| Objective: Our previous study showed that retinoic acid receptor-beta (RAR P ) gene transcription decreased in human tongue squamous carcinoma cell line (Tca8113) was associated with abnormal cell apoptosis and differentiation . The treatment with all-trans retinoic acid (ATRA) for 14 days induced cell apoptosis by 80.7% of Tca8113 cells transfected with RAR P gene . The purpose of this study was to determine the antitumor effect of RAR 3 gene and ATRA on human oral squamous carcinoma cell xenograft in athymic nude mice and to investigate the mechanism of growth inhibition of tumor in vivo,which was depended on the basis of in vitro study.Materials and methods: JM-109 bacteria were transfected with pSGSRAR P plasmids. The transfected bacteria were cultured in LB media at 37.The pSGSRARP plasmids were extracted from bacteria using plasmid mini extraction kit after the proliferation of the transfected bacteria. The purified plasmid DNA was digested with restriction endonuclease BamH- I and Sac- I ,then were confirmed using 0.8% agarose electrophoresis. The Tca8113 cells were routinely cultured in RPMI-1640 media supplemented with 10% bovine calf serum,2mM 1-glutamine ,100units/ml penicillin and 100 units/ml streptomycin in 95% air and 5% CO: Cells were harvested by trypsinization and resuspended in RPMI-1640 serum-free midia at a concentration of 1 X 107cells/ml .Athymic nude mice (6-7 weeks old, about 20 g in weight) were implanted with 2 X106 cells bilaterally into the buttocks with a 24 gauge needle/Ice tuberculin syringe. After two weeks the animals were divided randomly into three groups: control group, ATRA group and ATRA +RAR P group. There were 6,8 and 6 mice in each group, respectively. The lipofectamine-DNA compounds were prepared by mixing 75 u 1 lipofectamine and 45 u g pSGSRAR P plasmids after both of them were diluted in 500 u 1 RPMI -1640 serum-free media. The compounds were placed at room temperature for thirty minutes, so that the lipofectamine-DNA compounds could be formed. At the 14th and 21th day after the implanting of Tca8113 cells, selected three points around the tumor, there was an interval of 120?between two points .The compounds of lipofectamine-DNA (50 n 1) were injected into the tumor at each point, So that the compound can diffuse better in the tumor. The same volume of normal saline was injected into the tumors of mice in control group and ATRA group. At the same time, ATRA were administered p.o to mice in ATRA and ATRA +RAR 0 group with a 20 gauge intragastric feeding tube daily ,5 days per week, in 0.1 ml of super-refined and sterilized sesame oil at a dose of 15 mg/kg/d for 4 weeks. The sizes of the tumors were recorded every 4 days. The formula for the volume of the tumor was v- x /6(ab2) (a representing the longest diameter of the tumor ; b representing the longest diameter vertical to a). At the end of the treatment, all the animals were killed. Tumors were surgically excised. Some samples were fixed in 10% formalin for 24 to 48 hours before being embedded in paraffin. Hematoxylin and erosin staining were performed for regular pathological examination. Some samples were also prepared for electronic microscopic obseration and flow cytometry test. Lysis precedure was used to analyze the apoptotic ratio.Results: One small fragment of DNA was released from pSGSRAR 3 plasmid after digestion. The size of this cDNA fragment was about 1.4 kb consistent with RAR P cDNA. Tumors were formed in mice after subcutaneous injection of 2X106Tca8113 cells with 80% success rate . The average volumes of the tumors in control group, ATRA group and RAR 0 +ATRA group were 41.4mm3,21.6mm3 and 29.8mm3 respectively. At the end of the treatment, the average volumes of each group were 3111.2mm3, 1428.8mm3 and 653.9mm3, respectively. The Volumes of each group increased 75.1, 66.1 and 21.9 times, respectively. The average weights of the tumors for each group were 2.79g, 0.9Ig and 0.57g, respectively. SAS6.02 version statistical procedure was used for the statistic analyse. The difference of the...
|
PDF Full Text Request