Dissertation > Excellent graduate degree dissertation topics show

In Vitro Experimental Study of HSV1-TK/GCV Suicide Gene Therapy for Hepatocellular Carcinoma Mediated by Ultrasound Contrast Agent

Author: TangYong
Tutor: LiuChangAn
School: Chongqing Medical University
Course: Surgery
Keywords: Hepatic carcinoma Ultrasound contrast agent HSV1-TK/GCV Gene therapy
CLC: R735.7
Type: Master's thesis
Year: 2010
Downloads: 32
Quote: 0
Read: Download Dissertation

Abstract

Gene therapy has opened up new avenues for the treatment of liver cancer, to become one of the effective ways of treatment of liver cancer, more and more attention has been paid, medical biologists. One of the keys of the gene therapy is safe and effective to obtain a carrier-mediated gene transfected target cells. However, the application of viral vectors and non-viral vectors are limitations, can not have security at the same time, the targeting and effectiveness, clinical application is limited. Suicide gene therapy is one of the most effective programs in cancer gene therapy, including herpes simplex virus type 1 thymidine kinase gene / ganciclovir (HSV1-TK/GCV) suicide gene system is the most mature in liver cancer gene therapy . Its principle is HSV1-TK gene to import to the tumor cells, by expression of its encoded thymidine kinase, the low toxicity of the antiviral Ganciclovir phosphorylation, thus preventing the synthesis of DNA, to kill tumor cells. Due to the lack of specificity of the targeting vector system alone transfection efficiency is very low, poor efficacy. Recent studies have found that the accuracy of ultrasound-targeted microbubble destruction can enhance gene transfection, the principle is the use of ultrasound monitoring and crushed to reach the microbubbles within the target tissue, the release of the target gene, gene therapy and targeting. The ultrasonic the microbubbles generated cavitation effect and mechanical effects of the target tissue to increased permeability of cell membrane structure induced the diameter ≤ 7μm microvascular rupture, vascular endothelial cell gap widened, making the exogenous gene more easily penetrate the capillary blood vessels into the tissues and cells; remote targeted microbubbles as gene transfection accelerator, also protective gene in the gene transfer process; and low frequency ultrasonic irradiation conditions, to be transfected gene will not be destroyed. Therefore, the experiments by constructing the enhanced green fluorescent protein (EGFP) and herpes simplex virus type Ⅰ thymidine kinase gene (HSV1-TK) eukaryotic co-expression plasmid vector (pIRES2-EGFP-TK), then the use of low-frequency ultrasonic irradiation microbubble destruction mediated HSV1-TK gene transfection of hepatoma cells to observe the TK gene transfection efficiency and expression in hepatoma cells and the ultrasound microbubble mediated HSV1-TK/GCV killing effect of suicide gene system of liver cancer cells, liver targeted gene therapy in order to provide experimental evidence. The research is divided into the following two parts. The first part of plasmid pIRES2-EGFP-TK construct and its expression in the human hepatoma cell line HepG2 Objective To construct the enhanced green fluorescent protein, and herpes simplex virus type I thymidine kinase eukaryotic co-expression plasmid vector (pIRES2-EGFP-TK) and detect it in the hepatoma cell line HepG2 transfection and expression. HSV1-TK gene fragments obtained from a plasmid containing the gene cDNA pORF-HSV1tk connected to the vector pIRES2-EGFP recombinant plasmid pIRES2-EGFP-TK enzyme digestion, sequencing. The recombinant plasmid with liposome transfected hepatoma cell line HepG2 EGFP expression in cells was observed under a fluorescence microscope, TK protein expression was detected by Western Blot. Results recombinant plasmid digestion and sequencing results expected exactly match the sequence of the herpes simplex virus type 1 thymidine kinase gene HSV1-TK sequences in GenBank report, and inserted into the right direction. The recombinant plasmid transfected tumor cells under a fluorescence microscope to observe the bright green fluorescence, with blank plasmid pIRES2-EGFP transfection fluorescence expression was no significant difference. Western-blot results showed that the recombinant plasmid transfected HepG2 HSV1-TK gene is highly expressed little TK expression in the control group, there is statistical difference between the two, P lt; 0.01. The conclusion of this experiment was successfully constructed EGFP and TK eukaryotic co-expression plasmid, effective expression in hepatoma cells, an experimental basis for further research of liver cancer gene therapy. The second part of ultrasound microbubble mediated HSV1-TK/GCV suicide gene system Objective To investigate the killing effect of human hepatoma cell line HepG2 ultrasound microbubble-mediated destruction efficiency of the human hepatoma cell line HepG2 HSV1-TK/GCV suicide gene system provide experimental evidence for targeted gene therapy for liver cancer. Method HepG2 inoculated culture plates, were randomly divided into the following five groups: (1) blank control group; (2) pure plasmid group; (3) the ultrasound microbubble plasmid group; (4) ultrasound plasmid group; (5) ultrasound ultrasound microbubbles plasmid group. 24 h after transfection, fluorescence microscopy to observe the expression of green fluorescent cells, flow cytometry transfection efficiency of the cells in each group, the MTT assay transfection of HepG2 active cells in each group was detected by Western Blot TK protein expression; 24 hours after transfection, the cells in each group adding different concentrations of ganciclovir (GCV) after 72 hours by MTT assay the survival rate of the cells in each group. Results Ultrasound Ultrasound microbubbles the plasmid group of green fluorescence intensity, cell transfection and TK protein expression were higher than the other groups (p lt; 0.05); transfected HepG2 cell activity had no significant impact (p gt; 0.05) ; the GCV concentration above 50ug/mL for 72h, low-frequency ultrasound ultrasound microbubble plasmid cells almost all the killing, the strongest killing effect (p lt; 0.05), the rest of the group is almost non-killing effect. Conclusion Ultrasound destruction of microbubble contrast agent can improve the HSV1-TK gene transfection and expression in HepG2 hepatoma cell killing effect enhanced HSV1-TK/GCV suicide gene system, ultrasonic irradiation and microbubbles in transfected no effect on cell activity. Our results provide a good foundation for gene therapy for liver cancer targeting.

In Vitro Experimental Study of HSV1-TK/GCV Suicide Gene Therapy for Hepatocellular Carcinoma Mediated by Ultrasound Contrast Agent

Abstract

Related Dissertations