Monitoring Gene Expression Of Tyrosinase Using Magnetic Resonance Imaging

Objective: (1) To study the gene expression of pcDNA3.0-tyr in GM cells (GM0639) from the skin of the normal subject in vitro. (2) To evaluate the factors related to the imaging of the tyrosinase gene expression. (3) To evaluate the use of clinical 1.5-T magnetic resonance (MR) imaging equipment to depict the gene expression of pcDNA3.0-tyr in a mouse model with brain tumor (SHG44) in vivo. Methods: (1) Escherichia coli DH5αwere transfected with pcDNA3.0-tyr eukaryotic expression vector that encodes human tyrosinase by using a CaCl2-mediated transfection protocol, and grown in medium that contained ampicillin. The entire plasmid was isolated and purified by using acidic phenol, and its integrity was determined by means of sequencing and restriction analysis. The plasmids containing human tyrosinase gene cDNA were transfected into GM cells by electroporation. Then cells were lysed and total RNA was isolated. Reverse transcription PCR was performed in transfected cells that showed definite evidence of high levels of tyrosinase mRNA. PCR products were subjected to agarose gel electrophoresis together with size standards. The tyrosinase gene expression and its levels in transfected cells were confirmed by measuring absorption spectrum at 490 nm and staining with the Fontana stain. (2) With the same kind and amounts of cells, the experimental groups comprised mock-transfected cells, cells transfected with 5 μg of pcDNA3.0-tyr, cells transfected with 10 μg of pcDNA3.0-tyr, cells transfected with 15 μg of pcDNA3.0-tyr, and cells transfected with 20 μg of pcDNA3.0-tyr, and their MR signal intensity were compared. (3) With the same amounts of plasmids (10 μg of pcDNA3.0-tyr), but different amounts of cells (1×10~6, 5×10~5, 2.5×10~5, 1.25×10~5, and 6.25×10~4, respectively), MR signal intensity were compared between each group. (4) With the same amounts of plasmids (10 μg of pcDNA3.0-tyr) and cells (106 per well), cells were grown in media that contained additional iron at concentrations of 0.5, 2.5, and 5.0 μg Fe/ml respectively, MR signal intensity were compared among 3 groups. (5) Under the same experimental conditions, in one group cells were cultured for 24 hours after transfection, and exposed to medium contained Fe2(SO4)3 for 48 hours. While in another group cells cultured for 60 hours before exposed to medium for 12 hours. We also compared the MR signal intensity between the two groups. (6) To compare the difference of the tyrosinase activity and the intensity of MR signal between GM cells, SHG44 cells and T98 cells with the same amounts of cells and pcDNA3.0-tyr. (7) Ten athymic Balb/c mice were divided into two groups. SHG44 cells transfected pcDNA3.0-tyr by electroporation were inoculated to subcutaneous tissue at each right foreleg of 6 mice in experimental group. In another group 4 animals were injected mock-transfected cells as a control. Animals were underwent 1.5T MR imaging 4 weeks after inoculation. The axial and coronal plane T1 weighted images and T2 weighted images were obtained with 5-inch coil and self-made device which could improve the signal noise ratio. Finally, the animals were sacrificed after MR examination, and tumor samples were sent for histological and immuno-histochemical study. Results: (1) The result of sequencing confirmed that the eukurytoic expression vector induced contains the full-length human tyrosinase cDNA after checking with the published sequence in Genebank. The plasmid was digested with restriction enzymes and subjected to agarose gel electrophoresis together with size standards. Data from the experiment confirmed two bands. One is a band at 1.8 kb, which coincides with that of tyrosinase. Another band coincides with that of mammalian expression vector pcDNA3.0 at 5.4 kb. Reverse transcription PCR product of transfected cells shows a band that coincides with that of reverse primers at 600 bp, whereas the non-transfected cells don't show this band, which proves that tyrosinase gene is present in transfected GM cells. The value of A490 in transfected cells is lager than that in mock-transfected and non-transfected cells (p<0.001), which indicates a strong activity of tyrosinase. Fontana stain shows that the brownish color granules in pcDNA3.0-tyr transfected cells, whichrepresent induced melanin, and the absence of these in non-transfected cells. (2) The activity of tyrosinase and signal intensity in MR T1WI sequence become higher and higher with the amount of plasmids increasing. There is a close correlation between MR signal intensity in T1WI and tyrosinase activity(r=0.904, p<0.001). (3) With the same amounts of plasmids (10 μg of pcDNA3.0-tyr), but different amounts of cells (1×106, 5×105, 2.5×105, 1.25×105, and 6.25×104, respectively), there is no significant difference in the intensity of MR signal between each group(p>0.05), but the less amount of the transfected cells, the smaller area of the high signal in T1WI sequence. (4) With the same amounts of plasmids (10 μg of pcDNA3.0-tyr) and cells (106 per well), MR signal intensity of cells grown in media that contained additional iron at concentrations of 5.0 μg Fe/ml were higher than that of 0.5 and 2.5μg Fe/ml. (5) Under the same experimental conditions, the transfected cells, cultured for 24 hours before exposed to the metal, show higher MR signal intensity than that cultured for 60 hours before adding the metal(p<0.001). (6) There are no significant difference in tyrosinase activity and the intensity of MR signal between GM cells, SHG44 cells and T98 cells with the same amounts of cells and pcDNA3.0-tyr. (7) The experimental group comprised 4 animals. The tumor tissue manifested hyper intensity in T1WI and hypo intensity in T2WI with the shape varying from homogeneous (1 case), heterogeneous (2 cases) to dotting (1 case). The melanin granules and their front bodies could be found in transfected tumor cells by electric microscopy and immunohistochemical study. Whereas in control group comprising 2 mice there are no hyper signal in T1WI within the tumor and no melanin granules in transfected tumor cells. Conclusion: (1) The plasmids of pcDNA3.0-tyr could be transfected into human GM0639 cells and express and synthetize a large amount of melanin in human. (2) There is a close correlation between the amount of plasmid and signal intensity of transfected cells in MR T1WI sequence, but not a simple linear relation. It is appropriate and economical to transfect 10 μg of pcDNA3.0-tyr into the cells. (3) Less cells transfected can lead to less area of high signal intensity in MR T1 weight images.(4) Under the sameamount of cells and plasmids, the signal intensity of transfected cells depends on the iron concentration and when exposure to the iron occurs. (5) Under the same experimental conditions, GM cells, SHG44 cells and T98 cells show higher signal in MR T1WI sequence, but there is no significant difference between them. (6) In view of convenience in cell culture, SHG44 cell is suit for evaluating the response to gene therapy with MR in human brain glioma models. (7) 1.5T clinical MR unit can be used to image tyrosinase gene expression in small-animal with self-made device.