| Objective To establish A549 human nonsmall cell lung cancer cell line that coexpresses human somatostatin receptor subtype-2 and E.coli cytosine deaminase and construct the tumor bearing nude mice model. Observing the characteristics of hSSTR2 mediated reporter gene radionuclide imaging through 99Tcm-RC-160, and the antitumor effect through 131I-RC-160.Methods RT-PCR was used to amplify hSSTR2 cDNA from human embryonic kidney 293 cells. A fusion sequence of CD gene, internal ribosome entry site (IRES) and hSSTR2 gene was generated by gene splicing through overlapping extension (SOE) and PCR, and the expression vector was constructed. The vector was further transfected in vitro to A549 human nonsmall cell lung cancer cells which were then selected. The stable expression of hSSTR2 in established pCIS-A549 cell line was validated by indirect immunofluorescence assay, RT-PCR, Western Blot and Flow Cytometer. The transfected cells were then inoculated into BALB/C male nude mice and tumor bearing mice model was established. Using stannous tartrate as reductant and 99Tcm directly labeled RC-160, the reporter gene in tumor bearing mice were imaged mediated by hSSTR2. Nude mice bearing uninfected CD-IRES-hSSTR2 A549 tumor cells were taken as control. Imaging characteristics were observed at different time points of 30, 60, 120, 240, 480, 720, and 1440min. Then the antitumor effects were observed by intravenous injection of NS, Na131I, 5-FC,131I-RC-160 and 131I-RC-160 and 5-FC together respectively. After the observation, the tumors were dissected from mice under pentobarbital anesthesia for immunofluorescence analysis.Results hSSTR2 gene was successfully cloned and a bicistronic vector of CD and SSTR2 was constructed. pCIS-A549 cell line with stable transfection of the above vector was generated, and the expression of SSTR2 was validated by indirect immunofluorescence assay, RT-PCR, Western Blot and Flow Cytometer. The uptake of 99Tcm-RC160 at tumor lesions was first visualized 30min after the injection, and a significant enrichment was detectable during 4-8h. The image was still detectable at 12h, while tumor lesions in the control group were not detectable all along. In the pCIS-A549 transplanted tumors,contrast to normal sodium groups,the growth of the transplanted tumors therapied by 131I-RC-160 combined with 5-FC , 131I-RC-160 and 5-FC was inhibited significantly,and the inhibition rate is (82.75±4.2)%,(70.69±2.9)% and (66.36±3.7)% respectively, HE stain showed that bulk lamellar cellular necrosis occurred in the transplanted tumors therapied by 131I-RC-160 combined with 5-FC, was up to about 80 percent of tumor tissues. The study on immunohistochemistry showed the brown zones of 131I-RC-160 combined with 5-FC group are less than the other groups.Contrast to the A549 transplanted tumors,salient inhibition effect of 131I-RC-160 combined with 5-FC on the tumors has statistical significance(P<0.1).Na131I has no inhibition effent on transfected or non-transfected transplantation tumors.Conclusions The present study established Human nonsmall cell lung cancer cell line (pCIS-A549) which coexpresses hSSTR2 gene and E.coli CD"suicide gene". 99Tcm-RC160 can be used in reporter gene imaging of pCIS-A549 tumor bearing mice. The present study made an experimental foundation for in vivo study of hSSTR2 mediated radiolabeled ligand targeted killing and tumor therapy when combined with CD/5-FC. The antitumor effects Coupled with the CD suicide gene therapy and SSTR2 targeted radionuclide therapy are have an advantage compared with CD suicide gene therapy or SSTR2 targeted radionuclide therapy single. Using the CD-IRES-hSSTR2 gene and Coupled with the CD suicide gene therapy and SSTR2 targeted radionuclide therapy and/or imaging will be of great benefit for the therapy of tumor. And it may be a novel way to the theraphy of non small cell lung carcinoma and others tumors with no SSTR2 gene expressing.
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