| Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis. It causes nearly 1 million deaths annually worldwide. But only a minority of patients is eligible for surgical therapies due to advanced tumors or extrahepatic diseases at primary diagnosis. Recently, the methods of gene delivery are gradually developing and have made great progresses. So the gene therapy of HCC is a hot spot of the biology and clinic medical currently. Suicide gene therapy can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma due to direct, reasonable and effective effects. HSV-TK/GCV system is a method that applied extensively and achieved favorable effects.The HSV-TK enzyme is almost 1000-fold more efficient at monophosphorylating ganciclovir(GCV) than the mammalian cellular thymidine kinase. Normal mammalian guanylate kinase metabolizes the monophosphate to the diphosphate form (GCV-DP) and then to the triphosphate form (GCV-TP). The triphophate form is highly toxic to cells by inhibiting DNA polymerase and by incorporation into DNA with chain termination.However, HCC frequently occurs in patients with chronic hepatitis and liver cirrhosis, and prognosis of patients is influenced by hepaticreserve. The potentially toxic effects of gene transfer may have marked deleterious consequences. So the target regulation of suicide gene therapy of HCC, that is, the target gene transfer or the specific expression of transfected genes to hepatoma cells, which can kill HCC cells furthest but not damage hepatic function, is vital for curative effect and feasibility.HCC cells are known to express specifically an oncofetal antigen called a-fetoprotein (AFP). The expression of AFP is promoted by combining the specific transcriptional regulation protein (TRP) and transcriptional regulation elements of AFP. Therefore, combining the transcriptional regulation elements of AFP and suicide gene can induce the specific expression of suicide gene only in HCC cells, and the level of expression of the suicide gene is in direct proportion to the level of AFP expression in HCC cells.Several investigators have used the enhancer/promoter of AFP to achieve tumor-specific expression of foreign gene in AFP-producing carcinoma cells. Moreover, when the enhancer of AFP was used alone to regulate the foreign gene driven by non-specific promoter, the expression of foreign gene was not only increased but also specific only to HCC cells. However, the transcriptional activity of the AFP promoter was low.Based on the above progresses, we combined AFP enhancer core sequences and HSV-TK gene in the AFP positive HCC gene therapy and examined the feasibility of increasing the target of HSV-TK/GCV system.AFP is over expressed in about 70% of HCC cases, so it is potential for constructing the suicide gene therapy strategy of HCC targeting AFP-producing cells.In this study, we firstly used indirect immune-fluorescence assay and Western blot to analyze the expression of AFP in the experimental cells. After amplifying the AFP enhancer core sequences, we constructed recombinant plasmid pAFP-LUC that expressed the Luciferase driven by AFP enhancer and CMV promoter. We found that AFP enhancer could greatly increase the expression of Luciferase in the AFP positive cells and well conserve the HCC cell-specific transcriptional activity using report genes analysis. Then we constructed pAFP-TK in which the expression of TK was driven by AFP enhancer and CMV promoter, and hoped that it could provide the experimental data for targeting the gene therapy of HCC.The gene delivery vectors and vectors associated immune response, cell toxicity and safety are the bottleneck of research and clinic application of gene therapy. The traditional gene delivery vectors including viral vectors and non-viral vectors have some disadvantages that are hard to overcome. Nanoparticle is developing as a new non-viral gene delivery vector. Gene therapeutical molecules including DNA, RNA, and so on are encapsulated into or...
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