| Objective: To study the value of F-FDG coincidence imaging and registration with integrated CT on diagnosis and treatment of head and neck tumors and the role of glucose transporter proteins in the mechanism of increased uptake glucose of malignant tumor and the correlation of expression of glucose transporter proteins with the biological behaviors of head and neck cancers.Methods: From April 2003 to February 2005, 25 patients with head and neck tumors were examined by CT or MRI and underwent 18F-FDG coincidence imaging use of Gehawkeye coincidence SPECT at 40-60 min after the intravenous injection of 259~298MBq in three weeks, comparing the results. All patients were followed up from four months to twenty months. The results of coincidence imaging were judged in double blind by two doctors of Department of Nuclear medicine.From 2003 to 2005, 38 patients(ages 28-74 years) with a historically verified malignant tumor of the head-and-neck region. None had diabetes. Thirty-six of the patients had squamous cell carcinomas (SCC) with a variety site presentations. Two other patients were adenoid cystic carcinoma (ACC). Multiple tissue specimens of 38 carcinomas and adjacent tissue specimens of 20 patients and normal tissue specimens of 21 patients were examined. The tissue specimens were taken during surgery on different types of head and heck tumors. Tumors were histo-pathologically classified according to the WHO-TNM and clinical stage (Union Internationale Contre Le Cancer, UICC, 2002). All specimens were snap frozen immediately after surgical removal in liquid nitrogen and stored at -80℃ until use. Total cellular RNA was extracted by Rneasy Mini kit method, and cDNA was synthesized by using random hexamers. The reverse-transcribed cDNA from each sample was PCR-amplifed with primers based on the GLUT-1, GLUT-3 and P-actin (internal control) gene sequences. After pre-denaturation at 94 ℃ for 5 min, the cDNA was added to 10 μlof PCR Master mix, comprising 2 μl of 5 X QIAGEN OneStep RT-PCR buffer, 2μl RNase-free water, 0.4 μl of dNTP mix(containing 10 mM of each dNTP), 0.4μl QIAGEN OneStep RT-PCR enzyme mix, 0.5 μl of 50μ M forward primer, 0.5 μl of 50 μ M backward primer. We synthesized the GLUT-1, GLUT-3, and P-actin primers and sequenced them: GLUT-1, forward primer 5'-CAACTGTGTGGTCCCTACGTCTTC-3', reverse 5' -TCACACTTGGGAATCAGCCCC-3'; GLUT-3, forward 5' -AAAGTCCCTGAGACCCGTGGCAGG-3', reverse 5' -AAGATCCAAACCGCAGCCTTG-3'; We used the p-actin gene as the internal control. The sequences of its primers were: forward 5' -CGC TGC GCT GGT CGT CGACA-3' , and reverse 5' -GTC ACG CAC GAT TTC CCG CT-3' . All PCR products were subcloned and sequences were identical to the corresponding partial sequences. Amplification was done in thermal cycler under the following conditions:pre-denaturation at 95℃ for 3 min, denaturation at 94℃ for 30min, annealing at 60 ℃ for 30 min, and extension at 72 ℃ for 10min, followed by a final incubation at 72 ℃ for 7min. The lengths of the PCR products were at 201bp(GLUT-1), 314bp(GLUT-3), and 619bp(p-actin). 10 μl of the PCR products were electrophored and the bands were scanned by a digital gel imaging analyzer (Kodak EDAS290). The gene expression in each sample was expressed as the yield of the target gene relative to that of the p-actin gene.All remain specimens were defrosted and embedded in paraffin. The sections were immunostained with the anti-Glut-1 antibody (1:400) and anti-Glut-3 (1:400), which are rabbit polyclonal antibodies (sera) generated against 13-amino acid synthetic peptides corresponding to the COOH terminus of human Glut-1 and against 12-amino acid synthetic peptides corresponding to the COOH terminus of human Glut-3. A standard Streptavidin-Peroxidase(SP) method was employed to determine the location of Glut-1 and Glut-3 according to the manufacture's instructions. For Glut-1, the internal positive controls were erythrocytes of the same section, and the negative controls were the normal tissue and fibrocytes. For Glut-3, the internal positive controls were inflamma...
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