Intraarterial IL-12 Gene Therapy In Combination With TACE: Experiment Study In An Animal HCC Model

ObjectiveThe first purpose of this study was to construct a recombinant eukaryotic expression plasmid pEGFP-C1_IL-12 containing report gene (EGFP) and recombinant human interleukin 12 double-submit(P40 and P35), and to research its anti-tumor effect in vitro. The second purpose was to prepare the cationic product of a polysaccharide isolated from Chinese medicinal herb Bletilla striata, and to investigate the characterization and the possibility of being a non-viral gene vector in vitro and in vivo. The third purpose was to evaluate the anti-tumor and anti-angiogenesis effect of intraarterial IL-12 gene therapy alone and in combination with transarterial chemoemboliztion (TACE) in a rabbit VX2 liver cancer model.Methods and MaterialsTotal RNA was extracted from human umbilical cord blood lymphocytes, from which the total cDNA of human interleukin 12 double-submit was amplified by RT-PCR. The linkaged double-submit connected by overlap PCR was inserted into the MCS of vector pEGFP-C1 to construct a recombinant plasmid, which was identified by PCR and sequencing analysis. Then the recombinant plasmid was transfected into HepG2 cells lipidosome and its expression was detected by fluorescent microscopy, RT-PCR and Western blot.Bletilla striata polysaccharide was prepared by ethanol precipitation followed the hot water extraction Bletilla striata stem and deproteinized with Sevag method. It was further isolated by ion-exchange chromatography on DE-52 column and gel filtrationon Sephadex G-100 column. Cationic polysaccharide was synthesized by conjugation of spermine to oxidized polysaccharide. The ability of the caionic polysaccharide to incorporate and protect plasmid DNA was measured. The ability of the BSP+/pDNA complex to transfect into HepG2 cells was measured. Then, we investigated the transfected effect of the BSP+/pDNA complex in vivo using green fluorescence protein (GFP) gene as a reporter gene administered through the hepatic artery. Rabbits developed VX2 tumor in the liver were randomized into four groups, eight of each. After laparotomy and placement of a self-made 30-gauge needle into the proper liver artery, the following protocols of the interventional procedure were applied: 0.9% saline solution (group A), TACE (group B), intraarterial interlukin-12 gene infusion (group C) and intraarterial interlukin-12 gene infusion in combination with TACE (group D). Growth ratio was estimated by computed tomography. Interferon-gamma level of blood was analyzed by ELISA before and 3day,7day,14day after intervention. All animals were sacrificed 14day after procedure, and the tumor tissue were explanted to analyze vessel endothelial growth factor (VEGF), microvessel density (MVD) and apoptotic index of tumor tissues using immunohistochemistry.ResultsIdentification of pEGFP-C1_IL-12 by PCR and sequencing analysis showed that the length, inserted location and direction was correct, and the expression of reported gene and target gene was observed by fluorescent microscopy, RT-PCR and Western blot.There is no cationoid primary amine and reductive carbonyl in the polysaccharide isolated from the Chinese medicinal herb Bletilla striata. The structure of cationic Bletilla striata polysaccharide contained primary amine group was confirmed by FT-IR. The synthesized cationic product incorporated and protected plasmid DNA to avoidenzymolysis.Tumor growth and angiogenesis were significantly suppressed in group D compared with other groups, characterized by lower tumor growth rate, VEGF, MVD, and higher apoptotic index of tumor cells. Interferon-gamma were higher in group C and group D 3day and 7day after intervention compared to group A and group B, but there is no significantly difference between group C and group D.ConclusionThe cationic Bletilla striata polysaccharide synthesized by grafted spermine to the polysaccharide isolated from the Chinese medicinal herb Bletilla striata could incorporate and protect plasmid DNA, which could be an ideal non-viral gene vector in gene transduction. The BSP+/pDNA complex could target transfect into liver cell in vivo when administered through the hepatic artery using the interventional method, which could produce a marked effect as a new type polycation gene vector in gene therapy.Intraarterial interlukin-12 encoded gene delivery could induce interferon-gamma expression, which may contributed to anti-angiogenesis effect of interlukin-12. Intraarterial interlukin-12 encoded gene delivery combined with chemoembolization could inhibit rabbit VX2 hepatocellular growth significantly, perhaps the anti-tumor and anti-angiogenesis effect were associated with high concentration IL-12 of tumor local when combined with chemoembolization.