To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme fo...To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods： PSA enhancer （PSAE） and promoter （PSAP） sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results： The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion： An expression vector containing the Smac gene （based on elements of the PSA gene regulatory sequences） has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.展开更多
Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and ...Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.展开更多
To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-m...To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound spe-cifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunc-tional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding pro-teins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA pro-moter.展开更多
目的 构建前列腺特异性膜抗原启动子增强子靶向性调控的 U PRT基因真核表达重组质粒 (p PS-MAenhancer/ promoter- U PRT)。方法 采用 PCR技术从大肠杆菌 JM10 9基因组中扩增 U PRT基因 ,通过分子克隆技术将其克隆到包括前列腺特异性...目的 构建前列腺特异性膜抗原启动子增强子靶向性调控的 U PRT基因真核表达重组质粒 (p PS-MAenhancer/ promoter- U PRT)。方法 采用 PCR技术从大肠杆菌 JM10 9基因组中扩增 U PRT基因 ,通过分子克隆技术将其克隆到包括前列腺特异性膜抗原启动子增强子靶向性调控的 p EGFP- 1的质粒。利用重组质粒 p PS-MAenhancer/ promoter- U PRT)转染 L Ncap细胞 ,MTT法检测 5 -氯尿嘧啶 (5 - FU)对转染 L Ncap细胞存活率的影响。结果 质粒 p PSMAenhancer/ promoter- EGFP双酶切去除 EGFP,连接上 U PRT基因 ,成功构建 p PSMAenhancer/ promoter- UPRT,并转染 L Ncap细胞 ,使 L Ncap细胞对 5 - FU的杀伤敏感性大大提高。结论 p PSMAenhancer/ promoter- U PRT的构建和表达 ,为进一步研究 UPRT基因对 5 - FU的靶向性杀伤前列腺癌细胞增强作用和对前列腺癌自杀基因系统 (CD/ 5 -FC系统 )的放大效应奠定了基础。展开更多
基金This work was supported by National Natural Science Foundation of China(30271301)
文摘To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods： PSA enhancer （PSAE） and promoter （PSAP） sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results： The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion： An expression vector containing the Smac gene （based on elements of the PSA gene regulatory sequences） has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.
文摘Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.
文摘To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found in-dispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound spe-cifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunc-tional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding pro-teins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA pro-moter.
文摘目的 构建前列腺特异性膜抗原启动子增强子靶向性调控的 U PRT基因真核表达重组质粒 (p PS-MAenhancer/ promoter- U PRT)。方法 采用 PCR技术从大肠杆菌 JM10 9基因组中扩增 U PRT基因 ,通过分子克隆技术将其克隆到包括前列腺特异性膜抗原启动子增强子靶向性调控的 p EGFP- 1的质粒。利用重组质粒 p PS-MAenhancer/ promoter- U PRT)转染 L Ncap细胞 ,MTT法检测 5 -氯尿嘧啶 (5 - FU)对转染 L Ncap细胞存活率的影响。结果 质粒 p PSMAenhancer/ promoter- EGFP双酶切去除 EGFP,连接上 U PRT基因 ,成功构建 p PSMAenhancer/ promoter- UPRT,并转染 L Ncap细胞 ,使 L Ncap细胞对 5 - FU的杀伤敏感性大大提高。结论 p PSMAenhancer/ promoter- U PRT的构建和表达 ,为进一步研究 UPRT基因对 5 - FU的靶向性杀伤前列腺癌细胞增强作用和对前列腺癌自杀基因系统 (CD/ 5 -FC系统 )的放大效应奠定了基础。
