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
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
目的构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系。方法采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真...目的构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系。方法采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真核表达载体pEGFP-N1,构建最终的表达载体pEGFP-N1-MICA,脂质体法转染Tca8113-Tb细胞,G418筛选,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定过表达MICA基因的Tca8113-Tb细胞系,RT-PCR、real time PCR和免疫细胞化学检测MICA在该细胞中的表达。结果通过PCR技术获取了MICA基因并成功克隆入载体,测序鉴定该序列与GenBank中的序列相同。转染的细胞可见绿色荧光蛋白表达,RT-PCR、real time PCR及免疫细胞化学检测到目的基因MICA在转染细胞中为过表达。结论 pEGFP-N1-MICA真核表达载体的成功构建与稳定转染Tca8113-Tb细胞系的建立,为进一步研究该基因的功能奠定了良好的实验基础。展开更多
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.
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.
文摘目的构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系。方法采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真核表达载体pEGFP-N1,构建最终的表达载体pEGFP-N1-MICA,脂质体法转染Tca8113-Tb细胞,G418筛选,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定过表达MICA基因的Tca8113-Tb细胞系,RT-PCR、real time PCR和免疫细胞化学检测MICA在该细胞中的表达。结果通过PCR技术获取了MICA基因并成功克隆入载体,测序鉴定该序列与GenBank中的序列相同。转染的细胞可见绿色荧光蛋白表达,RT-PCR、real time PCR及免疫细胞化学检测到目的基因MICA在转染细胞中为过表达。结论 pEGFP-N1-MICA真核表达载体的成功构建与稳定转染Tca8113-Tb细胞系的建立,为进一步研究该基因的功能奠定了良好的实验基础。
