Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five a...Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.展开更多
BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expr...BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.展开更多
目的研究PC-SPESⅡ对雄激素非依赖性前列腺癌(AIPCa)基因表达的影响。方法雄性Balb/c-nu/nu裸小鼠20只,随机分为对照、PC-SPESⅡ组,每组10只,接种AIPCa细胞DU145建立动物模型。接种第2天起给药,8周后取肿瘤组织,应用human androgen sign...目的研究PC-SPESⅡ对雄激素非依赖性前列腺癌(AIPCa)基因表达的影响。方法雄性Balb/c-nu/nu裸小鼠20只,随机分为对照、PC-SPESⅡ组,每组10只,接种AIPCa细胞DU145建立动物模型。接种第2天起给药,8周后取肿瘤组织,应用human androgen signaling and prostate cancer gene array基因芯片,分析PC-SPESⅡ引起的基因表达变化。为验证基因芯片结果,选择应用RT-PCR方法检测IGF-1mRNA变化。结果30个基因表达下调,包括AIPCa凋亡耐受基因EIF4EBP和FOXj、AIPCa转化基因ADM和PCNA,AIPCa细胞表面标记物PSCA和FAS,以及侵袭性相关基因IGF-1、Met-1、B-myb等。11个基因表达上调,包括AIPCa细胞生长抑制基因PMEPA1和SFRP4,雄激素依赖性细胞表面标记物TMPPSS2、KLK3(PSA)、DD3和STEAP等。结论PC-SPESⅡ可能通过抑制AIPCa凋亡耐受机制、改善AIPCa表型,恢复雄激素依赖性、降低转移能力等多种途径发挥抗AIPCa作用。展开更多
目的:研究抑癌基因KLF6对人前列腺癌细胞系PC-3细胞的生长增殖、细胞周期和对Bc l-2和Cyc lin D1蛋白表达的影响及其可能的作用机制。方法:利用RT-PCR法克隆目的基因KLF6,采用阳离子脂质体介导将含或不含KLF6的pEGFP-C1质粒转染入PC-3细...目的:研究抑癌基因KLF6对人前列腺癌细胞系PC-3细胞的生长增殖、细胞周期和对Bc l-2和Cyc lin D1蛋白表达的影响及其可能的作用机制。方法:利用RT-PCR法克隆目的基因KLF6,采用阳离子脂质体介导将含或不含KLF6的pEGFP-C1质粒转染入PC-3细胞,分别作为转染组和对照组。分别进行噻唑蓝(MTT)法观察PC-3细胞的生长抑制率,流式细胞仪检测细胞周期比例变化和凋亡率,免疫组化法观察PC-3细胞Bc l-2和Cyc lin D1的表达水平变化。结果:转染了抑癌基因KLF6的前列腺癌PC-3细胞生长抑制率为(30.0±5.4)%(对照组为0%,P<0.01);细胞周期比例表现为G2/M期减少为(11.2±0.9)%[对照组为(25.2±2.8)%,P<0.05],G0/G1期比例增加为(80.0±9.8)%[对照组为(58.6±7.3)%,P<0.05];细胞凋亡峰为(24.3±2.3)%[对照组为(5.2±0.7)%,P<0.01];Bc l-2的表达率为(18.7±3.2)%[对照组为(41.8±5.9)%,P<0.01];Cyc lin D1的表达率为(25.3±3.7)%[对照组为(38.5±4.6)%,P<0.05]。结论:抑癌基因KLF6的转染可以明显抑制前列腺癌PC-3细胞的生长增殖,并诱导其凋亡,其作用机制可能与下调Bc l-2和Cyc lin D1的表达有关。展开更多
文摘Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.
基金Key Discipline Key Projects in Guangdong Province (9808)
文摘BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.
文摘目的研究PC-SPESⅡ对雄激素非依赖性前列腺癌(AIPCa)基因表达的影响。方法雄性Balb/c-nu/nu裸小鼠20只,随机分为对照、PC-SPESⅡ组,每组10只,接种AIPCa细胞DU145建立动物模型。接种第2天起给药,8周后取肿瘤组织,应用human androgen signaling and prostate cancer gene array基因芯片,分析PC-SPESⅡ引起的基因表达变化。为验证基因芯片结果,选择应用RT-PCR方法检测IGF-1mRNA变化。结果30个基因表达下调,包括AIPCa凋亡耐受基因EIF4EBP和FOXj、AIPCa转化基因ADM和PCNA,AIPCa细胞表面标记物PSCA和FAS,以及侵袭性相关基因IGF-1、Met-1、B-myb等。11个基因表达上调,包括AIPCa细胞生长抑制基因PMEPA1和SFRP4,雄激素依赖性细胞表面标记物TMPPSS2、KLK3(PSA)、DD3和STEAP等。结论PC-SPESⅡ可能通过抑制AIPCa凋亡耐受机制、改善AIPCa表型,恢复雄激素依赖性、降低转移能力等多种途径发挥抗AIPCa作用。
文摘目的:研究抑癌基因KLF6对人前列腺癌细胞系PC-3细胞的生长增殖、细胞周期和对Bc l-2和Cyc lin D1蛋白表达的影响及其可能的作用机制。方法:利用RT-PCR法克隆目的基因KLF6,采用阳离子脂质体介导将含或不含KLF6的pEGFP-C1质粒转染入PC-3细胞,分别作为转染组和对照组。分别进行噻唑蓝(MTT)法观察PC-3细胞的生长抑制率,流式细胞仪检测细胞周期比例变化和凋亡率,免疫组化法观察PC-3细胞Bc l-2和Cyc lin D1的表达水平变化。结果:转染了抑癌基因KLF6的前列腺癌PC-3细胞生长抑制率为(30.0±5.4)%(对照组为0%,P<0.01);细胞周期比例表现为G2/M期减少为(11.2±0.9)%[对照组为(25.2±2.8)%,P<0.05],G0/G1期比例增加为(80.0±9.8)%[对照组为(58.6±7.3)%,P<0.05];细胞凋亡峰为(24.3±2.3)%[对照组为(5.2±0.7)%,P<0.01];Bc l-2的表达率为(18.7±3.2)%[对照组为(41.8±5.9)%,P<0.01];Cyc lin D1的表达率为(25.3±3.7)%[对照组为(38.5±4.6)%,P<0.05]。结论:抑癌基因KLF6的转染可以明显抑制前列腺癌PC-3细胞的生长增殖,并诱导其凋亡,其作用机制可能与下调Bc l-2和Cyc lin D1的表达有关。
