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Study on AF-hTERT-TK/GCV Targeted Gene Therapy and Bystander Effect on HCC

Author: YangChangQing
Tutor: DengZhiHua
School: Shanxi Medical
Course: Digestion within the science
Keywords: Cancer Hepatocytes Gene Therapy Bystander effect Suicide gene
CLC: R735.7
Type: Master's thesis
Year: 2008
Downloads: 29
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Abstract

Telomerase is a ribonucleoprotein complex, whose function is to increase the abundance of telomere repeats chromosome ends to maintain its stability and proliferation of malignant cells. Telomerase consists of two groups of wood components, telomerase RNA template hTR and the catalytic subunit hTERT (human telomerase reverse transcriptase). hTERT is the most important component of telomerase, the enzyme catalyzes the whole activity, which promoter is a core of this feature because it Instrument in tumor cells with telomerase activity expressed, it can be used as a more ideal hTERT tumor therapeutic targets, as a breakthrough point, to reach targeted suicide gene therapy for liver cancer purposes. Objective: To construct asialoglycoprotein (AF)-pGL3-human telomerase reverse transcriptase (hTERT) promoter shoulder - thymidine kinase (TK), ie AF-pGL3-hTERT-TK complexes via liposomes transfection methods transfected into tumor cells, observe its effect on HepG2 cell line growth and apoptosis. Method: 1. Cell culture; 2 by digestion product to construct luciferase reporter plasmid plasmid group and treatment group; 3 AF labeled liposomes prepared by liposome transfection method into human hepatoma cells HeptG2 and normal liver cells L-02; 4. using fluorescence microscopy, fluorescence was observed liquid scintillation counting instrument reporter plasmid transfected cells after fluorescence, as well as in cells hTERT promoter driving expression of fluorescence intensity; 5 by TUNEL method, the flow cytometry technique plasmid transfected cells was observed after treatment of cells in cattle on a long, apoptosis and bystander effect situation; 6.Westem blotting technique was transfected AF-pGL3-hTERT-TK hepatoma cell cycle proteins after expression regulatory factor . Results: 1. Successfully constructed luciferase reporter plasmid group: pGL3-basic (negative control), pGL3-control (positive control), pGL3-hTERT-Luc; therapeutic plasmid group: pGL3-basic-TK (negative control), pGL3-control -TK (positive control), pGL3-hTERT-TK. (2) detection using Luciferase Assay System Discovery, pGL3-hTERT-Luc after HepG2 cells transfected with pGL3-control the relative activity of 60%, but was significantly higher than the negative control pGL3-basic; but pGL3-hTERT-Luc transfected telomere L-02 enzyme-negative cells, the fluorescence activity of pGL3-Control meter 13%, indicating that in hepatoma cells hTERT promoter has a strong transcriptional activity; contrast, pGL3-hTERT-Luc plasmid in telomerase-negative The normal liver cells in L-02 medium weak expression of luciferase. 3 TK gene in liver cancer cells can be efficiently hTERT promoter driven expression, almost does not affect the normal liver cell L-02 growth; AF by identifying ASGPR receptor binding to HepG2 cell surface, it carries more TK gene into liver cancer cells, while enhancing TK suicide gene expression results in the bystander effect mechanism with the participation of a total of hepatoma cell apoptosis rate was 85% ± 3%, while the normal liver cells instrument was 16% ± 2%; its mechanism may be is through the cell cycle factor cyclinD1, Cdk2 and p21 regulation to inhibit proliferation of cancer cells on 10. Conclusion: AF-pGL3-hTERT-TK can attack targeting hepatoma cells, normal liver cells has almost no effect, the gene delivery system has the potential for targeted therapy of liver cancer.

Study on AF-hTERT-TK/GCV Targeted Gene Therapy and Bystander Effect on HCC

Abstract

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