Purpose: Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. Materials and Methods: A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. Results: The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, $T_{1/2}$ of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. Conclusion: A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene.
Park, So-Yeon;Kim, Sung-Jin;Lee, Won-Woo;Lee, Heui-Ran;Kim, Hyun-Joo;Chung, June-Key;Kim, Sang-Eun
Nuclear Medicine and Molecular Imaging
/
v.42
no.5
/
pp.394-400
/
2008
Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.
Purpose: A prospective comparison was made between imaging with Tc-99m pertechnetate (Tc-99m) and Ioine-131 (I-131) for the detection of residual and metastatic tissue after total thyroidectomy in patients with well-differentiated thyroid carcinoma. Materials and Methods: Initially our patients had imaging with Tc-99m, followed by I-131 within 3 days. The study included 21 patients who had ablation with high dose of I-131 ranging from 100 mCi to 150 mCi. Planar and pinhole images were acquired for both Tc-99m and I-131. Diagnostic images of Tc-99m and I-131 were compared with post-therapy images. Degree of uptake on Tc-99m and I-131 images was scored by four point scale and compared. Results: The results of the Tc-99m study were: 16 of 19 studies (84%) were positive on simple planar images, but 19 of 20 studies (95%) were positive on pinhole images. Conventional I-131 diagnostic imaging on the other hand showed that all studies (100%) were positive on both planar and pinhole images. There was a significant difference in degree of uptake between Tc-99m and I-131 planar images (p<0.05). Only one case of Tc-99m scintigraphy was negative on both planar and pinhole studies (false negative). There was no distant metastasis on the therapeutic I-131 images. Conclusion: Tc-99m scan using pinhole in certain clinical situations is an alternative to the I-131 scan in detecting thyroid or lymph node metastasis prior to the first ablative treatment after thyroidectomy for well-differentiated thyroid carcinoma.
Purpose: Both human NIS and mutant $D_2R$ transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor ($D_2R$) and compared its characteristics. Materials and Methods: The recombinant plasmid ($pIRES-hNIS/D_2R$) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter $pIRES-hNIS/D_2R$ was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND ($SK-Hep1-hNIS/D_2R$) cells stably expressing hNIS and $D_2R$ was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and $D_2R$ genes. The expressions of hNIS and $D_2R$ were measured by $^{125}I$ uptake assays and receptor binding assays. Specific binding of $D_2R$ to $[^3H]spiperone$ was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. $K_d\;and\;B_{max}$ values were estimated. The correlation between hNIS and $D_2R$ expression was compared by using each clone. Results: Similar quantities of hNIS and $D_2R$ genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. $^{125}I$ uptake in HEP-ND cells was completely inhibited by $KClO_4$, a NIS inhibitor Specific binding to HEP-ND cells was saturable and the $K_d\;and\;B_{max}$ values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and $D_2R$ binding was highly correlated. Conclusion: We developed a dual positron and gamma imaging reporter system of hNIS and $D_2R$ in a stably transfected cell line. We expect that $D_2R$ and hNIS genes can complement mutually as a nuclear reporting system or that $D_2R$ can be used as reporter gene when hNIS gene were used as a treatment gene.
In this study, the several chemical compositions, which include general components (moisture, fat, protein, sugar, fiber, ash, acid insoluble ash), minor components (sesamol, sesamolin, sesamin), the characteristics of oil (specific gravity, refractive index, iodine value, saponification value, unsaponificable matter, insoluble impurities), fatty acid components (analyzed by GLC), amino acid patterns (analyzed by autoanalyzer), of Korean whole white Sesamum indicum were investigated and were compared with decuticled sesame samples. The results were summarized as follows: 1) The crude fiber, total ash and acid insoluble ash contents of the decuticled sesame seed and it meal were significantly lower as compared to the whole sesame samples. 2) The specific gravity, refractive index, iodine value, unsaponificable matter and insoluble impurities contents of the whole sesame oil were greater than the decuticled samples. 3) The fatty acid contents of the whole and decuticled sesame oil were approximatly equal amounts. But unsaturated fatty acid contents of the decuticled sesame oil was significantly lower than the whole sesame oil. 4) The decuticled sasame meal was concentrated higher protein than the whole sasame meal. But amino acid contents of the protein in their was approximatly equal amounts and sesame proteins are found to be rich in methionine, cystine and tryptophan, they are deficient in lysine. 5) The sesamol, sesamolin and sesamin contents of the whole and decuticled sesame oil were approximatly equal amounts. 6) The oxalate and calcium contents of the decuticled sesame seed and its meal were also significantly lower as compared to the whole sesame samples.
A summary of the clinical data of the $^{131}I$-thyroid function tests and the therapeutic results of $^{131}I$ among the 2,658 patients of various thyroid diseases treated over the past 10 years from May 1960 to Oct. 1969 at the Radioisotope Clinic and Laboratory, SNUH were presented and dscussed. 1. The patients examined consisted of: 929 cases (34.9%) of diffuse toxic goiter, 762 cases (28.7%) of diffuse nontoxic goiter, 699 cases (26.3%) of nodular nontoxic goiter, 58 cases (2.2%) of nodular toxic goiter and 210. cases (7.9%) of hypothyroidism. 2. There were 300 (11.4%) male and 2358 (88.6%) female, showing a ratio of 1:8. 3. The majority of patients (79.1%) were in the 3rd-5th decades of their lives. 4. The normal ranges, diagonstic values of $^{131}I$ uptake test, 48 hrs, serum activity, BMR and main subjective symptoms of various thyroid diseases were discussed. 5. In the 579 patients among 867 cases with hyperthyroidism treated with $^{131}I$, 47.8% were confirmed to be cured completely after single therapeutic doses. 6. The complications of $^{131}I$ therapy were discussed and myxedema had developed in 6.75% of our patients. 7. The results of $^{131}I$ thyroid function tests were analysed among the 160 cases of thyroid diseases which were confirmed the diagnosis with histopathological measures.
