The Empirical Study Of Hepatocellular Carcinoma's Gene Therapy Using HTERTp-TK/GCV Which Was Mediated By Chitosan

Chitosan is the only cationic polysaccharide in nature.It is considered to be a good candidate for gene carrier due to its biocompatibility,biodegradability,low toxicity, bacteriostatic action,antitumous effect,reutilization,abundant ingredient and protection for DNA.Telomerase is a ribonucleoprotein complex whose function is to add repetitive sequences of telomere for chromosomal ends which make the telomere stable and make the cell immortal. Telomerase consists of two essential components,the telomerase RNA template(hTR) and the catalytic subunit(hTERT).hTERT is the most important part of telomerase and can catalyze the full-enzyme,so it is the core of the function.Because it only expresses in tumor cells which express telomerase activity,it can be used as a specific promoter of tumor and can be used to cure cancer specifically.HSV-TK is the most popular suicide gene in the gene therapy of tumor until now.Objective:The aim of this study was to observe the feasibility of chitosan vehicle and the target therapeutic function of the plasmid GC-pGL3-hTERTp-tk on HCC cell line HepG2.Methods:1.Cell culture;2.We constructed luciferase reporter plasmid group and therapeutic plasmid group through conjugating the restrictive enzyme digestive products;3.To make galactosylated chitosan and transfect the HepG2 and the normal hepatic cell L02 with galactosylated chitosan/DNA;4.We wanted to observe the fluorescence and the expression of luciferase gene using the fluorescent microscope and the scintillation counter;5.To observe the cell growth and apoptosis through the Caspase-3 and the flow cytometry.Results:1.We constructed two groups of plasmids.The fluorescence report plasmid group included three kinds of plasmids:pGL3 - basic(negative),pGL3 - control(positive),pGL3 - hTERT Luc +.The treatment group:pGL3 - basic - TK(negative),pGL3 - control - TK(positive),pGL3 -hTERT - TK.2.Through Luciferase Assay system,our results showed that the relative activity of Ch-pGL3-hTERTp-luc+ was 52%which compared to Ch-pGL3-control on HepG2.But it was obviously higher than which of Ch-pGL3-basic.The relative activity of Ch-pGL3-hTERTp-luc+ was 10%which compared to Ch-pGL3-control on L02.The relative activity of GC-pGL3-hTERTp-luc+ was 136%which compared to Ch-pGL3-control on HepG2. 3.Through the flow cytometry,the results showed that the apoptotic rate was 65.28%on HepG2,while it was 10.80%on L02.Conclusion:Chitosan was a good gene vehicle.GC-pGL3-hTERTp-TK could specially attack HCC cell line and almost had no influence on normal hepatic cells.The GC-pGL3-hTERTp-TK has the potential for HCC therapy.