前列腺特异启动子驱动的自杀基因对雄激素非依赖前列腺癌的杀伤作用.docVIP

前列腺特异启动子驱动的自杀基因对雄激素非依赖前列腺癌的杀伤作用研究 王洛夫，靳风烁，江 军，张 尧，兰卫华 （第三军医大学大坪医院野战外科研究所泌尿外科，重庆 400042） 摘 要：目的 将前列腺特异性启动子驱动的自杀基因导入到雄激素非依赖性前列腺癌（AIPC）细胞，观察其对AIPC细胞的杀伤作用。方法 将rPB-DR（6）启动子驱动的胞嘧啶脱氨酶（CD）基因融合分子插入到逆转录病毒载体pLNCX，构建pLN-rPB-DR(6)-CD重组逆转录病毒载体，PA317包装，获得重组逆转录病毒，后者感染前列腺癌细胞PC-3，获得G418抗性细胞PC-3CD。在培养基中加入维甲酸、5-氟胞嘧啶,观察其对PC-3CD的杀伤作用。结果 经酶切和测序证实成功构建了pLN-rPB-DR(6)-CD重组逆转录病毒载体，PCR证实在PC-3CD基因组DNA中扩增出了CD基因，RT-PCR证实PC-3CD有CD mRNA表达。在培养基中加入维甲酸、5-氟胞嘧啶后，PC-3CD细胞大量死亡，生长显著被抑制。结论 前列腺特异性启动子驱动的自杀基因可杀伤雄激素非依赖性前列腺癌细胞并显著抑制其生长。 关键词：前列腺癌；前列腺特异性启动子；自杀基因；逆转录病毒；基因治疗 中图法分类号： 文献标识码：A Antitumor effect of suicide gene driven by prostate specific promotor on androgen independent prostate cancer cells WANG Luo-fu, JIN Feng-shuo, JIANG Jun, ZHANG Yao, LAN Wei-hua (Department of Urology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China) Abstract：Objective To transduce the suicide gene driven by prostate specific promotor into androgen independent prostate cancer (AIPC) cells and observe its antitumor effect. Methods A fusional fragment of cytosine deaminase (CD) gene driven by rPB-DR(6) promotor was inserted into the retroviral vector pLNCX to reconstruct the retroviral vector pLN-rPB-DR(6)-CD which was packaged by PA317 cells. The supernatant containing recombinant retrovirus was used to infect PC-3 cells and G418 was employed to select resistant clones, PC-3CD cells. The antitumor activity of the CD gene on PC-3CD cells was observed by adding tretinoin and 5-fluorocytosin into the culture media. Cell growth curve was plotted and growth inhibitory rate was calculated. Results The retroviral vector pLN-rPB-DR(6)-CD was confirmed to be successfully reconstructed by enzyme digestion and DNA sequencing. A fragment containing the CD gene was amplified by PCR in the genomic DNA of PC-3CD cells. The CD mRNAs were present in PC-3CD cells confirmed by RT-PCR. After adding tretinoin and 5-fluorocytosin into the culture media