SAC重组腺相关病毒载体的构建及其对前列腺癌CAM移植瘤的抑制作用  

Construction of a recombinant adeno-associated viral vector expressing SAC and its inhibition on CAM xenograft prostate cancer

作　　者：于张健[1] 张士宝[1] 刘庆勇[1] 阮喜云[2] 杨广笑 王全颖 

机构地区：[1]山东大学附属济南市中心医院泌尿外科,济南250013 [2]山东大学附属省立医院神经内科,济南250021 [3]西安华广生物工程有限公司,西安710025

出　　处：《山东大学学报（医学版）》2012年第7期41-45,49,共6页Journal of Shandong University:Health Sciences

基　　金：济南市科技发展计划(200815028)

摘　　要：目的构建携带NT4-SAC-HA2-TAT融合基因的重组腺相关病毒载体,并感染前列腺癌鸡胚绒毛尿囊膜(CAM)移植瘤,观察目的基因前列腺凋亡反应基因-4(par-4)选择性癌细胞凋亡结构域(SAC)的表达及其对CAM移植瘤的生长抑制作用。方法利用磷酸钙共沉淀法将包装质粒pAAV/Ad、腺病毒辅助质粒pFG140及重组穿梭质粒pSSCMV/NT4-SAC-HA2-TAT共转染293细胞,制备重组腺相关病毒(rAAV),斑点杂交法检测病毒滴度。取孵育9 d龄健康鸡胚,CAM接种前列腺癌细胞系PC-3细胞,建立前列腺癌的CAM移植瘤模型,接种3 d后挑选成瘤鸡胚随机分为3组:空白对照组(PBS),空病毒组(AAV)和重组病毒组(rAAV),观察瘤体生长情况,上述处理4 d后测量比较不同组移植瘤体积,并切片染色病理检查。免疫荧光法检测移植瘤中SAC融合肽的表达。结果酶切鉴定结果证实成功构建pSSCMV/NT4-SAC-HA2-TAT,斑点杂交法检测重组病毒滴度约为3.34×1010～3.34×1011cfu/mL。免疫荧光法证实SAC融合肽表达。移植瘤能在CAM上生长,可见瘤周围丰富毛细血管汇集,rAAV组瘤体显著缩小,病理HE染色可见瘤细胞局限及侵袭抑制。结论成功构建SAC重组腺相关病毒载体并感染前列腺癌CAM移植瘤,重组病毒表达分泌的SAC融合肽对前列腺癌CAM移植瘤具有诱导细胞凋亡、抑制肿瘤细胞侵袭性增殖等作用。Objective To construct a recombinant adeno-associated virus expression plasmid containing a unique core domain of par-4 sufficient for selective apoptosis induction in cancer cells （SAC） and protein transduction domain TAT, and investigate the effect of the SAC on chick chorioallantoic membrane （CAM） xenograft prostate cancer. Methods Plasmids pAAV/Ad, pFG140 and pSSCMV/NT4-SAC-HA2-TAT were transfected into 293 cells by method of Ca3 （ P04 ） 2 to harvest rAAV-NT4-SAC-HA2-TAT whose titer was detected by dot blot. PC-3 cells were grafted onCAM in the 9~h day of the embryonic development to construct the CAM xenograft tumor model of prostate cancer. The growth of xenograft tumor and richness of CAM capillaries around tumor were observed and photographed. 3 days later, those chick embryos with tumor formation were randomly divided into three groups （the PBS group, the AAV group, and the rAAV group）. The tumors of different groups were compared on the volume 4 days after the treatment and fixed with formalin for further analyzation using standard histopathology. The expression of SAC was detected with immuno- fluorescence method. Results Recombinant plasmid pSSCMV/NTa-SAC-HA2-TAT was successfully constructed con-firmed by restriction enzyme digestion. The viral titer was about 3.34×10^10-3.34×10^11cfu/mL detected by dot blot. The expression of SAC fusion peptide in the tumor tissue was detected by immune-fluorescence test. The xenograft tumor could grow on the CAM, and a mass of capillaries around the tumor were observed. Tumors in the rAAV group significantly shrank. The invasiveness of PC-3 cells was inhibited and the tumor cells distributions were limited under the microscope. Conclusion The xenograft tumor model of prostate cancer on CAM is established successfully. Fusion peptide of SAC plays a significant role in inhibiting the invasiveness and inducting the apoptosis of PC-3 cells.

关 键 词：前列腺凋亡反应基因-4选择性癌细胞凋亡结构域 重组腺相关病毒 鸡胚绒毛尿囊膜 雄激素非依赖性前列腺癌 移植瘤 基因治疗 

分 类 号：R737.25[医药卫生—肿瘤]