In order to examine the effect of storage conditions on lipid and minor elements compositions of sesame seed, the changes in general characteristics and fractionation pattern o the lipid, and such minor elements as sesamol, sesamolin and sesamin were analyzed during the storage of sesame seeds for 4, 9, and 15 months at the four different storage conditions, that is, storage under light with linen pouch(LA), storage in dark with linen pouch(DA), storage under light with polyethylene pouch(LS) and storage in dark with poly ethylene pouch(DS). The acid value were increased significantly during the storage for 4 to 9 months then continued same acid value with prolonged storage until 15 months, but iodine value were increased continuously until 15 months in all the storage conditions, and the increasing tendency of the value at LA and DA was higher than that at LS and DS. Steryl ester was increased in the LA but decreased in DS with prolonged storage until 15 months. Diglyceride was increased during the storage in alt the storage conditions and increasing tendency in LA was higher than that in DS and LS. The free fatty acid contents were significantly increased until g months of storage, then similar value was continued with prolonged storage. Phospholipid was decreased with prolonged storage until 15 months and decreasing tendency in DA and LA was sharper than that in LS and DS. The content of free sesamol was ranged from 0.0021 to 0. 0058% of total lipid. Free sesamol was increased but bound sesamol was decreased during the storage for 15 months in all the storage conditions. Sesamolin was ranged from 0.11 to 0.19% of total lipid and decreasing tendency during the storage was similar to free sesamol.
Purpose : In gene therapy, tumor cells expressing the herpes simplex virus thymidine kinase are sensitive to prodrugs. Potential prodrugs IVDU and IVFRU were synthesized and radiolabeled with radioiodine for noninvasive imaging of herpes simplex virus type 1 gene expression. Material and Methods : 5-(2-trimethysilyl) vinyl-2'-deoxyuridine and 5-(2-trimethylsilyl)vinyl-2'-fluoro-2'-deoxyuridine, precursors of 5-(2--iodo)viny l-2'-deoxy uridine(IVDU) and 5-(2-iodo)-2'-vinyl-2'-deoxy-2'-fluororibofuranosyl uracil(IVFRU), were synthesized from reaction of trans-1-trimethylsillyl-2-tri-n-butylstannylethylene with 5-iodo-2'-deoxyuridine and 5-iodo-2'-fluoro-2'-deoxyuridine, respectively, on the condition of Pd catalyst. These precursors were separated from reaction mixture by silica gel column chromatography method. Each precursor was radioiodinated with radioiodine by mixing with ICI oxidizing agent. These radioiodinated compounds were purified with HPLC. Radiohalogen exchange has been shown to be effective for the synthesis of products with lower specific activity. Similarly, carrier-added and high specific activity products have been isolated in respectable radiochemical yields using ICI method. Results : Synthetic yield of precursors, IVDU and IVFRU were 43% and 18%, respectively. Radiochemical purity of both compunds was over 98%. Conclusion : We synthesized precursors of IVDU and IVFRU for monitoring of HSV1-tk gene expression. Radiotracers were radioiodinated with high radiolabeling yield by ICI method.
Edible hardened soybean oil is processed by hydrogenation of refined soybean oil in order to upgrade the heat and oxidation stability and to improve flavor and physical nature. This study aims to investigate the influences of various reaction conditions on iodine value, fatty acid composition and trans isomer formation in hydrogenating soybean oil. In case that hardening temperature is $180^{\circ}C$, trans acid formation increased by 6.2 times more under $3.0{\;}kg/cm^{2}H_{2}$ than under $0.5{\;}kg/cm^{2}H_{2}$, while linolenic acid decreased in contents. In case of $200^{\circ}C$ of hardening temperature trans acid formation showed 4.6% higher under $0.5{\;}kg/cm^{2}H_{2}$ than under $3.0{\;}kg/cm^{2}H_{2}$ while contents of linolenic and linoleic acids showed 0.51% and 2.5% lower respectively. It is concluded that $200^{\circ}C$ of hardening temperature under 0.5 and $3.0{\;}kg/cm^{2}H_{2}$ is better condition because trans isomers are little produced, and iodine value and linolenic acid content decreased in hardening soybean oil.
To evaluate the clinical utility of Tc-99m MIBI scan in the detection of residual thyroid tissue or metastatic lesion in postoperative thyroid cancer patients, we compared Tc-99m MIBI scan with I-131 diagnostic and therapeutic scan in 30 postoperative well-differentiated thyroid cancer patients. Thyroglobulin levels of both on and off thyroid hormone medication and antithyroglobulin antibody were tested [Tg(on), Tg(off)]. Positive rates for I-131 diagnostic and Tc-99m MIBI scan were 81% and 68% respectively. Concordance between I-131 diagnostic and Tc-99m MIBI scan was observed in 15 cases; 12 positive and 3 negative respectively. Among the 6 cases with negative I-131 diagnostic scan and positive Tc-99m MIBI scan, 4 were Positive in the therapeutic I-131 scan We concluded that Tc-99m MIBI scan maybe a useful complementary modality to the currently established method of I-131 scan and serum thyroglobulin level in the detection of recurrent or metastatic thyroid cancer, especially in the case of negative I-131 diagnostic scan.
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