BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the...BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.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
基金the National Key Basic Research Program of China,No. 2006cb500700the National Natural Science Foundation of China,No.30470904the Natural Science and Technology Foundation of Guangdong Province,No. 04009356, 2008B030301320
文摘BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.
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.
