Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

Lingping Kong; Lingzhi Wu; Jie Zhang; Yaping Liao; Huaqiao Wang

doi:10.3969/j.issn.1673-5374.2009.06.001

摘要：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 (Aβ25-35).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-Cdk5 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 μmol/L Aβ25-35 for 24 hours.Normal human embryonic cortical neurons treated with 10 μmol/L Aβ25-35 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.