Study On The Inhibition Of Breast Cancer Transplantation Tumor Of Nude Mice With Double Suicide Gene Transfected By Ultrasound Mediated Microbubble Destruction In Vivo

BackgroundBreast 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. Currently the most widely used systems are herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and cytosine deaminas/5-fluorocytosine (CD/5-Fc). Combined using of them has several advantages, such as improving anti-tumor effect, avoiding drug resistance, etc.The specific expression of suicide genes in the tumor cells so as to selectively damage tumor cells could be realized by taking advantage of some tumor specific transcription modulating elements(promoter or enhanser). So, the anti-tumor effect is quite satisfied with mild side-effects. It is the main dominance of suicide gene therapy in clinic application. So that KDR promoter (KDRp) could make genes of interest over-express in tumor cells and its vascular endothelial cells.In recent years, 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.The project planed to apply CD/TK fusion suicide gene system driven by VEGF promoter to MCF-7 tumor cells, transfecting by ultrasound mediated microbubble destruction. Then, the effect of the system therapy for breast cancer was observed. So, the study Was carried out to provide further experimental evidences to treat the breast cancer with double suicide genes.Objective 1.Construction of nude mice model bearing human breast cancer (MCF-7 in vivo).2.To transfect the pEGFP-KDRp-CD/TK into breast cancer model in nude mice by ultrasound mediated microbubble, and then investigate the anti-tumor effect of the treatment system on breast cancer in vivo.MethodsConstruction of nude mice model bearing human breast cancer,20 nude mice were randomly divided into 4 groups (n=5). GroupⅠ(control group):only injection of 5-FC and GCV. GroupⅡ:injection of naked plasmid,5-FC and GCV. GroupⅢ:injection of naked plasmid,5-FC and GCV, ultrasonic irradiation was given at the same time. GroupⅣ: injection of targeted ultrasound contrast agents,5-FC and GCV, ultrasonic irradiation was given at the same time. Measurement of tumor size from the day of treatment every 3 days to calculate the tumor' s size and observe the tumor growth of different groups. The nide mice were killed after end of the treatment, the tumor were removed and weighted to calculate the inhibition rate compared with the control group. The target gene transfer situation in vivo was observed by inverted fluorescence microscope to calculate the gene transfer rate. Target gene expression was detected by RT-PCR. Microvessel density was counted by immunohistochemistry. The growth of tumor and target gene transfection in vivo were observed by indicators above to assess the tumor inhibitory effection of the tumor in nude mice by ultrasound microbubble mediated double suicide gene transfer.Results1.The inhibition rate of groupⅡ, groupⅢ, groupⅣis 3.72%, 21.40%,47.13%, the tumor size between groupⅡand groupⅠshowed no statistically significant difference(P>0.05). The tumor size of groupⅢand groupⅣshowed statistically significant difference compared with groupⅠ(P<0.05). The inhibition of tumor size of groupⅣwas most significant.2. The gene transfection rate of group IV mediated microbubble was higher than that of groupⅢ, and there was statistically significant difference the two group (P＜0.001). The gene transfection rate of group Ⅲwas higher than that of groupⅡ, but there was no statistically significant difference the two group (P>0.05)3. CD/TK expressed in the tumor of groupⅢand groupⅣ, but not expressed in the tumor of groupⅠand groupⅡ.4.The tumor angiogenesis inhibition of group IV mediated microbubble was higher than that of groupⅢ, and there was statistically significant difference the two group (P<0.05). That of groupⅢand groupⅣcompared with control group, there were statistically difference (P<0.05). The MVD count of groupⅡwas lower than that of control group, but there was no statistically significant difference the two group (P>0.05).Conclusions1. Construction of nude mice model bearing human breast cancer with subcutaneous inoculation has the advantages of simply operation, stability, high success rate. The model is able to maintain some of the human breast tumor' s biological characteristics and pathological features.2. Construction of nude mice model bearing human breast cancer with MCF-7 cells, the growth of transplanted tumor could be observed conveniently.3.Ultrasonic irradiation can increase the microvascular permeability of transplanted tumor and promot transfection of genetic material into cells. Ultrasound mediated microbubble can transfer target gene more efficiently to enhance gene transfer rate.4. Ultrasound mediated microbubble can transfer target gene expressed in tumor tissue, which making a satisfactory tumor inhibition by the treatment system showed the inhibition of tumor growth and decline in tumor MVD.5.Ultrasound irradiate microbubble mediated double suicide gene system is a perfect strategy of gene therapy for breast cancer.