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目的构建含rPB-DR(6)启动子和CD自杀基因的重组溶瘤腺病毒,观测其对雄激素依赖性和非依赖性前列腺癌细胞的杀伤作用,探索雄激素非依赖性前列腺癌治疗的新方法。方法应用分子生物学技术,构建并包装出重组溶瘤腺病毒Ad5-rPC,纯化并扩增,测定滴度。采用PCR方法对重组溶瘤腺病毒颗粒进行鉴定并测序,Western blot检测E1A、CD蛋白表达,TCID50法检测重组腺病毒感染癌细胞后的复制能力,应用CCK-8法检测重组病毒特异性杀伤能力。结果经PCR及测序鉴定证实成功构建了包含rPB-DR(6)启动子和CD基因的重组溶瘤腺病毒Ad5-rPC,测滴度为1.1×1011pfu/mL。Western blot检测到E1A和CD蛋白阳性表达。Ad5-rPC病毒感染前列腺癌细胞系细胞产生的子代病毒滴度均大于3.0×106pfu/mL,CCK-8法证实重组腺病毒对雄激素依赖性和非依赖性前列腺癌细胞均具有特异性杀伤能力,杀伤能力均大于65%。结论前列腺特异性启动子调控的溶瘤腺病毒能在前列腺癌细胞内靶向复制,并对雄激素依赖性和非依赖性前列腺癌细胞均有特异性杀伤作用。
Objective To construct recombinant oncolytic adenovirus containing rPB-DR (6) promoter and CD suicide gene and to observe its cytotoxicity on androgen-dependent and non-dependent prostate cancer cells and to explore the role of androgen-independent prostate cancer new method. Methods Using molecular biology techniques, recombinant oncolytic adenovirus Ad5-rPC was constructed and packaged, purified and amplified to determine the titer. The recombinant adenovirus was identified by PCR and sequenced. Western blot was used to detect the expression of E1A and CD protein. The TCID50 method was used to detect the replication ability of recombinant adenovirus. The CCK-8 method was used to detect the specific virulence of recombinant adenovirus . Results The recombinant adenovirus Ad5-rPC containing rPB-DR (6) promoter and CD gene was confirmed by PCR and sequencing. The titer was 1.1 × 1011 pfu / mL. Western blot detection of E1A and CD protein expression. Ad5-rPC virus-infected prostate cancer cell lines produced more than 3.0 106 pfu / mL titers of virus, and the CCK-8 method confirmed that the recombinant adenovirus had specific killing on androgen-dependent and independent prostate cancer cells Ability, ability to kill more than 65%. Conclusion The oncolytic adenovirus regulated by prostate-specific promoter can target the replication in prostate cancer cells and has specific killing effect on androgen-dependent and non-dependent prostate cancer cells.