Molecular Imaging Of MRI Reporter Of Ferritin Heavy Chain In Vivo And In Vitro

Background MR reporter genes have the potential to monitor transgene expression non-invasively in real time at high resolution. These genes can be applied to interrogate the efficacy of gene therapy, to assess cellular differentiation, cell trafficking,and specific metabolic activity, and also assess changes in the microenvironment. MRI-based approaches are now emerging that rely on:1) enzyme-based cleavage of functional groups;2) expression of surface receptors that enable binding of specific MR contrast agents;3) expression of para- and anti-ferromagnetic (metallo)proteins involved with iron metabolism, such as tyrosinase, transferrin receptor, and ferritin. Thus far, all the reporter genes developed for MRI rely on exogenous administration of contrast material; therefore, delivery barriers and clearance must be considered.Ferritin is a ubiquitous and highly conserved iron-binding protein that consists of variable amounts of heavy (21 kDa H-Ferritin) and light (19 kDa, L-Ferritin) chains and plays a major role in iron homeostasis. Protein binding of iron is generally about 4000 iron ions to form a superparamagnetic iron proteins, causing changes in MRI signal. The studies showed that the expression of ferritin can shorten the T2 of MRI.As the iron protein-based reporter gene is not dependent on exogenous contrast agent, so there are great advantages and broad prospects for development. In this paper, constructed mouse ferritin heavy chain plasmid and transfected C6 glioma cells in vitro and in vivo imaging showed ferritin is a novel MR reporter gene that generates significant contrast in the absence of exogenously administered substrates opens new possibilities for noninvasive molecular imaging of gene expression by MRI.Part One Construction of plasmid of ferritin and transfection of C6 CellsObjective To investigate the impossibility and superiority of a new endogenous MRI reporter-ferritin by construction of the plasmid of heavy chain of murine ferritin and transfection of glioma C6 cells.Methods The fragment of heavy chain of ferritin (Fth) was amplified from murine muscle mRNA using RT-PCR with a set of primers containing digestive sequences of restriction enzyme Not I and Bam HI. The Fth fragment was inserted into pcDNA3.1 vector after digested with NotI and BamHI and ligated with T4 ligase. After identification with restrictive endonuclease analysis, the recombinant plasmid was transfected into C6 cells with the liposome as the transfection reagent. The expression was confirmed by immunohistochemical staining of transfected C6 cells with a specific antibody of anti-H chain of murine ferritin.Results The constructed plasmid was confirmed by digestion with restrictive enzymes and DNA sequencing. Ferritin protein was highly expressed in transfected C6 cells.Conclusion The plasmid of heavy chain of murine was successfully constructed and transfected into C6 cells. Part TWO MR imaging of H-chain of murine ferritin in vivo and in vitroObjective To investigate the feasibility and characteristics of ferritin as a new endogenous MRI reporter by MR imaging of C6 cells transfected with H-chain of murine ferritin in vitro and in vivo.Methods C6 cells transfected with ferritin plasmid and control C6 cells were suspended in 0.2 ml of agarose (1% in PBS) in two eppendorf tubes (1.5×109/tube).T2 and T2* value of different locations near the central voxels were measured on pseudo-color pictures. Statistical analyses were performed using SPSS software. The two independent samples t test was used to determine the statistical significance of the differences cells. Prussian blue staining showed that transfected C6 cells accumulated more iron compared to parental C6 cells. C6 cells transfected with ferritin plasmid and control C6 cells were inoculated bilaterally into high iron model SD rats (n= 8) in the hind limbs. C6 cells were inoculated in left hind limb, while C6-Fth cells in the right hind limb. When diameter of tumors was about 0.8～1.0cm, T2-weighted imaging on a 3 Tesla MRI was performed using a GRE. The analysis and comparison of signal intensity (SI) on T2WI of tumors implanted in the right and left hind limbs of rat by using two independent samples t-test. Prussian blue iron stain of tumor tissue slices showed that ferritin transfected C6 cells accumulated more iron compared to parental C6 cells.Results T2 and T2* values of C6 cells and C6-Fth cells showed statistically significant difference (t=-13.273, P<0.001, t=-3.284, P<0.05). T2 and T2* values of C6 cells were higher than those of C6-Fth cells. Prussian blue stain showed C6 over exprssing ferritin accumulated significantly more iron than C6 control cells. Ferritin transfencted C6 cells and parental C6 cells were inoculated bilaterally into SD rat in the hind limbs. The signal intensity of T2-weighted imaging was measured and analyzed using two independent samples t-test. Ferritin over expressed C6 cells and parental C6 cells showed statistically significant difference (t=-5.292, P<0.001). Ferritin over expressed C6 cells significantly reduced signal intensity in T2-weighted images.Conclusion Our data indicated that over-expressed ferritin can accumulate more iron and reduce signal intensity in T2-weighted images of MRI. This novel cell tracking in vivo with MRI may have potential benefits for research in cellular and gene therapy.