The Study Of Ultrasound Mediated Microbubble Destruction Combined With Liposome Transfecting Double Suicide Gene Into MCF-7 Cells And The Killing Effects

Background:Breast cancer is one of the most common malignant tumor, threatening the health of women. With the knowledge improvement of tumorous pathogenesis, and the rapid development of molecular biology, gene therapy represents a novel treatment model in cancer therapy. And the suicide gene therapy as a potential strategy for clinical application, is the hotspot of tumor gene therapy in recent years.Suicide gene system kills tumor cells by direct cytotoxic effect and bystander effect, and every system has its characteristic, therefor combined using of different systems can make up for each other and enhance efficacy. Currently the most widely used systems are herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and cytosine deaminas/5-fluorocytosine (CD/5-Fc), and they have highly complementary. Combined using of them has several advantages, such as improving anti-tumor effect, avoiding drug resistance, etc.Targeted expression of gene is related to the security of gene therapy, suicide gene should specifically express only in tumor than normal tissue, that could be realized by making use of specific promoter. Most of the studies demonstrated that kinase domain-containing receptor (KDR) over-expresses in the majority of the solid cancer cells and neogenetic vascular endothelial cells of neoplasma but not in normal cells, so that KDR promoter (KDRp) could make genes of interest over-express in tumor cells and its vascular endothelial cells.In recent years, the focuse of research shift to non-viral vectors, because of viral vectors'security risks. Liposome is the most common non-viral vector, safe and convenient, but its transfection efficiency is low. Microbubble contrast agent is a novel gene vector, with the ultrasound irradiation it can not only transfect naked plasmid into target cells, but also enhance the transfection efficiency of liposome, considering the important part of non-viral vectors research.Objective: 1.To construct the tumor specific vector containing KDRp and CD/TK suicide genes.2.To transfect the pEGFP-KDRp-CD/TK into MCF-7 cells in vitro by ultrasound mediated microbubble destruction combined with liposome, and then investigate the transfection efficiency and the killing effect after given the prodrugs.Methods:1.Fristly, the KDRp, CD and TK gene fragments were amplified with polymerase chain reaction (PCR) technique. Secondly, every PCR product was cloned to T-vector respectively, establishing recombinant T-plasmid T-KDRp, T-CD, and T-TK. Finally, the target gene fragments of recombinant T-plasmids were subcloned to expression vector pEGFP-C2 gradually. The fragments and plasmid vectors were identified by cataphorerising and sequence analysising.2.The optimized ultrasound and microbubble paramaters were used. MCF-7 cells were dividcd into 5 groups,①naked plasmid group,②liposome group,③ultrasound irradiated microbubble group,④ultrasound irradiated liposome group,⑤ultrasound irradiated microbubble+liposome group. The transfection efficieny of pEGFP-KDRp-CD/TK in MCF-7 cells was detected by fluorescence microscope and flow cytometry. The killing effects of double suicide genes on MCF-7 cells (transfected and untransfected) that treated with 0.1mg/ml GCV,2mg/ml 5-Fc or combination GCV and 5-FC were determined by the method MTT.Result:1.The gene fragments amplified with PCR were equal to the expection. The sequence of every recombinant plasmid was identical with the array published in GeneBank. And the length of KDRp, CD and TK gene fragments was 563bp,1184bp and 1131bp respectively.2.The GFP of fifth group was the most and strongest observed by fluorescence microscope. The results of flow cytometry showed that the transfection efficiency of the fifth group was the highest one (39.59±1.19)% compared with the other groups (P<0.05), the fourth group was the next one (27.72±1.21)%, the third group (21.84±1.04)% was as near as the second group (22.96±0.93)%(P>0.05), and the first group is the lowest one (0.78±0.03)%(P<0.001).3.The results of MTT showed that the inhibition ratios of transfected MCF-7 cells were significantly higher than untransfected groups (P< 0.001), the inhibition ratio of transfected MCF-7 cells treated with GCV and 5-Fc was (90.77±2.68)%, obviously higher than the single prodrug group GCV or 5-Fc (P<0.05), furthermore the value of CDI was less than 1, both of these prompt that it has the synergistic killing efficacy between the two suicide gene systems.4.Every inhibition ratio of transfected groups was obviously higher than the transfection efficiency of MCF-7 cells (39.59±1.19)%(P<0.05), showing a significant bystander effect.Conclusions:1.The recombinant plasmid vector pEGFP-KDRp-CD/TK containing KDRp and CD/TK suicide genes was constructed successfully.2.Ultrasound mediated microbubble destruction combined with liposome can enhance the efficiency of gene transfection. The transfection efficiency is much higher than ultrasound irradiated liposome group, ultrasound irradiated microbubble group and liposome group.3.It has the synergistic efficacy between CD/5-Fc and TK/GCV suicide gene systems. The combination of both prodrugs were more effective than either one on the MCF-7 cells transfected with double suicide genes.4.Bystander effect to some extent makes up the weakness of low transfection efficiency, enhancing the feasibility of the transfection method of ultrasound mediated microbubble destruction combined with liposome in suicide gene therapy.5.Ultrasound mediated microbubble destruction combined with liposome transfecting double suicide genes is an ideal strategy for breast cancer gene therapy.