超声微泡联合pDsRed2-N1-CD/UPRT融合自杀基因对肺癌细胞的杀伤作用

Therapy of lung cancer by ultrasound microbubble combined with pDsRed2- N1-CD/UPRT fusion suicide gene in vitro

摘要:

目的：探讨超声微泡联合pDsRed2-N1-CD/UPRT融合自杀基因在肺癌A549细胞中的表达水平及其杀伤作用。方法：提取前期构建的pDsRed2-N1-CD/UPRT质粒，转染肺癌A549细胞，分为A组：空白对照组；B组：脂质体+质粒；C组：超声照射+脂质体+质粒；D组：超声照射+微泡+脂质体+质粒。荧光显微镜下观察质粒的转染情况，免疫细胞化学法及Western blot法检测胞嘧啶脱氨酶（Cytosine deaminase，CD）、尿嘧啶磷酸核糖转移酶（Uracil phosphoribosyltransferase，UPRT）在各组细胞中的蛋白表达水平，MTT法检测超声微泡联合CD/UPRT质粒对肺癌A549细胞的生长抑制作用。结果：超声微泡能提高A549细胞中CD/UPRT质粒转染率达（54.8±2.59）%。免疫细胞化学与Western blot结果均显示联合超声微泡后CD、UPRT蛋白表达明显增加（P<0.05）。在前体药物５－氟尿嘧啶（５－Fluorouracil，5-FU）作用下，CD/UPRT质粒转染对细胞有一定的杀伤作用，联合超声微泡后，细胞的生长抑制率明显升高（P<0.05）。结论：超声微泡能提高CD/UPRT质粒在肺癌A549细胞中的转染率及CD、UPRT在细胞中的蛋白表达，从而增强CD/UPRT对肺癌A549细胞的杀伤作用。

Abstract:

Objective：To explore the killing effect and enhancing transduction efficiency of ultrasound microbubble combined with pDsRed2-N1-CD/UPRT fusion suicide gene on lung cancer A549 cells in vitro. Methods：Pre-constructed recombinant plasmid pDsRed2-N1-CD/UPRT was extracted，and then the recombinant plasmid was tranfected to A549 cells. There were four test groups treated by different agents，Group A:blank control；Group B：plasmid with lipofectamine transfection；Group C：plasmid with lipofec－tamine transfection combined with ultrasound irradiation；Group D：plasmid with lipofectamine transfection combined with ultrasound microbubble. The transduction efficiency of target cells was detected by fluorescent microscope. The immunocytochemical method and western blot were used to observe the protein expression of CD/UPRT in A549 cells. Meanwhile，the direct killing effect of CD/UPRT gene on lung cancer A549 cells was measured by MTT assay respectively. Results：Ultrasound microbubble could improve the trans－duction efficiency of CD/UPRT gene into A549 cells to （54.8±2.59）%. Immunocytochemical method and western blot showed the protein expression of CD/UPRT in A549 cells increased significantly when combined with ultrasound microbubble（P<0.05）. CD/UP－RT had slight killing effect on A549 cells treated by 5-fluorouracil. The inhibitory rate against growth of transfected A549 cells in－creased significantly when combined with ultrasound microbubble（P<0.05）. Conclusion：Ultrasound microbubble can enhance the killing effect of CD/UPRT gene on lung cancer A549 cells through promoting the transduction efficiency and protein expression of CD/UPRT，thereby shows curative effect on lung cancer.