The Empirical Study That VHL-gene Transfection Mediated By Ultrasound-targeted Microbubble Destruction Intervenes Development Of Renal Cell Carcinoma

Objective:VHL gene which named after Von Hippel-Lindau's disease, is a typical tumor suppressor gene. It can inhibit cell growth, inhibit angiogenesis, regulate cell cycle effects, and inhibit the development of tumor in various ways. Multiple genes are involved in the pathogenesis of renal cell carcinoma, both genetic and sporadic patients can find the abnormity of VHL gene, Therefore, VHL gene is considered the major oncogene of renal cell carcinoma. Our preliminary results show that in renal cell carcinoma VHL gene widespread existences mutation and hypermethylation, And related with Clinical stage, histological type, lymph node metastasis. We believe that the VHL gene's inactivation is closely related to the development of kidney cancer. And that needs to be discussed more in depth.Gene therapy has broad application prospects in cancer treatment. In research of gene therapy,gene transfer methods are the key step to success in gene therapy. Traditional virus gene vector has high transfection efficiency, but the safety and long-term effect of such methods are questioned. Those limited its clinical application. In recent years, Non-viral gene vector is in the researcher's attention and has been extensively studied. Ultrasound-targeted microbubble destruction is a new method of gene transfection which is relatively safe and effective. The cell's cavitation effect which is generated by the microbubble destruction irradiated by ultrasonic can make the permeability of membrane be reversible increased, and promotes gene to transfer into cells. The UTMD has a lot of merits,such as,safety, high performance, strong targeting.In this study,we will further study the VHL gene's effects on regulating the proliferation and apoptosis of renal cell carcinoma,and we also study safety and transfection efficiency of UTMD. We use renal carcinoma cell line in vitro study.We use UTMD to transfect the VHL gene into renal cancer cell line(786-0), then we determine expression of VHL gene in renal cancer cell lines.simultaneously,we also determine proliferation and apoptosis of 786-0.we will elucidate the depressant effect of VHL gene in renal cell carcinoma.And then we use respectively UTMD and Liposome gene transfection technique to transfcet pEGFP-VHL into 786-0. We use immunofluorescence method to determine transfection efficiency. we use respectively UTMD and Liposome gene transfection technique to transfect empty vector into 786-0,and evaluate the cell growth activity with MTT method.We want to prove that the UTMD is a safe and effective strategy of gene transfectiong,and provid a new target and strategy for gene therapy of the renal cell carcinoma.Methods:1.A eukaryotic expression vector pcDNA3.1-VHL was constructed and transfected into 786-0 cells;pcDNA3.1(+) was also transfected as control.After transfection,the expression of VHL in 786-0 cells was detected by RT-PCR.The changes of cell proliferation and apoptosis were observed by MTT and FCM assay.2.A fusion gene expression vector pEGFP-VHL was constructed. Then we used respectively UTMD and Liposome gene transfection technique to transfect pEGFP-VHL into 786-0.We used immunofluorescence method to determine transfection efficiency. we used respectively UTMD and Liposome gene transfection technique to transfect empty vector into 786-0,and evaluated the cell growth activity with MTT method.Results:1.The mRNA expressions of VHL gene in pcDNA3.1- VHL-transfected 786-0 cells were significantly increased than those in untreated and pcDNA3.1(+)-transfected cells. However, there was no difference in VHL expression between untreated and pcDNA3.1(+)- transfected 786-0 cells. The inhibitory rate of pcDNA3.1-VHL-transfected 786-0 cells was significantly higher than those in untreated and pcDNA3.1(+)-transfected cells. Apoptosis analysis by flow cytometry showed that the Apoptosis of pcDNA3.1-VHL-transfected 786-0 cells was significantly higher than those in untreated and pcDNA3.1(+)-transfected cells.2.The pEGFP-VHL gene was transfected in 786-0 cells. After 48h,we detected transfection efficiency. The results of each group were as follows:control group 0, liposome group(35.55±2.77)%,UTMD group(18.27±2.83)%; there was no significant difference in the influence of Cell viability between liposome group and UTMD group.Conclusions:1. After transfecting the VHL gene into 786-0, compared with the negative control group, VHL gene can express in 786-0 cells,And the expression levels of VHL gene's mRNA significantly increase.By MTT method and flow cytometry detection,we find that the cell growth inhibition rate of 786-0 increase significantly,and the apoptosis rate of 786-0 increase significantly too. The results show that the expression of VHL gene can inhibit obviously the proliferation capacity of renal cell carcinoma 786-0,and can promote 786-0 apoptosis.VHL gene is a potential target for gene therapy of renal cell carcinoma.2. Compared with the control group,transfection efficiency of UTMD group elevate in a certain range. Compared with the liposome group,the UTMD group has no significant difference in the influence of Cell viability. The transfection efficiency of UTMD is still lower than that of Liposome transfection.Both specific transfection methods and transfection conditions need to be further optimized.There are still many problems for the UTMD. We need to continue to explore.