The Experimental Study Of Construction Of Group Ⅰ Intron Ribozyme Mediated The Dual-Reporter Gene Imaging Of Carcinoembryonic Antigen

PurposeCarcinoembryonic antigen (CEA) is overexpressed in a majority of carcinomas. Therefore, CEA has been widely used as a serum cancer marker especially for a colorectal tumor, breast tumor and lung carcinoma. Reporter gene imaging potentially allows noninvasive visualization of the evaluation of the tumor therapy, the expression of the tumor gene and the endogenous biological processes. What we focused on was to find a novel reporter system to noninvasively and specially monitor the expression of the mRNA of CEA in real-time. In this study, we developed a specific trans-splicing intron ribozyme type1-based, which can target CEA mRNA and reprogram the mRNA with new transcripts and mediate bioluminescence nuclide dual reporter gene system (Rib53-Fluc-tk). We hope it could achieve a goal that the dual-reporter can directly, specially and noninvasively monitor the expression of CEA mRNA in real-time. Simultaneously, it can be applied into monitor the CEA mRNA after the therapy of siRNA and therefore can evaluate the therapeutic effect of the therapy.MethodsThe novel CEA-targeting trans-splicing ribozyme with the downstream reporter system (Rib53-Fluc-tk) was constructed by the PCR and the sequence was tested. The CEA-expressing cancer cells were transfected with Rib53-Fluc-tk and293T cells were co-transfected with Rib53-Fluc-tk and pCDNA3.1-CEA. The trans-splicing reaction was evaluated using the dual-luciferase reporter assay system, RT-PCR and the determination of131I-FIAU cellular uptake rates. The cell bioluminescence was acquired two days after the transfection of Rib53-Fluc-tk and CEA. Whole-body bioluminescence was acquired24 hours after the co-transfected293T cells were injected into the right hind leg of nude mice. The data were compared between the groups by the independent t test. A probability value P<0.05(two-sided) was considered to be statistically significant.ResultsMCF-7cell showed a high ratio of firefly luciferase/renilla luciferase (P<0.01), while Hela and SK-OV-3cell had a minimal bioluminescence over background levels. After the transfection with Rib53-Fluc-tk and CEA in293T cell, the activity of luciferase showed an increased level along with the increase of the CEA. A product of520bp of band existed, which matched with the predicted size generated with the same primers using the RNA from cells transfected with Rib53-Fluc-tk in MCF-7. No specific product was detected in mock-transfected cells or in SK-QV-3transfected with Rib53-Fluc-tk. The uptake of131I-FIAU in293T co-transfected with CEA and Rib3-Fluc-tk increased with the incubation time and reach1.40%at4.5h, which was three times higher than non CEA transfected293T cell (P<0.01). It showed somewhat weak signal in the cell bioluminescence image, but there were no signal in bioluminescence images or uptake of131I-FIAU in SPECT imaging in nude mice.ConclusionTaking advantage of trans-splicing reaction of the ribozyme, it can somewhat improve the binding property of the reporter gene and target gene. In this study, we succeeded establishing a novel and specific reporter gene imaging methodology but limited with the sensitivity. We hope the trans-splicing efficiency could be evaluated by the genetically code system or modification of the structure of ribozyme. Therefore we can get better images by the dual-reporter gene imaging and move to the next step to evaluate the therapeutic effect of siRNA during the cancer gene therapy. PurposePositron emission tomography (PET) as a unique tool in vivo for imaging biological events has successfully been applied in research and clinic for years. Recently, small animal imaging with PET is developing toward a powerful tool in research offering the possibility to visualize small molecules in living animals. L-[methyl-11C] methionine positron emission tomography ([11C] Met PET) is the most popular amino acid imaging modality in oncology. However, there is limited research work about the kinetic modeling of [11C] Met PET in human and rodents study. Here, the aim of this study was to provide additional information which containing the kinetic parameters and focusing on the best mathematical models to describe the biodistribution of [11C] Met in rodents and the ability to penetrate the blood-brain barrier in rodents.MethodsFour nude-rats and two nude-mice were injected with U87MG cells and YUMAC cells separately. Then1.85～18.5MBq of [11C] Met was injected and a dynamic PET scan was initiated three weeks after the injection. An arterial input function and brain tissue curve were obtained by delineation of a ROI on the left heart ventricle and brain tissue. The kinetic parameters of one-tissue compartment model, two-tissue compartment model and Patlak model were analyzed separately. All reported values are expressed as mean±SD. P values<0.05(two-sided) were considered to be statistically significant. Each of K1(ml/min/mg), k2(min-1), k1/k2(ml/mg), Ki (min-1), VT and SUV were analyzed.ResultsThe two compartment model (one-tissue compartment model,1TCM) gives the best results for the rat brain. In nude rats, the Vt and K1, k2are3.894±0.149(n=4) and 0.157±0.014ml/cc/min (n=4),0.041±0.004/min (n=4) in rat. The three compartment model (two-tissue compartment model,2TCM) gives the best fitting for the mouse brain. In nude mice, the mean value of K1, k2,k3and k4are0.175±0.046ml/cc/min (n=2) and0.115±.0032/min (n=2),0.407±0.095/min (n=2),0.567±0.139/min (n=2); the mean VD is1.535+0.029(n=2), the mean Ki is0.019±0.0001ml/cc (n=2)。ConclusionThe one-tissue compartment model can be considered as a good fitting to describe the uptake of tracer in the brain, while the two-tissue compartment model as a good fitting in the mouse. The VT and K1obtained from one-tissue compartment model can be good parameters for the tracer distribution in the brain tumor. K1can be considered as a predominant value, representing the cerebral flow influx of [11C] Met, while k2corresponds to slow passive efflux out of the brain.