Objective To investigate the expression of telomerase reverse transcriptase (TERT) and cell apoptosis in neonatal rats with hypoxia ischemia brain damage (HIBD). Methods A total of 42 7-day-old SD rats (12-18 g, male or female) were randomly allocated into sham-operation group (n=6) and hypoxia-ischemia (HI) group (n=36). In HI group, the rats were anesthetized with ethylether. The right common carotid artery (CCA) was exposed and permanently l igated with a 7-0silk suture through a midl ine cervical incision. A duration of 2.5 hours of hypoxia (8%O2 / 92%N2) was used to produce HIBD model. For sham-operation group, the CCA was exposed without l igation or hypoxia. The brain tissues were harvested at 4, 8, 12, 24, 48, and 72 hours after completion of an HI insult. The expressions of TERT and CC3 were detected by immunohistochemical staining. The apoptosis cells were detected with TUNEL staining method. Results The expression of TERT was increased at 4 hours after HI injury, significantly increased at 24-48 hours and then decreased at 72 hours. The expression of CC3 was increased at 4 hours after HI injury, significantly increased at 24 hours and still maintained high expression at 48 hours and 72 hours. However, in the sham-operation group, both the expressions of TERT and CC3 were extremely low. The expression of TERT and CC3 were higher in the HI group than in the sham-operation group at different time points, and the differences were significant (P lt; 0.05). The TUNEL staining showed that the positive cells in hippocampus and cortical areas were increased at 4 hours after HI injury, significantly increased at 24-48 hours and maintained a high level at 72 hours. However, there was few positive cells in the sham-operation group. There were significant differences between the HI group and the sham-operation group at different time points (P lt; 0.05). Conclusion TERT could be induced by HI in neonatal rats, and might have a protective role in regulating the cell apoptosis in the neonatal HIBD.
Objective Telomerase reverse transcriptase (TERT) is the key factor to determine cell growth and l ifespan. Meanwhile, it is tightly related to resistance of cell to stress and apoptosis. However, up till now l ittle 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 cultivationof AS. Then AS were transfected with pcDNA3-TERT through l iposomes mediation, and positive clones were selected by G418 and expanded for continuous culture to establ ish the plamid pcDNA3-TERT transfection group. Meanwhile, the empty plasmid pcDNA3 transfection group and the non-transfection group were establ ished as control. The expression of gl ial 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 plamid 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 TERTACM 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 ......（余见正文）