SYBR GreenⅠ实时荧光定量逆转录聚合酶链反应（RT-PCR）技术，建立检测前列腺癌抗原3（DD3）mRNA的标准，定量检测人体不同组织中该基因的表达水平。方法 从LNCaP细胞中采用RT-PCR法扩增特异性DD3基因片段，将其与pMD 18-T载体连接，转化宿主菌JMl09，对质粒标准进行聚合酶链反应（PCR）检测及测序。纯化后检测质粒拷贝浓度，制备梯度浓度标准品。应用Light Cycler荧光定量PCR仪对标准品进行检测。取正常人乳腺组织、膀胱、结肠、尿道、肺、睾丸、精囊、卵巢、正常前列腺组织、前列腺增生及前列腺癌组织，定量检测DD3mRNA表达。结果 建立稳定的检测DD3 mRNA的标准，正常人乳腺、膀胱、结肠、尿道、肺、睾丸、精囊、卵巢中无DD3 mRNA表达，在正常前列腺及前列腺增生组织中低表达，两者间差异无显著性（P〉0．05）；在前列腺癌中高表达（P〈0．05）。结论 应用SYBR GreenⅠ实时荧光定量RT-PCR技术检测DD3mRNA表达水平简便、可行、经济。DD3基因的表达具有良好的前列腺癌特异性。

英文摘要：

Objective To establish the quantitative detection method by SYBR Green Ⅰ real time quantitative RTPCR. DD3 mRNA copies in the cDNA obtained from different tissues were determined by real-time PCR. Methods The 406bp fragment of DD3 mRNA was amplified from total RNA of LNCaP cells using RT-PCR method, and was ligated by pMD 18-T Simple vector. Combined vectors were transformed into JM109. PCR detecting and sequencing were performed for standard plasmid. Then, we detected the copy concentration of stock solution and standardized serial concentrations by SYBR Green Ⅰ real time quantitative RT PCR. Then, the expression level of DD3 mRNAs in normal tissues of breast, bladder, colon, urethra, lung, prostate, seminal vesicle, testis, ovary, as well as prostate cancer were quantitatively determined. Results The 406 bp fragment of DD3 cDNA was successfully cloned into the pMD 18-T simple vector and was verified by sequence analysis. The stable detecting standards were constructed, all samples, except prostate, were negative for DD3 mRNA. The DD3 mRNA expression level had no statistical difference between normal prostatic tissues and BPH. As compared with the normal prostate and BPH tissues, the DD3 mRNA expression level of prostate cancer showed significant statistical difference. Conclusion Quantitative detection of DD3 mRNA by the method of SYBR Green Ⅰ real time quantitative RT-PCR is stable and reliable. The expression of DD3 mRNA is prostate-specific and has great potential for development of a new molecular diagnostic tool.