超声微泡联合pDsRed2-N1-CD/UPRT融合自杀基因治疗肺癌的机制

目的研究超声微泡联合pDsRed2-N1-CD/UPRT融合自杀基因治疗肺癌的机制。方法采用PCR法扩增大肠杆菌CD和UPRT基因,克隆入pDsRed2-N1载体,构建pDsRed2-N1-CD/UPRT质粒,转化感受态大肠杆菌BL21(DE3),挑取阳性菌,提取质粒,酶切及测序鉴定。将质粒与超声微泡融合,将混合物转染A549细胞,测定CD/UPRT基因的直接杀伤效应和旁效应;并检测不同强度的超声对超声微泡与质粒混合物中质粒结构、转染细胞形态及转染效率的影响。结果 pDsRed2-N1-CD/UPRT质粒经酶切及测序证明构建正确。荧光显微镜下可观察到质粒在超声微泡中的分布。转染A549细胞后,随着5-FU浓度的增加,细胞生长抑制率也增加,可高达58%;在相同剂量的5-FU作用下,增加转染细胞的比例,相应的细胞生长抑制率也随之增加,可高达78%。低能量的超声波对pDsRed2-N1-CD/UPRT质粒结构无明显改变,电泳条带变化不大,对转染细胞形态也无明显影响,超声介导基因转染率最高可达30%。结论超声微泡能增强CD/UPRT基因对肿瘤细胞的直接杀伤和旁效应,起到治疗肿瘤的作用;低能量的超声微泡能提高CD/UPRT基因对细胞的转染效率,且不会损伤细胞及目的基因的DNA。

Mechanism of Therapy of Lung Cancer by Ultrasound Microbubble Combined with pDsRed2-N1-CD/UPRT Fusion Suicide Gene

Objective To study the mechanism of therapy of lung cancer by ultrasound microbubble combined with pDsRed2N1-CD /UPRT fusion suicide gene.Methods The cytosine deaminase(CD)and uracil phosphoribosyltransferase(UPRT)genes of E.coli were amplified by PCR and cloned into vector pDsRed2-N1.The constructed recombinant plasmid pDsRed2-N1-CD /UPRT was transformed to E.coli BL21(DE3),and the positive recombinants were screened,from which plasmid was extracted for restriction analysis and sequencing.The recombinant plasmid was fused with ultrasound microbubble,and the mixture was tranfected to A549 cells to observe the direct killing and side effects of CD /UPRT gene.The effects of ultrasound at various strengths on the plasmid structure in mixture of ultrasound microbubble and recombinant plasmid,morphology of transfected cells and tranfection efficacy were determined.Results Both restriction analysis and sequencing proved that recombinant plasmid pDsRed2-N1-CD /UPRT was constructed correctly.The distribution of recombinant plasmid in ultrasound microbubble was observed by fluorescent microscopy.The inhibitory rate to growth of transfected A549 cells increased with the increasing concentration of 5-Fu,which reached 58%.However,at the same 5-Fu dosages,the inhibitory rate increased with the increasing proportion of transfected cells,which reached 78%.The electrophoretic bands of plasmid pDsRed2-N1-CD /UPRT showed no significant change after treatment by ultrasound with low energy,indicating no significant effect on plasmid structure.The ultrasound with low energy showed no significant effect on the morphology of transfected cells,and the ultrasound-mediated gene transfection efficacy reached 30% at most.Conclusion Ultrasound microbubble enhanced the directly killing and side effects of CD /UPRT gene on tumor cells,thereby showed curative effect on tumor.The ultrasound microbubble with low energy increased the tranfection efficacy of cells with CD /UPRT gene while showed no damage to cell and DNA of target gene.