[Protective effect of conditioned medium from astrocytes transfected with telomerase reverse transcriptase on hypoxia-ischemia neurons].
OBJECTIVE
Telomerase reverse transcriptase (TERT) is the key factor to determine cell growth and lifespan. Meanwhile, it is tightly related to resistance of cell to stress and apoptosis. However, up till now little is known about the role TERT plays in nervous system. To investigate the effect of conditioned medium from astrocytes (AS) transfected with TERT on neurons subjected to hypoxia-ischemia-reperfusion (HI-RP) through construction of in vitro HI-RP model of neurons.


METHODS
An eukaryote expression plasmids containing rat full length TERT gene was constructed as pcDNA3-TERT. Twenty newborn rats at age of 3 days were sacrificed and their cerebral cortex were collected for isolation and cultivation of AS. Then AS were transfected with pcDNA3-TERT through liposomes mediation, and positive clones were selected by G418 and expanded for continuous culture to establish the plasmid pcDNA3-TERT transfection group. Meanwhile, the empty plasmid pcDNA3 transfection group and the non-transfection group were established as control. The expression of glial fibrillary acidic protein (GFAP), which was the specific marker of the AS, was detected by immunocytochemistry, as well as the expression of TERT. Astrocyte conditioned medium (ACM) of the plasmid pcDNA3-TERT transfection group was collected as TERT-ACM, while the ACM of the empty plasmid pcDNA3 transfection group and the non-transfection group were collected respectively as p-ACM and ACM. Next, 60 rats at age of 1 day were sacrificed and their cerebral cortex were collected for isolation and cultivation of neurons. The neurons were randomly divided into experimental group and normal group, the experimental group were further divided into 4 groups including control group, ACM group, p-ACM group, and TERT-ACM group. The neurons of control group were subjected to HI damage in serum-free DMEM, and the neurons of ACM group, p-ACM group, and TERT-ACM group were subjected to HI damage in different medium which contained ACM, p-ACM, and TERT-ACM, respectively. After duration of HI for 3 hours under the environment with 5%CO2, 1%O2, and 94%N2; the neurons of experimental groups were placed in CO2 incubator to imitate RP for 3, 6, 18, 24, and 36 hours in vitro. The neurons of normal group were not subjected to HI and RP treatment. During the treatment of HI-RP, the survival ratio of neurons was detected by means of MTT, the lactate dehydrogenase (LDH) activity of neuron medium with LDH detection kit, and the neuronal apoptosis by means of TUNEL.


RESULTS
The percentages of GFAP positive cells were 98%, 99%, and 98% in non-transfection group, plasmid pcDNA3-TERT transfection group, and plasmid pcDNA3 transfection group, respectively. There was no expression of TERT in no-transfection group and plasmid pcDNA3 transfection group, and the percentage of TERT positive cells in plasmid pcDNA3-TERT transfection group was 98%. Compared with normal group, the survival ratio of the neurons in experimental group were significantly decreased (P < 0.05); the LDH activity of neuron medium and the neuronal apoptosis in experimental group were significantly increased (P < 0.05) throughout the duration of HI-RP. Compared with control group, the survival ratio of the neurons in ACM group and p-ACM group were significantly increased (P < 0.05); the LDH activity of neurons medium and the neuronal apoptosis in ACM group and p-ACM group were significantly decreased (P < 0.05) during the early stage of HI-RP (HI 3 hours and RP 3 hours), but at later time points, there was no significant difference in the above indexes (P > 0.05). Compared with control group, ACM group, and p-ACM group, the survival ratio of the neurons were significantly increased (P < 0.05), and the LDH activity of neurons medium and the neuronal apoptosis were significantly decreased (P < 0.05) in the TERT-ACM group throughout the duration of HI-RP; compared p-ACM group with ACM group, none of the indexes were changed significantly (P > 0.05) throughout the duration of HI-RP.


CONCLUSIONS
Neurons cultured in the conditional medium of the AS transfected with TERT showed stronger tolerability against HI damage; so the conditional medium of AS transfected with TERT may have protective effect on neurons subjected to HI damage.
Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu Sichuan 610041, PR China.
Zhoujin
Z
Duan
Yi
Y
Qu
Fengyan
F
Zhao
Binzhi
B
Tang
Jiao
J
Li
Dezhi
D
Mu
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D007511:Ischemia; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019098:Telomerase / Q000235:genetics*; D014162:Transfection
D017077:Culture Media, Conditioned; D019098:Telomerase