| Objectives:Based on the cloning of human sodium/iodide (hNIS) and human thyroid peroxidase(hTPO), we constructed their recombinant expression plasmids, then transfected human glioma cell lines and human ovarian cancer cell lines with Iipofectamine2000-plasmid complexes respectively, we accordingly obtained stably expressing hNIS cell lines TJ-905-t and ES-two-t, transient co-transfection of hNIS and hTPO cell lines TJ-905-co and ES-two-co, subsequently we investigated their radioiodide uptake function and 1311I inhibitory effect on cell proliferation; at last we established xenografted ovarian cancer nude mice model and investigated radioactive isotope 99mTcO4- imaging and radioiodine 131I treatment effect on xenografted ovarian cancer in vivo, thereby to provide an objective evidence for radioiodine therapy in nonthyroid tumor. Methods:1. Molecular biology experiment: After design PCR primer personally, total RNA was isolated from the thyroid tissue sample of GD patient by TRIzol reagent, the full length of hNIS gene and hTPO gene were amplified by RT-PCR, the target gene were inserted into cloning and expressive vector pcDNA3.1/D-V5-His by TOPO clone methods, then transformed into B. coli TOP10 by calcium chloride methods, after positive bacterium clone's screening and amplifying, the recombinant expressive plasmids pcDNA3. 1D/FLhNIS and pcDNA3.1D/FLhTPO were isolated , then restrictive enzyme digested and sequenced.2. Cellular biology experiment: The human glioma cell lines and humanovarian cancer cell lines were tranfected with recombinant expressive plasmid-pcDNA3.1D/FLhNIS by lipofectamine 2000 respectively, we obtained two kinds of transient expressive cell lines, after their biologic activities were identified primary, screened them with G418 for 2 weeks, we obtained two kinds of stably expressing hNIS cell clones TJ-905-t and ES-two-t, the TJ-905-t and ES-two-t were again tranfected with recombinant expressive plasmid pcDNA3. lD/FLhTPO by lipofectamine 2000 respectively, we also obtained transient co-transfection of hNIS and hTPO cell lines TJ-905-co and ES-two-co, subsequently we investigated their biologic function including radioiodide uptake assay, l25I influx-course, 125I efflux-course and l2SI organification degree assay, furthermore we also investigated l3lI inhibitory effect on kinds of cell proliferation by MTT and cell clonogenic assays in vitro.3. Animal model experiment: Xenografted ovarian cancer were established in nude mice by subcutaneous injection.of 10V0. lmL ES-two-t on the shoulder blade, and controls were injected 107/0. lmL wild type ES-two on the same position. Two weeks after injection, nude mice were received L-T4 (5mg/L) supplementation in their drinking water to maximize radioiodine uptake in the tumor and reduce uptake by the thyroid gland. By 4 weeks after injection, Xenografts had reached 10mm in diameter, intraperitoneal injection of 0. 15mCi 99BTc04-, 40 minutes later, radioactive isotope 99mTc04- imaging was performed using ECT imaging system; for quantitative analysis of the amount of 125I in the tumors or other tissues, their radioactivity were quantified using a well-gamma counter for 1 minute. For 131I therapy studies , mice were divided into large dose group and small dose group, each groupincluding transfection subgroup and control subgroup, intraperitoneal injection of 6raCi or 3mCi 131I respectively, then Xenografted tumor's volume, weight and pathologic morphology change were observed in order to investigate l3lI treatment effect on xenografted ovarian cancer in vivo. Results:1. The full length of hNIS gene and hTPO gene were amplified by RT-PCR, then their recombinant expressive plasmids pcDNA3.1D/FLhNIS and pcDNA3. 1D/FLhTPO were successfully constructed by TOPO clone methods respectively, and the sequences were the same as the results of SM. Jhiang and Kimura S. by restrictive enzyme digested and sequencing analysis.2.Stably expressing hNIS human glioma cell clones TJ-905-t and transient co-transfection of hNIS and hTPO cell lines TJ-905-co were...
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