Summary：To investigate the effects of ATRA, acitretin and tazarotene on the growth and apoptosis of human tongue squamous cell carcinoma cell line Tca8113. The effect of retinoids on growth of Tca8113 cells in vitro ...Summary：To investigate the effects of ATRA, acitretin and tazarotene on the growth and apoptosis of human tongue squamous cell carcinoma cell line Tca8113. The effect of retinoids on growth of Tca8113 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis with double staining with Annexin V-FITC and PI, and active caspase-3 analysis with the staining of FITC-conjugated monoclonal rabbit anli-active caspase-3 antibody were made by flow cytometer. Streptavidin-biotin complex （SABC） immunocytochemical assays were employed for the detections of Bax/Bcl-2 proteins expressions. Our results showed that the retinoids inhibited growth of Tca8113 cells in a dose-and time-dependent manner with maximal inhibition 24 h after treatment of 10 5 mol/L. 10^-5 mol/L retinoids altered cell cycle distribution of Tca8113 cells, revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. 10^-5 mol/L retinoids significantly induced apoptosis of Tca8113 cells （all P〈0.05）, elevated the cells population with detectable active caspase-3 （P〈 0.05 for all）, increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly （all P〈0.05）. Acitretin played a most prominent role among the retinoids. It is concluded that the inhibition of cell cycle progress of Tca8113 cells by ATRA, acitretin and tazarotene is one of the possible mechanisms for proliferation arrest of TcaS113 cells elicited by the retinoids. The retinoids mediate apoptosis in TcaS113 cells that may be caspase-dependent through mitochondria pathway. High concentration retinoids inhibit growth of Tca8113 cells in vitro by interfering with proliferation and inducing apoptosis of cells. Acitretin may be an alternative medicine for the prevention and treatment of tongue squamous cell carcinoma.展开更多
Some aspects of current research have focused on its relationship with malignant diseases and on its chemothearapeutic role. An attempt was made to determine the inhibitory effects of selenium on Tca8113 cells in vivo...Some aspects of current research have focused on its relationship with malignant diseases and on its chemothearapeutic role. An attempt was made to determine the inhibitory effects of selenium on Tca8113 cells in vivo. In the experimental study, Na<sub>2</sub>SeO<sub>3</sub> effectively limited the growth and proliferation of Tca8113 cell in vivo. The response was dependent on the dose, the starting administered time, exposed length of selenium and the concentration of inoculated Tca8113 cells. The morbidity of transplanted tumors was remarkably decreased with enhancing dose of selenium. At 60μg ip dose, the weight of nude mice were not remarkably reduced, and the pathological changes in liver and kidney had not been found in this experiment.展开更多
Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retrovira...Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.展开更多
Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell ...Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.展开更多
文摘Summary：To investigate the effects of ATRA, acitretin and tazarotene on the growth and apoptosis of human tongue squamous cell carcinoma cell line Tca8113. The effect of retinoids on growth of Tca8113 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis with double staining with Annexin V-FITC and PI, and active caspase-3 analysis with the staining of FITC-conjugated monoclonal rabbit anli-active caspase-3 antibody were made by flow cytometer. Streptavidin-biotin complex （SABC） immunocytochemical assays were employed for the detections of Bax/Bcl-2 proteins expressions. Our results showed that the retinoids inhibited growth of Tca8113 cells in a dose-and time-dependent manner with maximal inhibition 24 h after treatment of 10 5 mol/L. 10^-5 mol/L retinoids altered cell cycle distribution of Tca8113 cells, revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. 10^-5 mol/L retinoids significantly induced apoptosis of Tca8113 cells （all P〈0.05）, elevated the cells population with detectable active caspase-3 （P〈 0.05 for all）, increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly （all P〈0.05）. Acitretin played a most prominent role among the retinoids. It is concluded that the inhibition of cell cycle progress of Tca8113 cells by ATRA, acitretin and tazarotene is one of the possible mechanisms for proliferation arrest of TcaS113 cells elicited by the retinoids. The retinoids mediate apoptosis in TcaS113 cells that may be caspase-dependent through mitochondria pathway. High concentration retinoids inhibit growth of Tca8113 cells in vitro by interfering with proliferation and inducing apoptosis of cells. Acitretin may be an alternative medicine for the prevention and treatment of tongue squamous cell carcinoma.
基金Supported by the Chinese National Nature Scientific Foundation
文摘Some aspects of current research have focused on its relationship with malignant diseases and on its chemothearapeutic role. An attempt was made to determine the inhibitory effects of selenium on Tca8113 cells in vivo. In the experimental study, Na<sub>2</sub>SeO<sub>3</sub> effectively limited the growth and proliferation of Tca8113 cell in vivo. The response was dependent on the dose, the starting administered time, exposed length of selenium and the concentration of inoculated Tca8113 cells. The morbidity of transplanted tumors was remarkably decreased with enhancing dose of selenium. At 60μg ip dose, the weight of nude mice were not remarkably reduced, and the pathological changes in liver and kidney had not been found in this experiment.
文摘Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.
文摘Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.
