PSA启动子调控并荷载hTERT-siRNAs的条件增殖型腺病毒的构建及生物学活性  

Construction and biological activity of conditionally replicative adenovirus expressing hTERT- siRNA in regulation of PSA promoter

作　　者：孙方浩[1] 郑骏年[2] 魏晋[1] 刘星[1] 徐觉见 陈伟[1] 

机构地区：[1]徐州市第一人民医院泌尿外科,江苏徐州221002 [2]徐州医学院肿瘤生物治疗实验室,江苏徐州221002

出　　处：《徐州医学院学报》2015年第7期471-473,共3页Acta Academiae Medicinae Xuzhou

基　　金：徐州市科技局项目（XZZD1332）

摘　　要：目的构建前列腺特异抗原（PSA）启动子调控并荷载hTERT-siRNAs的条件增殖型腺病毒，研究其对前列腺癌的特异性抗肿瘤作用。方法EcoRV和Xba1分别酶切pZD55-hTERT-shRNA和pZXC2-PSA-E1A，将目的片段连接重组，获得质粒pPSA-ZD55-hTERT，将其与质粒pBHGE3共转染293细胞。PCR鉴定正确者即为条件增殖型腺病毒PSA-ZD55-hTERT。结晶紫染色观察其对前列腺癌细胞毒性。RT-PCR、Westernblot检测前列腺癌细胞中hTERT基因沉默效果。Westernblot检测E1A在前列腺癌细胞中的表达。结果成功构建PSA启动子调控并荷载hTERT-siRNAs的条件增殖型腺病毒PSA-ZD55-hTERT，初步证实PSA-ZD55-hTERT复制具有前列腺癌靶向性和特异的hTERT沉默效果。结论成功构建条件增殖型腺病毒PSA-ZD55-hTERT，为进一步体内外研究其对前列腺癌的治疗作用奠定基础。Objective To construct conditionally replicative adenovirus expressing hTERT- siRNA in regulation of prostate- specific antigen （PSA） promoter and evaluate its specific activities against prostatic cancer. Methods The plasmids pZD55 -hTERT- shRNA and pZXC2 -PSA- E1A were enzymatically digested with EcoRV and Xba I , producing a recombinant plasmid pPSA - ZD55 - hTERT. The obtained plasmid and adenovirus packaging plasmid pBHGE3 were transfected into 293 cells, resulting in a product which was then determined as conditionally replicative adenovirus PSA - ZD55 - hTERT by PCR. Its toxicity on LNCaP human prostate cancer ceils was measured by crystal violet stai- ning. The quantity of hTERT mRNA in adenovirus - infected LNCaP cells after gene silencing was determined by RT - PCR and Western blotting. The level of E1A was detected by Western blotting. Results The conditionally replicative adenovirus expressing hTERT - siRNA in regulation of PSA promoter was successfully constructed. The recombinant plas- mid PSA - ZD55 - hTERT produced specific activities against prostatic cancer and blocked the expression of hTERT in LNCaP cells. Conclusion The construct of conditionally replicative adenovirus expressing hTERT - siRNA in regulation of PSA promoter provides experimental evidence for further research to treat prostatic cancer.

关 键 词：前列腺癌 前列腺特异抗原启动子 HTERT基因 RNA干扰 腺病毒 

分 类 号：R733[医药卫生—肿瘤]